Lamp primers

Lamp primers DEFAULT

Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers

Abstract

Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.

Introduction

In molecular diagnostics, isothermal nucleic acid amplification methods are an attractive alternative to benchmark polymerase chain reaction (PCR)1 strategies because of the low cost equipment required to initiate and maintain the reaction. This is of particular relevance to low resource settings and point-of-care applications. Numerous methods have been described2,3,4 for isothermal amplification of which loop-mediated amplification (LAMP)5 is the most widely reported. LAMP based assays have been used for numerous applications including the detection of pathogens such as malaria6 and salmonella7, viral RNA rapid detection for HIV with reverse transcriptase LAMP (RT-LAMP)8, GM crop contamination9 and in forensic science to specifically detect human DNA10.

The LAMP reaction is initiated by strand invasion of the DNA template by hairpin-forming LAMP primers which anneal and extend catalysed by a strand displacing DNA polymerase. These annealed LAMP primers are in turn displaced by displacement primers in the initiation of amplification, and lead to the formation of a dumbbell-like structure. This structure forms the basis of cycle amplification and elongation, with only LAMP primers, into cauliflower-like formations of single stranded DNA loops. The LAMP primers FIP and BIP (forward and backward inner primer), are designed to hybridise to the complementary and reverse complementary target sequences of F2/B2 and F1/B1 (Fig. 1). Displacement primers are referred to as F3 and B3. The addition to the reaction of Loop primers11 and STEM primers12 accelerates the DNA amplification by hybridising and extending from the hairpin loops or the region between loops respectively. Faster reaction times have been shown with the addition of swarm primers13. LAMP amplification is typically achieved at 60 to 65 degrees C for a time period dependent on the concentration of the template. Highly desirable characteristics of LAMP includes high sensitivity and specificity with rapid reaction times and there is also evidence that LAMP amplification will proceed in the presence of PCR inhibitors9,14,15 permitting less stringent DNA extraction procedures.

LAMP amplification initiation, cycling and elongation. Strand invasion of double stranded DNA with LAMP and displacement primers initiates the LAMP reaction. Positions of primer recognition sites are shown in red for displacement primers, green for LAMP primers and blue for Loop primers. LAMP initiation leads to the formation of dumbbell-like structures with cycling between the two forms and elongation into cauliflower-like structures with multiple loops. The fluorophore is shown unquenched attached to the FIP primer and quenched by proximity to guanine bases due to fluorescence resonance energy transfer (FRET).

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PCR has been developed to increase the specificity of the two primer amplification method. Real time PCR16 developments have included amplicon specific TaqMan probes17 which are quenched when unbound, but the fluorophore is unquenched by release from the probe because of the 5′ to 3′ exonuclease activity of Taq polymerase. Molecular beacons18 are self-quenching oligonucleotides with a template specific loop structure which when bound to the target sequence unquenches the fluorophore, unbound molecular beacons remain quenched. The increased specificity of these example methods, combats the false positive results that can affect indirect methods of amplification detection such as intercalating dyes and gel electrophoresis.

Although LAMP isothermal amplification with Loop acceleration utilises six primers targeting eight target DNA sequences, the incidence of false positive results has been reported19. LAMP assays may use indirect methods of amplification detection and these, whether real-time or end-point, rely on careful primer design, optimal reaction conditions and robustness testing to negate false positives. Such indirect detection strategies include the addition of intercalating dyes for fluorescence or colorimetric determination, turbidity20 from pyrophosphate precipitation, observations of LAMP ladder patterns with agarose gel electrophoresis5, quenched calcein21, hydroxy-naphthol blue22 as well as a number of fluorescent probe based techniques15,23,24. Real-time methods include bioluminescent reporting (BART)25 whereby the increase in inorganic pyrophosphate during nucleic acid amplification is converted to ATP which is converted into detectable light using a thermostable luciferase and luciferin substrate. The incorporation of intercalating dyes such as SYBR Green26,27 and the SYTO family28,29 generate increased fluorescence with increasing double-stranded DNA propagation. BART and SYTO9 real time LAMP amplification detection are used in this report. Other real time methods include turbidimetry30, colour change with pH sensitive dyes31 and amplifying with the nucleic acid specific dye berberine32.

To improve the specificity of LAMP, a number of solutions involving fluorescent labelled probes and primers have been proposed. Fluorescent probes used for quantitative PCR such as for example molecular beacons33, have been adapted to LAMP, however the displacement activity of the polymerase and lack of thermal cycling make such adaptations problematic. Fluorescently labelled oligonucleotide probes targeting the LAMP loop structures34 offer increased specificity when visualised after precipitation with polyethylenimine (PEI) which forms an insoluble fluorescent complex. Proximity to guanine nucleotides had been previously shown to quench certain fluorophores35 and forms the basis of a number of specific LAMP detection methods. Alternately binding quenching probe competitive LAMP (ABC-LAMP)15 is another oligonucleotide probe method for increased specificity, whereby the AB-Q probe with a 5′ fluorophore hybridises to the target loop and specially designed competitor DNA which has a number of guanines replaced with cytosines in proximity to the fluorophore. Real time LAMP amplification will therefore show a reduction in fluorescence in the presence of specific loop sequences. Fluorescent labelling of the loopF primers for fluorescence quenching during LAMP amplification by proximity to guanine36,37 enabled different target LAMP amplifications to be detected. Further multiplex detections were shown with melt LAMP (mLAMP)38,39 designed to use fluorescent labelled FIP primers for end point detection following real time turbidimetry of multiple targets.

Fluorescence resonance energy transfer (FRET) methods such as duplex LAMP23 utilise an acceptor intercalating dye and 5′ end FAM labelled BIP primer for one gene target sequence and no fluorophore for another. Increase or decrease in fluorescence is indicative of the gene present. A FRET LAMP assay19 utilises a combination of donor probe, acceptor probe and a hybridisation probe to detect white spot syndrome virus by fluorescence. Another probe method for increased specificity is the detection of amplification by release of quenching technique (DARQ)40 whereby a quencher linked to the 5′ end of a FIP primer quenches a hybridised fluorescent probe which is released during amplification. Assimilating probe24 employs a similar methodology. One step strand displacement LAMP amplification (LAMP-OSD)41 utilises a quenched reporter consisting of Reporter F and Reporter Q probes. Reporter F has an additional 11 nucleotide toehold sequence and the probe hybridises to a specific target loop of the amplification. The probe is unquenched and fluorescence can be detected. Graphene Oxide based FRET LAMP42 is another approach that uses a fluorescent probe, but with graphene oxide quenching the DNA fluorophore complex. Quenching of unincorporated amplification signal reporters (QUASR)43 offers an alternative approach for increased specificity using temperature to determine the presence of target. Labelling FIP and BIP primers with various fluorophores and dot-ELISA44 was used in a further specific multiplex LAMP assay and recently fluorescence of Loop primer upon self dequenching (FLOS LAMP)45 used modified thymine residues towards the 3′ end of primers with linked fluorophores so that during LAMP amplification the probes were unquenched.

In our work we have combined the 5′ fluorescent labelling of LAMP primers with quenching by LAMP amplification to provide a simple and specific method of DNA quantification and detection. The LAMP primers are ideally suited to this purpose because they are both essential and lead to the formation of complex looped structures. The attachment of a fluorophore does not appear to compromise LAMP target sensitivity and detection times remain rapid when compared to qPCR. We further show how the labelling of different primers, combined with non-specific detection of amplification with SYTO9 can be used to inform the primer optimisation process in designing LAMP assays. Furthermore we show that multiplex assays are possible with this method.

Results

The ‘cauliflower-like’ structures which are rapidly generated in LAMP amplification potentially bring into proximity the 5′ end of a Loop primer with the 5′ end of a LAMP primer (Fig. 1). We investigated using a FAM labelled LoopF and a JOE labelled FIP to explore whether fluorescence resonance energy transfer (FRET) could be observed between these two fluorophores by excitation of FAM transferring energy to JOE for increased fluorescence (Fig. 2). However we observed a decrease in fluorescence with time from JOE and upon further investigation it was clear that the fluorescence of both of these fluorophores was quenched during the LAMP amplification. Furthermore the time at which quenching occurred was proportional to the original concentration of the DNA template, suggesting it was linked to the increasing amount of LAMP amplicon.

Quenching of fluorescence. LAMP amplification with 35S promoter primers substituted with a FIP primer labelled with JOE and LoopF primer labelled with FAM. Dilution series of a linear plasmid template and with additional no template control. Quenching of the FAM signal from the FAM-LoopF primer (A) and quenching of the JOE signal from the JOE-FIP primer (B). Four replicates for each dilution of template and for NTCs. Average Ct (cycle threshold set at − or −) against logarithmic scale of the template dilutions for JOE-FIP (C) and FAM-LoopF assays (D). Three out of four samples were positive at 50 copies, one out of four samples were positive at 5 copies. The R2 values with the highest concentration of template removed were and for JOE-FIP and FAM-LoopF respectively.

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The results in Fig. 2 show that the labeling of either the FIP primer or the LoopF primer produced similar results in terms of degree of signal quenching and the timing of the quenching. The non-template controls remained constant throughout the assay time of  minutes. All four replicates of samples with copies or more of linear plasmid template were detected, but only 75 percent of the samples with 50 copies and 25 percent of those with 5 copies. However the positive results at these low concentrations of template were closely aligned with quantitative semi-logarithmic trend lines (Fig. 2C,D).

The concentration of primers and the ratio of primer combinations which contain the fluorescently labelled FIP were then optimised and shown in Fig. 3. Best results were obtained with to micromolar JOE-FIP (Fig. 3A,E).

LAMP primer combinations with JOE labelled FIP. LAMP amplification and fluorescent detection of copies per 5 microlitres linear plasmid DNA using 35Sp primers; (A) micromolar JOE-FIP with 1x other primers ( micromolar BIP, micromolar LoopF and LoopB, micromolar F3 and B3), (B) micromolar JOE-FIP, micromolar FIP, 1x other primers, (C) micromolar JOE-FIP, micromolar FIP, 1x other primers, (D) no fluorescently labelled primers, 1x primers, (E) micromolar JOE-FIP, 1x other primers, (F) micromolar JOE-FIP, 1x other primers, (G) micromolar JOE-FIP, 1x other primers, (H) micromolar JOE-FIP, micromolar BIP, 1x other primers, (I) micromolar JOE-FIP, 2x other primers. Red lines: no template control; blue lines: samples with copies of target.

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The clearest separation between the detection of amplified template and the negative template controls (NTC) coupled with low variation between the replicates was observed with micromolar JOE-FIP and the standard concentration of the other primers (Fig. 3A). The concentration of micromolar JOE-FIP with standard concentration of the other primers (Fig. 3E) was better separated with less replicate variation than micromolar JOE-FIP with micromolar FIP (Fig. 3B). However micromolar JOE-FIP with micromolar FIP (Fig. 3C) was also better than micromolar JOE-FIP and FIP. The higher concentration of JOE-FIP of micromolar (Fig. 3G,H,J) all showed reduced initial quenching when compared to the lower concentration of JOE-FIP with a further reduction in quenching towards the end of the assay.

Using the optimised ratio and concentration of primers in Fig. 3A the quenching LAMP method was compared to LAMP detection with the intercalating dye SYTO9. The SYTO9 was used in the presence or absence of JOE-FIP primer to gauge the impact of the labelled primer on the assay kinetics. In Fig. 4,1, the assay with the JOE-FIP primer in the presence of SYTO9 was acquired in the yellow channel to detect the fluorescence signal from JOE, and the assay with template copies had an average Ct of (SD ) and for 50 copies an average Ct of (SD ), at a defined cycle threshold of − In Fig. 4,2, the JOE-FIP primer with SYTO9 assay was acquired via the green channel to detect the fluorescent signal from SYTO9. The template copies assay had an average Ct of (SD ) and for 50 copies this figure was (SD ) at a cycle threshold of The replicate assay with unlabelled FIP and SYTO dye had more rapid Ct values of (SD ) for the copies and (SD ) for the 50 copies at the cycle threshold of (Fig. 4,3).

Detection with JOE-FIP and SYTO9. LAMP amplification with 35S promoter primers of a linearised plasmid template (1) quenched fluorescence in presence of JOE-FIP primer with SYTO9 using yellow channel detection of JOE, (2) fluorescence of same samples with green channel detection of SYTO9, (3) fluorescence with unmodified primers with SYTO9 only (green channel). (A) Normalised fluorescence truncated from 60 minute assay, (B) melt curve analysis, (C) average Ct plotted against template concentration on a log scale and (D) summary of replicate Ct values, average Ct and standard deviation (SD) for each dilution. NTCs (red lines) were negative. Light blue lines: copies; orange: copies; dark blue: copies; brown: copies; green: 50 copies.

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The linearity for the dilution range to a semi-log model with each method was high (R2 to ), however it is apparent that the variation between replicates increases with decreasing concentration as previously observed46. The variability between replicates at each dilution for the SYTO9 detection method was lower than that of the quenched fluorophore technique and increased variation and slower detection times were shown with SYTO9 detection using the primer combination that included the fluorophore labelled FIP. Melt curve analysis showed almost identical profiles for the two assays with SYTO9 detection and the melt curve using quenched fluorophore detection clearly separates the positive from the NTC replicates.

The quenched fluorophore method using labelled LoopF or FIP primers could provide an enhanced specificity when compared to detection methods that rely solely on increases in double stranded DNA or pyrophosphate production, because only the products of LAMP amplification will be detected and not ‘false positives’ from non-specific primer interactions. To investigate this possibility LAMP primers with minimal post-synthesis purification were subjected to extended assay times of  minutes to encourage amplification from non-specific primer interactions (Fig. 5,2A–C).

Detection with SYTO9 and JOE-FIP positive and negative samples. (1) LAMP amplification of copies linear plasmid template and NTCs with detection by both (1A) and JOE-FIP quenched fluorescence (1B) in separate tubes. Four replicates of template in blue and NTCs in red. Melt curve analysis (1C,D) showing amplification of template only in the 90 minute assay. (2) Amplification with LAMP primers of 72 replicate NTCs for  minutes. (2A) SYTO9 detection of amplification between and  minutes showing 5 of 72 giving a positive signal. (2B) JOE-FIP detection of the same samples using the yellow channel (excitation at  +/− 5 nm with emission at  nm +/− 5 nm). (2C) FAM-LoopF detection using the green channel (excitation at  nm +/− 10 nm with emission at  nm +/− 5 nm). (2D–F) Melt curve analyses.

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Using SYTO9 for detection, five of the seventy two NTC replicates gave a positive signal between and  minutes (Fig. 5,2A). The melt curve analysis (Fig. 5,2D) showed a higher Tm than for the true LAMP products formed from template with the same primers (Fig. 5,1C) with SYTO9 detection. The LAMP assay with JOE labelled FIP supplemented for the basic purification FIP (Fig. 5,2B) showed no positive melt curve results from the same seventy two NTCs over the  minute assay time, when compared with the corresponding LAMP assay with template and NTCs (Fig. 5,1B). The supplementing of LoopF with FAM labelled LoopF showed one of the seventy two NTCs as a positive (Fig. 5,2C). We conclude that combining SYTO9 detection with quenched fluorophore detection provides two methods of detection in one tube at the same time increasing overall specificity.

We expanded this concept to see if we could observe simultaneous non-specific detection with SYTO9 and specific detection with the quenched fluorophore attached to the FIP LAMP primer. Using JOE to label the FIP primer, fluorescence acquisition can be achieved with excitation and emission of SYTO9 at and nanometers separated from JOE excitation and emission at and nanometers. Assay times were extended to  minutes to facilitate non-specific primer interactions.

The detection of the fluorescence from the JOE labelled FIP primer in the LAMP amplification showed three positive results from the seventy two NTCs. These positives were detected after  minutes (Fig. 6C). Detection of the same LAMP amplification from the seventy two NTCs with SYTO9 (Fig. 6A) showed a higher number of positives with the three positives detected by the quenched fluorophore method also positive with SYTO9 detection (highlighted in blue). Melt curve analysis showed a spread of melt temperatures for these amplicons between 85 and 90 degrees C. However agarose gel electrophoresis (Fig. S1) showed LAMP ladder patterns for the three positives detected using the JOE labelled FIP primer in contrast to the other putative positives detected only using SYTO9. The detection of the three ‘false positives’ which produced a ladder pattern similar to that seen from LAMP amplicons suggest that the JOE labelled FIP primer was one of the primers involved in the non-specific primer interaction.

Non-specific primer interactions detected with SYTO9 and JOE-FIP. Panel (A) shows the normalised fluorescence from SYTO9 detection and (C) shows the consecutive JOE-FIP fluorescence for NTCs. Corresponding melt curve analysis shown in panels (B,D). Negative results and SYTO9 detected positives are highlighted in red, JOE-FIP detected positives are highlighted in blue. One negative and all positives were examined on an agarose gel (E) for amplicons and the JOE-FIP detected positives showed a ladder pattern associated with LAMP amplification.

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At higher concentrations of template the importance of non-specific primer interactions is likely to be less significant due to the domination of ‘true’ LAMP amplification. However at very low concentrations of template, and for example the template dilutions used in digital quantification47,48, the prevalence of ‘false positives’ will be detrimental to accurate quantifications. We therefore investigated dual detection of dilute samples of denatured maize genomic DNA with a low proportion of transgenic targets to compare sensitivity to bioluminescent detection of pyrophosphate production (BART) and to identify incidence of non-specific amplification (Fig. 7).

Detection of LAMP amplification with SYTO9 and JOE-FIP reporting compared to LAMP-BART results. Panels (1A) to (1F) represent amplification with the 35S promoter primers of a low concentration of denatured maize genomic DNA ( percent maize GM event Bt11). Panels (2A) to (2F) represent amplification of the same sample with the NOS terminator primers. Panels (1A and 2A) show LAMP amplification with BART detection with corresponding frequency distribution of positive results (1B and 2B). Panels (1C and 2C) show detection using SYTO9 and (1E and 2E) show the simultaneous results from JOE-FIP detection. The melt curve analysis for these results are shown in panels (1D,1F,2D,2F).

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LAMP BART assays of a denatured maize genomic DNA sample showed five positive results between 10 and 20 minutes and a further two positive results after 40 minutes within the 90 minute assay period, with two different primer sets targeted to the 35S promoter and NOS terminator. The dual detection using 35Sp primers and JOE-FIP highlighted six positives before 90 minutes with the majority between 10 and 40 minutes. One of the positives was detected by SYTO9 only and is therefore likely to be a false positive. The sensitivity between the LAMP BART and the dual detection methods appeared to be similar. With the NOSt LAMP dual detection the quenched fluorescence detected four positive results all between 10 and 40 minutes. The SYTO9 method detected numerous other positives but the melt curve (Fig. 7,2D) indicated that these products have a higher melt temperature than the ones identified with the quenched fluorescence method.

Carrier DNA is often used in LAMP assays to improve results9, however the mechanism by which this happens is unclear. We therefore used the dual detection method to investigate whether the addition of carrier DNA increases the incidence of non-specific primer interactions, thereby only apparently increasing the amplification frequency.

The amplification frequency of both LAMP primer interrogations of the same template showed the frequency increased using JOE-FIP quenched fluorescence detection from 18 percent without carrier DNA to approximately 25 percent with nanograms per partition of salmon sperm carrier DNA (Fig. 8A). There was a general trend downwards of ‘false positives’ (in red) with increasing concentrations of carrier DNA indicating that non-specific primer interactions are not contributing to the improved amplification frequency seen with carrier DNA inclusion. For both primer sets with this template the optimum concentration of carrier DNA is nanograms per partition. However, the average Ct values for both primer sets with JOE-FIP detection are faster without carrier or with 50 nanograms per partition of carrier DNA than the nanogram level. The fastest Ct value at each concentration of carrier DNA with both LAMP primer sets remains fairly constant below the 20 minutes level. This result taken in conjunction with the average Ct values suggests that carrier DNA produces greater variability between replicates.

Carrier DNA in LAMP amplification with SYTO9 and JOE-FIP detection. Panels (1A to C) represent amplification with the 35S promoter primers of a low concentration of denatured maize genomic DNA ( percent maize GM event Bt11). Panels (2A to C) represent amplification of the same sample with the NOS terminator primers. Panels (A) show the amplification frequency for increasing concentrations of carrier DNA. Cycle threshold set for all assays at − Panels (B) show the average Ct values and panels (C) shows the fastest Ct values with increasing concentrations of salmon sperm carrier DNA. Green indicates SYTO9 derived values, blue indicates JOE-FIP derived values and red indicates ‘false positive’ results derived from the difference between the SYTO9 and JOE-FIP values. In panels 1C and 2C the SYTO9 and JOE-FIP values are identical.

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With non-specific LAMP detection techniques that report on concentration changes of DNA or pyrophosphate production, there is limited potential for multiplexing. The specific detection of quenched fluorescence through labelling of FIP primers was investigated further to see if the technique can be multiplexed effectively. The FIP primer in the 35S promoter primer set was replaced with a TAMRA labelled FIP and ADH1 with FAM. Denatured maize genomic DNA with a transgenic proportion was assayed with each LAMP reaction individually and as a duplex. Displacement and Loop primers were not included to reduce non-specific primer interactions (Fig. 9).

Duplexed LAMP reaction with ADH1 and 35Sp detection. LAMP amplification and detection of 20 copies of denatured Bt11 maize genomic DNA with ADH1 LAMP primers with FAM labelled FIP and 35Sp LAMP primers with TAMRA labelled FIP. Blue indicates template amplification and orange denotes the NTCs. The (DUPLEX) column shows the detection of two LAMP reactions in each tube. Other columns show the individual LAMP reactions and the detection channels of the thermocycler; FAM-FIP detected on green channel only and TAMRA-FIP by the orange channel.

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In the duplex reaction ADH1 and 35Sp LAMP amplifications from the template are successfully detected by excitation and detection of the various fluorophores attached to the FIP primer in each set. The signal from FAM-FIP in the ADH1 LAMP amplification showed the least quenching of template amplification compared to the NTCs. The strongest signal was derived from the TAMRA-FIP used in the 35Sp LAMP amplification. Singleplex LAMP amplifications showed the correct identification in the green channel of FAM-FIP in the 35Sp LAMP amplification and appropriate negative results for the ADH1 LAMP amplifications. The orange channel selected the TAMRA labelled FIP in the 35Sp LAMP amplification and not the other LAMP amplifications.

Discussion

We show here that FIP and LoopF labelled with JOE and FAM respectively can be successfully incorporated into LAMP reactions replacing their unlabelled counterparts and are quenched as a result of amplification. The mechanism by which the fluorescently labelled FIP and LoopF primers are quenched is unclear. One theory is that the formation of cauliflower-like concatamers in the elongation stage of LAMP amplification tightly packs the fluorophores to nucleotide residues in sufficiently close proximity for fluorescence resonance energy transfer (FRET) to quench the fluorescence as previously observed35. This phenomenon was observed with labelling of both the LoopF and FIP primers and with three different fluorophores; FAM, JOE and TAMRA. The FIP and BIP primers are the most fundamental to LAMP amplification because no other primers are required during LAMP cycling and elongation phases. We therefore used the FIP primers in our investigations, seeking to make use of the phenomenon.

The fold dilutions of linearised plasmid template in Fig. 2 demonstrated the potential of the quenching primer method for high sensitivity and quantification by detecting 5 copies per microlitre in approximately 40 minutes and 5 million copies in approximately 20 minutes. However, at the lower concentration of DNA template the amplification frequencies were low for the two assays. Also variation of the quenched signals was evident for some of the template concentrations and the separation between non-template fluorescence and quenched fluorescence in the presence of template amplification was poor for some concentrations. Optimisation of the primer combinations and concentrations of the JOE labelled FIP in LAMP amplification was therefore carried out (Fig. 3), highlighting the optimal concentration from these experiments of micromolar. At this concentration the variation between replicates was low and the difference between quenched and unquenched signals was clear.

We compared the quenching of the JOE-FIP primer to detection of LAMP amplification with SYTO9 including JOE-FIP in the primer set or with unlabelled FIP substituted (Fig. 4). All three methods showed high linearity to the semi logarithmic trendline, but it was observed that moderately faster times and lower variation were evident using the unlabelled FIP primer compared to the JOE-FIP assay. Nevertheless the observation that LAMP amplification with a fluorescently labelled FIP primer was successful with SYTO9 detection suggests that amplification could be monitored in real time on the green channel for SYTO9 and yellow channel for JOE of a qPCR thermocycler as a dual detection method.

Theoretically, the labelling of a FIP or BIP primer will have absolute specificity for the LAMP amplification. In the absence of template there would be no LAMP amplification and therefore no quenching of the signal from the fluorophore labelled LAMP primer. We sought to produce false positive results with a combination of a low purity LAMP primer set with extended reaction times. Detection of increases in double stranded DNA using the intercalating dye SYTO9 showed five positive partitions out of the 72 tested. The same assay monitored with JOE-FIP did not show quenching from any of the partitions. All false positives occurred after  minutes at 60 degrees C and are highly unlikely to be caused by contamination; positive template results were rapid and reaction times were similar for the two assays (Fig. 5).

By combining SYTO9 detection with JOE-FIP quenching in the same tube (Fig. 6) over an extended time period we could characterise the false positives generated in the absence of template by melt curve analysis and gel electrophoresis of the amplicons. Three false positives were evident in both the green channel for SYTO9 detection and the yellow channel for JOE-FIP quenching which indicated that the FIP primer was involved in the generation of the false positives. This was supported by a shift in melt temperature for these three and a ladder pattern on the agarose gel typical for LAMP amplification indicating concatamers of varying size. The closed tube format of this dual testing restricts amplicon contamination which is a risk when opening tubes for gel electrophoresis. The results also show the synchronicity of the false positives between the two methods reporting on a single partition.

We then explored the uses of quenching as a tool to optimise LAMP assays. Bioluminescent reporting of LAMP amplification with BART25 used to detect low concentrations of transgenes in genomic DNA samples shows a spread of positive results. Our investigation of the samples with quenching dual detection showed that the fastest groups of positive results are likely to be ‘true’ positives, but later results are likely to be false positives (Fig. 7). This was also supported with the melt curve analysis which showed a shift in melt temperatures between the ‘true’ positives and the false positives. This indicates that particular sets of LAMP primers are therefore susceptible to false positives at low copy number and would benefit from further investigation and possible redesign of certain primers. Displacement, LAMP and Loop primers can be designed using Primer Explorer (Eiken, Japan), commercial software and bioinformatic programmes49,50. However, the interactions between primers, target sequences, non-target sequences and amplicons are difficult to predict in silico. This demonstrates how quenched-based dual detection of amplification can serve as an optimisation tool in the development of LAMP assays.

Carrier DNA has previously been shown to enhance sensitivity, reduce variation between replicates, and increase reaction times and amplification frequency9 when added at nanograms per reaction. We used the dual detection method with 35S promoter and NOS terminator LAMP amplification of a transgenic maize DNA sample to demonstrate that amplification frequency increased at nanograms per reaction without an increase in false positive detection (Fig. 8). The fastest partition at each concentration of carrier DNA were of similar values whereas amplification frequency was highest at nanograms reaction, possibly indicating that carrier DNA has a positive impact on initiating LAMP amplification from available target copies, but does not affect subsequent amplification.

Many fluorescent probe and modified primer technologies with enhanced specificity also describe adaptations for multiplexing. We have shown a duplex system here with JOE labelled LAMP primers and SYTO9, and we also investigated a duplex approach with the detection of reference gene ADH1 and transgene 35S promoter (Fig. 9). The FIP primers of each of these LAMP primer sets was linked to FAM and TAMRA respectively. Our results showed that two LAMP amplifications could occur in the same tube with quenching detection of the two labelled FIP primers. The excitation:emission for FAM is and for TAMRA. This duplex reaction shows that multiplexing with this technique could be developed.

We conclude that the simplicity of using quenching of a suitable 5′ labelled LAMP primer is an attractive proposition for specific LAMP detection and can be used with an intercalating dye to assess the extent to which false positives may affect a new LAMP primer assay. This will be an important factor in developing LAMP primers for digital LAMP assays due to the impact a false positive result could have on quantification calculations, and will enable existing indirect LAMP amplification detection methods to be employed with well designed, high quality primers.

Methods

DNA templates

A linearised plasmid pART751, of defined copy number from calculations based on initial quantification by NanoDrop spectrophotometry and Agilent Bioanalyzer, was used as a DNA template. The plasmid contains a variant of the cauliflower mosaic virus 35S promoter sequence (35Sp). Genomic DNA was extracted from percent w/w maize event Bt11 certified reference material (CRM) supplied by Fluka GmbH (Buchs, Switzerland). The transgenes in Bt11 are regulated by the 35S promoter from the cauliflower mosaic virus and the nopaline synthase terminator (NOSt) from Agrobacterium tumefaciens. The genomic maize DNA contains the alcohol dehydrogenase 1 gene sequence (ADH1). The Promega Wizard genomic DNA purification kit (Madison, United States) was used to extract the DNA from maize event Bt11 according to the manufacturer’s instructions for plant tissue. The final pellet was hydrated with 50 microlitres of rehydration buffer and stored at 4 degrees C. A further percent Bt11 CRM, percent NK CRM and percent Mon maize sample were extracted using the Promega Wizard kit and quantified using the Qubit dsDNA BR assay kit (Thermofisher, Massachusetts USA) in accordance with the instructions for the Qubit fluorometer.

Copy number calculations

The initial quantification values from NanoDrop, Agilent Bioanalyzer and Qubit were converted from nanogram per microlitre to copies per microlitre using the following formula:

$${\rm{Copies}}\,{\rm{of}}\,{\rm{target}}=\frac{{\rm{ng}}\,{\rm{of}}\,{\rm{double}}\,{\rm{stranded}}\,{\rm{DNA}}\times {\rm{Avogadro}}\mbox{'}{\rm{s}}\,{\rm{constant}}\,({\rm{}}\times {10}^{23})}{{\rm{Length}}\,{\rm{in}}\,{\rm{base}}\,{\rm{pairs}}\times {{\rm{10}}}^{{\rm{9}}}\times {\rm{}}\,{\rm{Daltons}}}$$

The size of the linearised plasmid pART7 was assumed to be base pairs and  × 10(9) base pairs was used in calculations for the maize genome.

The online calculator was used at http://cels.uri.edu/gsc/cndna.html.

The calculations of copy number for the CRM materials for maize events Bt11 and NK, and the seed stock of Mon are made in accordance with Hardinge et al.46 by adjusting transgenic copy numbers by / Futhermore Bt11 has two copies of the 35Sp promoter which doubles the available target sequence.

Primer design and synthesis

Oligonucleotide primers for LAMP DNA amplification (Table 1) were synthesized and HPLC purified by Sigma Aldrich (Poole, UK). Primers were hydrated with molecular grade water to micromolar and stored at minus 20 degrees C. The unmodified LAMP primers used to target 35Sp, NOSt and ADH1 sequences have previously been described and optimised9,46,52. An additional set of LAMP primers to target the 35S promoter was designed using Primer Explorer v4 from Eiken (Japan) for displacement, LAMP and loop primers. The stem primers were designed using the Primer 3 online software at http://primer3.ut.ee/. The fluorophores attached without additional modification to the 5′ end of FIP or LoopF primers were selected based on published evidence of nucleotide base quenching35. FAM, JOE and TAMRA were selected for LAMP quenching. Modifications to the 5′ terminal nucleotides were not made due to the NOSt JOE labelled FIP adjacent to a guanine base remained functional in amplification and LAMP quenching.

Full size table

Fluorescent LAMP amplification

DNA samples were amplified using LAMP and detected using SYTO9 in real time on a Qiagen (Hilden, Germany) RotorGene thermal cycler acquiring to the green channel unless otherwise specified. All reagents were supplied by Sigma Aldrich (Poole, UK) unless otherwise stated. The reaction chemistry for LAMP and amplification detection was 1X isothermal buffer (New England Biolabs Inc, Massachusetts, United States), micromolar each deoxynucleotide triphosphate (dNTP), micromolar Betaine, units per microlitre Bst polymerase v warm start (NEB), micromolar SYTO9 Green, micromolar each LAMP primer, micromolar each Loop primer, micromolar each displacement primer and molecular grade water for a reaction volume of 20 microlitres. The parameters were set for 60 cycles of 60 seconds at 60 degrees C unless otherwise stated. Temperature melt analysis provided data between 60 and 95 degrees C. Results were analysed on RotorGene software v and Microsoft Excel. The threshold was set at the mid point between the background fluorescence and quenched fluorescence from the positive samples. The default threshold value was − on the normalised fluorescence scale. Standard normalisation was used in the RotorGene software to take the average background from the first five cycles to provide a value by which sample data points are divided.

Quenched fluorescence LAMP amplification

Oligonucleotide LAMP primers with JOE, FAM or TAMRA fluorophores attached to the 5 prime end were incorporated into the fluorescent LAMP amplification method without the inclusion of SYTO9. For dual detection SYTO9 was included with JOE labelled FIP primers. The LAMP reaction was monitored in real time on the RotorGene thermocycler according the emission wavelength of the fluorophore. The multiplex and dual detection reactions monitored 6′FAM or SYTO9 using the green channel (source  +/− 10 nanometers, detection  +/− 5 nanometers), JOE on the yellow channel (source  +/− 5 nanometers, detection  +/− 5 nanometers) and TAMRA with the orange channel (excitation at  +/− 5 nanometers, detection aat  +/− 5 nanometers) in the same reaction. Melt temperature analysis followed amplification between 60 and 95 degrees C. Results were analysed RotorGene software v and Microsoft Excel.

Real-time LAMP-BART detection

LAMP-BART chemistry was previously described by Gandelman et al.25 and Kiddle et al.9. Salmon sperm carrier DNA at nanograms per partition was used in partitions of 5 microlitres. The LAMP-BART reaction mix contained 1x Thermopol buffer (NEB), 10 millimolar dithiothrietol (DTT), milligrams per millilitre polyvinylpyrrolidone (PVP), 60 millimolar potassium chloride (KCl), micromolar each dNTP, micrograms per millilitre D-luciferin (Europa, Ipswich, UK), micromolar adenosine-5′-O-phosphosulfate (APS; Biolog, Bremen, Germany), micrograms per millilitre Ultra-Glo luciferase (Promega, Madison, United States), units per microlitre ATP sulfurylase (NEB), units per microlitre Bst polymerase v, micromolar each LAMP primer, micromolar each Loop primer, micromolar each displacement primer and molecular grade water. Each partition was layered with mineral oil and sealed with a clear adhesive film. The bioluminescent signal was detected and analysed using React IVD software (Synoptics, Cambridge, UK) in a ‘Lucy’ device developed by ERBA MDX (Ely, UK). The LAMP-BART assays were set for 90 minutes at 60 degrees C.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Saiki, R. K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, – ().

    ADSCASArticle Google Scholar

  2. 2.

    Gill, P. & Ghaemi, A. Nucleic acid isothermal amplification technologies: a review. Nucleosides Nucleotides Nucleic Acids27, – ().

    CASArticle Google Scholar

  3. 3.

    Asiello, P. J. & Baeumner, A. J. Miniaturized isothermal nucleic acid amplification, a review. Lab Chip11, – ().

    CASArticle Google Scholar

  4. 4.

    Haleyur Giri Setty, M. K. & Hewlett, I. K. Point of care technologies for HIV ().

  5. 5.

    Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res28, E63 ().

    CASArticle Google Scholar

  6. 6.

    Poon, L. L. et al. Sensitive and inexpensive molecular test for falciparum malaria: Defecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clinical Chemistry ().

  7. 7.

    Ohtsuka, K., Yanagawa, K., Takatori, K. & Hara-Kudo, Y. Detection of Salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of Salmonella isolates. Applied and Environmental Microbiology ().

  8. 8.

    Curtis, K. A., Rudolph, D. L. & Owen, S. M. Rapid detection of HIV - 1 by reverse - transcription, loop – mediated isothermal amplification (RT - LAMP). Journal of Virological Methods ().

  9. 9.

    Kiddle, G. et al. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnology12, 15 ().

    CASArticle Google Scholar

  10. Watthanapanpituck, K., Kiatpathomchai,W., Chu, E. & Panvisavas, N. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe. International Journal of Legal Medicine ().

  11. Nagamine, K., Hase, T. & Notomi, T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes16, – ().

    CASArticle Google Scholar

  12. Gandelman, O., Jackson, R., Kiddle, G. & Tisi, L. Loop-mediated amplification accelerated by stem primers. Int J Mol Sci12, – ().

    CASArticle Google Scholar

  13. Martineau, R. L. et al. Improved Performance of Loop-Mediated Isothermal Amplification Assays via Swarm Priming. Analytical Chemistry ().

  14. Kaneko, H., Kawana, T., Fukushima, E. & Suzutani, T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods70, – ().

    CASArticle Google Scholar

  15. Tani, H. et al. Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification. Anal Chem79, – ().

    CASArticle Google Scholar

  16. Kubista, M. et al. The real-time polymerase chain reaction ().

  17. Holland, P. M., Abramson, R. D., Watson, R. & Gelfand, D. H. Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA88, – ().

    ADSCASArticle Google Scholar

  18. Tyagi, S. & Kramer, F. R. Molecular Beacons: Probes that Fluoresce Upon Hybridization. Nature Biotechnology14, – ().

    CASArticle Google Scholar

  19. Chou, P. H., Lin, Y. C., Teng, P. H., Chen, C. L. & Lee, P. Y. Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes. J Virol Methods, 67–74 ().

    CASArticle Google Scholar

  20. Mori, Y., Nagamine, K., Tomita, N. & Notomi, T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun, – ().

    CASArticle Google Scholar

  21. Tomita, N., Mori, Y., Kanda, H. & Notomi, T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc3, – ().

    CASArticle Google Scholar

  22. Goto, M., Honda, E., Ogura, A., Nomoto, A. & Hanaki, K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques46, – ().

    CASArticle Google Scholar

  23. Kouguchi, Y., Fujiwara, T., Teramoto, M. & Kuramoto, M. Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates. Mol Cell Probes24, – ().

    CASArticle Google Scholar

  24. Kubota, R. et al. Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica. Chin Sci Bull58, – ().

    CASArticle Google Scholar

  25. Gandelman, O. A. et al. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time. PLoS ONE5 ().

  26. Iwamoto, T., Sonobe, T. & Hayashi, K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol41, – ().

    CASArticle Google Scholar

  27. Parida, M. M. Rapid and real-time detection technologies for emerging viruses of biomedical importance. J Biosci33, – ().

    CASArticle Google Scholar

  28. Seyrig, G. et al. Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics. J Microbiol Methods, – ().

    CASArticle Google Scholar

  29. Njiru, Z. K. et al. Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis2, e ().

    Article Google Scholar

  30. Mori, Y., Kitao, M., Tomita, N. & Notomi, T. Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods59, – ().

    CASArticle Google Scholar

  31. Tanner, N. A., Zhang, Y. & Evans, T. C. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques58, 59–68 ().

    CASArticle Google Scholar

  32. Fischbach, J., Xander, N. C., Frohme, M. & Glökler, J. F. Shining a light on LAMP assays–a comparison of LAMP visualization methods including the novel use of berberine. Biotechniques58, – ().

    CASArticle Google Scholar

  33. Liu, W. et al. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Scientific Reports7 ().

  34. Mori, Y., Hirano, T. & Notomi, T. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol6, 3 ().

    Article Google Scholar

  35. Torimura, M. et al. Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base. Anal Sci17, – ().

    CASArticle Google Scholar

  36. Zerilli, F. et al. Methylation-specific loop-mediated isothermal amplification for detecting hypermethylated DNA in simplex and multiplex formats. Clin Chem56, – ().

    CASArticle Google Scholar

  37. Spinelli, O. et al. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology. Oncoscience2, 50–58 ().

    Article Google Scholar

  38. Aonuma, H. et al. A single fluorescence-based LAMP reaction for identifying multiple parasites in mosquitoes. Experimental Parasitology ().

  39. Stratakos, A. C., Linton, M., Millington, S. & Grant, I. R. A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces. Journal of Applied Microbiology ().

  40. Tanner, N. A., Zhang, Y. & Evans, T. C. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. Biotechniques53, 81–89 ().

    CASArticle Google Scholar

  41. Jiang, Y. S. et al. Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons. Analytical Chemistry ().

    CASArticle Google Scholar

  42. Waiwijit, U. et al. Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection. J Biotechnol, 44–49 ().

    CASArticle Google Scholar

  43. Ball, C. S. et al. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses. Anal Chem88, – ().

    CASArticle Google Scholar

  44. Nkouawa, A., Sako, Y., Okamoto, M. & Ito, A. Simple identification of human taenia species by multiplex loop-mediated isothermal amplification in combination with dot enzyme-linked immunosorbent assay. American Journal of Tropical Medicine and Hygiene ().

  45. Gadkar, V. J., Goldfarb, D. M., Gantt, S. & Tilley, P. A. Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes. Scientific Reports ().

  46. Hardinge, P., Kiddle, G., Tisi, L. & Murray, J. A. H. Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART). Scientific Reports ().

  47. Vogelstein, B. & Kinzler, K. W. Digital PCR. Proc Natl Acad Sci USA96, – ().

    ADSCASArticle Google Scholar

  48. Gansen, A., Herrick, A. M., Dimov, I. K., Lee, L. P. & Chiu, D. T. Digital LAMP in a sample self-digitization (SD) chip. Lab Chip12, – ().

    CASArticle Google Scholar

  49. Torres, C. et al. LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures. BMC Bioinformatics12, ().

    CASArticle Google Scholar

  50. Salinas, N. R. & Little, D. P. Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification. ISRN Bioinformatics ().

  51. Gleave, A. P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol20, – ().

    CASArticle Google Scholar

  52. Lee, D., La Mura, M., Allnutt, T. R. & Powell, W. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences. BMC Biotechnol9, 7 ().

    Article Google Scholar

Download references

Acknowledgements

This work was supported by BBSRC Stand Alone LINK Grant BB/L/1, a BBSRC Industrial CASE Award, and part funded by the European Regional Development Fund.

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  1. Cardiff School of Biosciences, Biomedical Science Building, Museum Avenue, Cardiff, CF10 3AX, UK

    Patrick Hardinge & James A. H. Murray

Contributions

P.H. and J.A.H.M. conceived the experiments, P.H. conducted the experiments, P.H. and J.A.H.M. analysed the results and wrote the manuscript. Both authors reviewed the manuscript.

Corresponding author

Correspondence to Patrick Hardinge.

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Design Primers for Loop Mediated Isothermal Amplification

Home >> Products >> LAMP Designer >> Overview

LAMP Designer

LAMP Designer designs efficient primers for Loop-Mediated Isothermal Amplification assays, that amplify DNA and RNA sequences at isothermal conditions, eliminating the necessity of a PCR setup. The technology relies on auto-cycling and DNA polymerase mediated strand displacement DNA synthesis, amplifying a few copies of DNA to 109 in less than an hour. Reverse transcription coupled LAMP can be applied for amplification of RNA sequences.

LAMP employs four specially designed primers (two inner and two outer) that recognize six distinct regions in the target DNA. Hybridization of the four primers to the target DNA is a very crucial step for the efficiency of LAMP. The design of these four primers is therefore critical for a successful assay.

Avoid Cross Homologies

LAMP Designer automatically interprets BLAST search results and avoids those regions to design primers that have significant cross homologies with the database.

Verification BLAST

The primers can be BLAST searched against a database to verify their specificity.

Multiplex LAMP Primer Set

The primers designed for a sequence can be checked for multiplexing. The free energies of the most stable cross homologies between the primers designed, is displayed by the program.

Export Result

The designed primer sets, along with their properties, can be exported in standard csv or excel formats.

Data & Database Management

Multiple projects can be created. Data of multiple experiments can be easily managed by creating a separate project for each experiment. It maintains a local database for sequence information and search results.

Sours: http://www.premierbiosoft.com/isothermal/lamp.html
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GLAPD: Whole Genome Based LAMP Primer Design for a Set of Target Genomes

Introduction

Loop-mediated isothermal amplification (LAMP) is a simple-operating, effective and reliable method to amplify DNA sequence (Notomi et al., ; Parida et al., ; Mori and Notomi, ). The amplification is under a constant temperature (about 62°C) and the running time is short (within 1 h). In many application scenarios, LAMP is a better option than polymerase chain reaction (PCR) because the reaction can be in small and portable devices (Curtis et al., ; Chaumpluk et al., ). A basic LAMP primer set contains four synthetic primers derived from six primer regions (Supplementary Figure S1). Therefore, LAMP primer design is more complex than PCR primers. Designing a LAMP primer set to specifically identify a group of genomes (group-specific) at the same time has a high demand in many application fields like foodborne harmful bacteria detection, clinical pathogen identification, agricultural pathogen identification, and so on.

The group-specific primers mean that they can be applied to many target genomes belonging to a group (the primers are common) and at the same time the primers can&#x;t amplify any other genomes not included in this group (the primers are specific) (Jarman, ; Kalendar et al., ). For example, there are 16 strains of white spot syndrome virus with complete genomes and more than thousand other viruses in NCBI nucleotide database (up to October 29th, ). A group-specific primer for white spot syndrome virus should only amplify the 16 strains but no other viruses. Traditionally, the group-specific primers are designed based on conserved genes (Peng et al., ), genome regions (Yao et al., ) or the multiple sequence alignment (MSA) of these genes or genomic regions (Kurosaki et al., ). But this method is limited by the small number of suitable genes and the difficulty to generate MSA for a large number of sequences (Chen and Tompa, ). In addition, the primers based on traditional methods often can&#x;t meet the requirements in practice. For example, the LAMP primer set from Wang et al. () targeting Staphylococcus aureus was not able to amplify some S. aureus strains and might amplify some unexpected genomes (more details in results part). With more whole genomes available (O&#x;Leary et al., ), it&#x;s a better method to design group-specific primers based on whole genomes (Treven, ; Demkin et al., ).

There are some systems for LAMP primer design now. The most popular one is PrimerExplorer V5, an online software. However, the maximum length of its input sequence is limited up to 2, bps. Therefore, designing primers based on the whole genome is not provided. In addition, common or specific primers can be designed by PrimerExplorer V5 using MSA results by clicking the Common or Specific button separately. However, these two buttons can&#x;t work at the same time, which makes it not straightforward to design group-specific primers. Another system is LAVA (Torres et al., ), which can design common primers for a group of target genomes. However, it requires MSA results as the input, which limits the target region to a gene or conserved genomic region. LAVA doesn&#x;t check the specificity of primers. FastPCR (Kalendar et al., ) is a system that can design LAMP primers using the whole genome. Similar as PrimerExplorer, it has an online version, and it designs common primers or specific primers separately. It can&#x;t design group-specific primers in one run. According to the authors&#x; best knowledge, no existing system can design group-specific LAMP primers using whole genomes.

Here we present GLAPD (whole genome based LAMP primer designer), a new system to design group-specific LAMP primer sets. By using the whole genome sequences as input data, GLAPD can ensure the specificity of the primers and increase the chance to design a successful primer set. A graphics processing unit (GPU) version of GLAPD is also provided.

Materials and Methods

The Genome Based LAMP Primer Design System GLAPD

The system diagram of GLAPD is listed in Figure 1. GLAPD has three steps: (I) identifying candidate single primer regions; (II) combining single primers into LAMP primer set; and (III) checking the LAMP primer set. The inputs, outputs and the computation steps are listed below in more details.

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Figure 1. The system diagram of GLAPD. The inputs of GLAPD include the target group of genomes, the reference genomes, and the background group of genomes. There are three steps to generate LAMP primer sets: (I) identifying candidate single primer regions; (II) combining single primers into LAMP primer sets; (III) checking the LAMP primer sets. The check part contains commonality check based on target group, specificity check based on the background group and the tendency check of binding among single primers. Parts I, II, and III could be accelerated by using GPU.

Inputs

In this step, two groups are defined first. The target group is defined as a group of genomes or genome regions which are expected to be amplified by the LAMP primer set. The primers generated by GLAPD are expected to identify each target genome. If it failed to generate a primer set for the group of target genomes, the system will output primers that can amplify the maximum number of genomes.

Similar to the target group, a background group is defined as a group of genomes or genome regions which are not expected to be amplified. Primers designed by GLAPD should not amplify any genome in the background group.

One genome from the target group needs to be selected as the reference genome which will be used as the temperate to generate primer sequences. The reference genome can be randomly picked from the target group.

Identifying Single Primer Regions

A basic LAMP primer set contains four synthetic primers from six primer regions, named F3, F2, F1c, B1c, B2, and B3. Sequences from F1c and F2 are synthesized into one primer FIP and sequences from B1c and B2 are synthesized into another primer BIP. The positions relationship among these single primers are showed in Supplementary Figure S1. In order to design a LAMP primer set, those candidate primer regions are identified first. They are then combined into LAMP primer sets.

Genome based LAMP primer designer identifies all candidate primer regions in the reference genome according to primer&#x;s length, GC-content, melting temperature (Tm), stability and so on (Supplementary Table S1). The secondary structure of primer is checked by GLAPD using the thermodynamical approach similar to Primer3 program (Untergasser et al., ). At the same time, the end of each primer is checked to exclude symmetric sequences and homopolymers. GLAPD uses customized parameters to identify primer regions according to the GC-content of the target region (between F3 and B3). If GC-content of the target region is high, the GC-content and Tm of primers are set to be high, vice versa.

Combining Single Primers Into Primer Sets

Primers from six regions are combined into one basic LAMP primer set. GLAPD uses the positional relationship (Supplementary Figure S1) among the six regions, GC-content relationship between primers and whole amplification region (Supplementary Table S1), Tm relationship among primers (the Tms of F1c and B1c are 3°C higher than other primers&#x;) to combine them. Then the combined LAMP primer set will be checked for commonality, specificity and tendency of binding among single primers.

Checking Commonality and Specificity of the LAMP Primer Set

An ideal LAMP primer set should be able to amplify all the target genomes but not genomes from the background group. In order to do this check, firstly, all single primers are aligned to the target genomes and the background genomes using Bowtie (Langmead et al., ). By default, no mismatch is allowed when a primer is aligned to the target group. If GLAPD fails to design LAMP primer sets to amplify all target genomes, a small number of mismatches are allowed when primers are aligned to the target group. However, if a primer can be aligned to a background genome within two mismatches (by default), this primer is considered as not specific. The more mismatches required to align the primer to the background genomes, the more specific is the primer. No matter how many mismatches in a primer, no mismatch is allowed in the 5&#x; of F1c and B1c, and the 3&#x; of F3, F2, B2, B3, LF, and LB primer. After the alignment, primers&#x; positions, strand information (plus or minus) and the number of mismatches in each genome are recorded.

Using the information generated above and the positional relationship of the six primer regions (Supplementary Figure S1), GLAPD checks the ability of a LAMP primer set to amplify genomes in the background group. If a LAMP primer set can amplify any background genome, this set will be discarded. Therefore, more flexible thresholds for positional relationship among primers can be used to improve the specificity in this step. After the LAMP primer set passes this specificity check, the number of genomes or genome regions in the target group amplified by the primer set is calculated using the same method in specificity check.

Checking the Binding Tendency of Any Two Primers

The LAMP primer set passed the commonality and specificity check will be checked for every single primer&#x;s tendency of binding to other single primers in this primer set. This check uses the thermodynamical approach similar as the Primer3 program does.

Outputting LAMP Primer Sets

The LAMP primer set passed all above check steps will be output. When GLAPD has designed 10 (by default) LAMP primer sets successfully, or GLAPD has checked all candidate LAMP primer sets, the system stops automatically. The outputs contain the sequences, positions, lengths of the primers and genomes which can be amplified. The LAMP primer sets are not overlapped with each other. By default, the shortest distance between two LAMP primer sets&#x; F3 regions is bps.

Loop Primers

In order to accelerate the amplification, two additional loop primers (LF and LB) can be added (Supplementary Figure S1). GLAPD can also design LAMP primer set with loop primers. The candidate primer regions are identified for loop primers from the reference genome first. Those candidate regions must meet the requirements listed in Supplementary Table S1. Then, GLAPD combines loop primers with other single primers into a LAMP primer set. A LAMP primer set could contain one or two loop primers. The Tms of loop primers are set to be 3°C higher than Tm of F3, F2, B2, and B3. At last, this LAMP primer set is checked for the tendency of primer annealing and its commonality.

GPU Version

Graphics processing units can be used to accelerate GLAPD in three steps. In the step of identifying candidate single primer regions, GLAPD can identifies them from many positions of the reference genome simultaneously. Many primers&#x; GC-content, stability, Tm and secondary structure can be calculated in parallel in GPUs. In the step of combining single primers, each thread of GPU is assigned with a different candidate F3 primer then GLAPD tries to design all LAMP primer sets containing this F3 primer in parallel. In the checking step, each thread calculates the number of target genomes and background genomes that can be amplified by the primer set designed in this thread, and every single primer&#x;s tendency of binding to other single primers. In each thread, only the LAMP primer set amplifying the maximum number of target genomes will be returned to CPU for output.

Databases

Three databases were generated. Database-1 was the database of complete genome sequences of bacteria and archaea, which was downloaded from NCBI&#x;s FTP on August 5th, It contained 4, sequence files from 2, strains (about 9GB).

Database-2 was the database of all complete mitochondrion sequences of suina, bovinae, and caprinae, which was downloaded from NCBI nucleotide database on June 14th, In this database, , , and mitochondrion sequences for suina, bovinae and caprinae, respectively were selected.

Database-3 was the database of complete genome sequences of bacteria and archaea downloaded from NCBI&#x;s Nucleotide database on September 19th, It contained 15, bacterial sequence files and archaea sequence files (about 60GB).

Database-4 was the database of complete genome sequences of viruses downloaded from NCBI&#x;s Nucleotide database on August 2th, It contained , viruses sequence files.

DNA Extraction

The activated strains were cultured in appropriate method (Supplementary Table S2) and collected when the cultures reached an optical density at nm (OD) between The genomic DNA of the strains were extracted using AxyPrepTM Multisource Genomic DNA Miniprep Kit (Axygen Bioscientific, Inc., United States). The samples were ground into powder with liquid nitrogen and homogenized with μl of cell lysis buffer, and all other steps followed the manufacturer&#x; s instructions.

LAMP Reaction

Loop-mediated isothermal amplification reaction was performed in a 25 μl reaction mixture. The mixture contained 1 × ThermoPol Buffer (contained 2 mmol/L MgSO4), 6 mmol/L MgSO4 (total 8 mmol/L), mmol/L of each dNTP, μmol/L of each inner primers (FIP and BIP), μmol/L of each outer primers (F3 and B3), 8 units of Bst DNA Polymerase (Large Fragment).

When test the LAMP primer designed for S. aureus, the mixture contained 20 ng of template DNA of each 29 bacterial strains. When test the LAMP primer&#x;s specificity designed for Vibrio vulnificus and Vibrio cholerae, the mixture also contained 20 ng of template DNA of each 29 bacterial strains and when test its commonality, the amount of template DNA was 50 ng, 5 ng, pg, 50 pg, 5 pg, fg, 50 fg, 5 fg and fg, respectively. In all NTC (no template control) reaction, template DNA was replaced by sterilized water.

The LAMP reaction was carried out at 62°C for 60 min using a VeritiTM Dx Thermal Cycler (Thermo Fisher, United States), then inactivated Bst DNA Polymerase at 80°C for 10 min. After the reaction, 1 μl × SYBR Green I was added into the solution to confirm whether the reaction occurred. In a positive reaction, the color of the solution was green and in a negative reaction, the color was orange.

Results

Experimental Validation of the Group-Specific Primers Designed by GLAPD

Group-Specific LAMP Primers of Staphylococcus aureus

Staphylococcus aureus is one main type of foodborne pathogens around the world (Kadariya et al., ; Paudyal et al., ). Traditionally, its nuc, mecA genes were used to design the LAMP primer sets (Wang et al., ; Chen et al., ). But the two genes are not conserved among all S. aureus (Hoegh et al., ; Karmakar et al., ) and may exist in other Staphylococcus spp. (Borjesson et al., ). Therefore, existing LAMP primer sets (Wang et al., ) for S. aureus are neither common nor specific enough (Supplementary Tables S3, S4).

In database-1 the S. aureus species had 43 strains (Supplementary Table S3). GLAPD used those 43 strains as the target group and the rest of the genomes in database-1 as the background group then designed several group-specific LAMP primer sets. One set (Table 1) was located in a predicted gene which codes 50S ribosomal protein L This LAMP primer set was common for all S. aureus and not for any other bacteria. It was validated by experiments (Supplementary Figure S2).

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Table 1. The LAMP primer set designed by GLAPD for S. aureus.

Vibrio Group-Specific LAMP Primer Set

Vibrio spp. is one of the main pathogenic bacteria in seafood (Huehn et al., ; Mizan et al., ). Most LAMP primers are designed only for the identification of V. cholerae (Okada et al., ) or V. vulnificus (Han et al., ). In spite that the V. vulnificus and Vibrio parahaemolyticus can be detected simultaneously in one reaction, it required two LAMP primer sets in the reaction (Wang et al., ).

In database-1, there were eight strains of V. cholerae and three strains of V. vulnificus (Supplementary Table S5). V. cholerae and V. vulnificus belonged to Vibrio genus. GLAPD used those 11 strains as the target group and the rest of the genomes in database-1 as the background group. Several group-specific LAMP primer sets were designed by GLAPD. One set (Table 2) was located in a gene which coded 30S ribosomal protein S This LAMP primer set was common for all V. cholerae and V. vulnificus, and was specific to these bacteria only. It was validated by experiments listed in Supplementary Figure S3.

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Table 2. The LAMP primer set designed by GLAPD for V. cholerae and V. vulnificus.

Besides the two group-specific LAMP primer sets mentioned above, we have applied GLAPD to design group-specific primers for more than ten other foodborne pathogens. GLADP could successfully design group-specific primers for most of these foodborne pathogens and more than half of those primers worked well in real experiments (Supplementary Table S6).

The flexible setting of target and background group.

When GLAPD is used to design group-specific primers, the target group and background group could be defined flexibly by users. Some examples are listed below to show this flexibility.

(1) To design group-specific primer for all genomes in a genus. Salmonella is a common bacterial pathogen responsible for salmonellosis, a common disease affects the intestinal trace and it can cause substantial socioeconomic burden (Liu et al., ). In database-3, complete genomes and 1 assembly genome with 66 contigs were Salmonella. GLAPD could design the genus-level salmonella-specific LAMP primer set successfully. One LAMP primer set designed by GLAPD (Supplementary Table S7) was in a gene coding (2E,6E)-farnesyl diphosphate synthase. It could amplify all genomes without any mismatches, and it was specific to all target genomes considering all other genomes in database-3 as the background genomes.

(2) To design group-specific primer for some genomes in a genus. Both V. cholerae species and V. vulnificus species belong to Vibrio genus. In database-3, 44 complete genomes were V. cholerae and 19 genomes were V. vulnificus. The primer set listed in Table 2 was still common in all 63 genomes without any mismatches, and is specific to the target genome considering the other genomes in database-3 as the background group (don&#x;t allow any mismatches).

(3) To design group-species primer for all genomes in a species. In database-3 there were complete genomes of S. aureus, the primer set in Table 1 was neither common nor specific in the new database. One new primer set (Supplementary Table S8) designed based on database-3 was overlap with two genes coding S4 domain-containing protein YaaA and DNA replication repair protein RecF. It was common for all genomes without any mismatches and specific to the target genomes as well.

(4) To design group-specific primer for some genomes in a species. Enterohemorrhagic Escherichia coli O:H7 was a major foodborne pathogen and it caused diarrhea (Tarr et al., ; Lim et al., ). The E. coli O:H7 was one group of E. coli species. In database-3, there were 59 complete genomes of E. coli and three were O:H7 (In order to exclude ambiguous group, only take care of part E. coli genomes, those accession number must start with NC ). One LAMP primer set (Supplementary Table S9) designed by GLAPD overlaps with two genes coding recombinase and peptide transporter. This primer set was common for all three genomes without any mismatch and specific to all these three genomes as well (with all other genomes in database-3 as the background group).

GLAPD Can Design Primers for Other Organisms

Genome based LAMP primer designer was first developed for rapidly detecting foodborne pathogens using LAMP technology. But group-specific primers for other organisms can also be designed by GLAPD. For example, we have tried to design group-specific primers to detect halal products using GLAPD. Pork is not allowed in halal products (Nakyinsige et al., ). The previous LAMP primer sets for pork identification were designed on DN1 gene and cytb gene located in mitochondria (Yang et al., ; Ran et al., ). But the LAMP primer set (Yang et al., ) couldn&#x;t cover all mitochondria genomes (Supplementary Table S10). In database-2, there were mitochondria sequences of sunia which were considered to be from pork products. We used GLAPD to design several LAMP primer sets with the sunia mitochondrion sequences as the target group and the rest in database-2 as the background group. One of these primer sets (Supplementary Table S11) was in a 16S rRNA gene. This LAMP primer set was common for all pork mitochondria and would amplify the pork mitochondria only.

We also tried to design group-specific primers for several types of aquatic animal viruses in database GLAPD could design group-specific primers for most aquatic animal viruses using default parameters (Supplementary Table S12). The group-specific primers of infectious hematopoietic necrosis virus and spring viraemia of carp virus were validated by experiments. If GLAPD couldn&#x;t design group-specific primers, it would output the most common primers.

Comparison With Existing Systems

PrimerExplorer V5 was an online LAMP primer design software. A candidate genomic region was required as the input. However, GLAPD did not require the prior knowledge about the candidate gene or genomic region. Using the same sequences from the candidate regions containing primer sets designed by GLAPD, the results of PrimerExplorer V5 and GLAPD were very similar. Most primers from two designers overlapped and some of them were identical (Figure 2). The main reason of differences was the different primer combination strategies.

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Figure 2. Comparison of the primer sets designed by GLAPD and PrimerExplorer V5. The sequence containing LAMP primer set designed by GLAPD was input to PrimerExplorer V5 to design primers. Four scenarios of LAMP primer sets were shown: (A) LAMP primer set for S. aureus based on database-3; (B) LAMP primer set for V. cholerae and V. vulnificus; (C) LAMP primer set for Salmonella based on database-3; (D) LAMP primer set for E. coli O:H7 based on database The primers designed by GLAPD were shown in green solid arrows and primers designed by PrimerExplorer V5 were in red blank arrows. The Salmonella&#x;s B2 primer designed by PrimerExplorer was the same as the B3 primer designed by GLAPD.

Existing systems such as PrimerExplorer, LAVA and FastPCR can design common primer set for a group of genomes without considering the specificity comparing to a background group (LAVA was not used to design primers because it was not downloadable anymore). Users can input a set of genomic regions considering the first region as the target and the rest as the background group to design specific primers. However, it is not straightforward to design primer sets directly with a given target group and a given background group at the same time. For example, the mecA and nuc genes were used as candidate regions (Wang et al., ; Chen et al., ) to design primers for S. aureus. In database-3, there were S. aureus genomes, among which genomes had mecA gene and all genomes had nuc gene. The non-redundant 19 mecA sequences and 44 nuc sequences were used as inputs for PrimerExplorer and FastPCR to design primers. For mecA gene, the MSA result was generated by clustalW (Thompson et al., ) with default parameter, and only % nucleotides were identical. PrimerExplorer couldn&#x;t design any LAMP primer sets using this MSA result and FastPCR also couldn&#x;t design any primers using the 19 mecA sequences. For nuc gene, the MSA alignment result was also generated by clustalW and only % nucleotides were identical. FastPCR couldn&#x;t design any common primers for the 44 sequences. PrimerExplorer could design two LAMP primer sets. However, because of the low similarity, there were many mutations in the designed primer regions in different target genomes (Figure 3, red underline), which indicated that the commonality of this primer set was not good. Therefore, using PrimerExplorer and FastPCR to design common primer set for S. aureus with the mecA and nuc gene regions as input was not successful. On the contrary, GLAPD could use the whole genome to design LAMP primer sets for the S. aureus genomes successfully. More examples are in Supplementary Material (Supplementary Figure S4 and Supplementary Table S14).

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Figure 3. The common primer sets of nuc gene designed by PrimerExplorer. The two primer sets are design by PrimerExplorer using MSA result of non-redundant nuc gene sequences. The red nucleotide with red underline means a mutation among those gene sequences.

More comparisons between GLAPD and those existing systems were listed in Table 3.

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Table 3. The comparison with existing LAMP primer designing systems.

Discussion

Why Do We Use Group-Specific Primers?

Detecting multiple foodborne pathogens simultaneously can help the food safety because it can speed the detection of pathogens. When use LAMP or PCR, the traditional method for this aim is adding multiple sets of primers into one assay. Each primer set can only be applied to one pathogen. This method has some disadvantages: (1) a large number of different primers in one assay may increase the risk of generating primer dimer, and the efficiency of one primer set could be inhibited by other primer sets (Xu et al., ; Zhao et al., ); (2) It&#x;s a challenge task to find a feasible combination of multiple primer sets due to the huge number of different combinations of candidate primer sets in addition to the test of each primer set for each target genome.

Genome based LAMP primer designer, on the other hand, can design group-specific primers, which can avoid those disadvantages. In one assay, only one primer set is needed. Less primers in assay can decrease the risk of interactions among primers and reduce the test workload as well.

Designing Primers Based on the Whole Genome

Traditionally, primers are designed for some conserved genes or a small genome region. Most regions of the whole genome are neglected. In addition, many primer design systems have a limited length of input sequences. If no primers could be designed by GLAPD, the chance to find suitable primers would be low.

Genome based LAMP primer designer can use whole genomes as input directly. It scans all candidate single primers derived from the whole genome, then all candidate primers are combined into primer sets and tested one by one. This can vastly improve the success rate of primer design.

A New Strategy Is Used to Design Group-Specific Primers

The group-specific primers can be designed based on conserved genes, genomic regions or MSA results. However, existing methods have limitations: (1) There are limited number of well-known conserved genes in each organism. This number will be much smaller for a slightly larger number of different target organisms. (2) The conserved genes may exist in some background organisms. For example, the 16S rRNA genes are conserved in many bacteria, therefore non-target organisms may also be amplified if they contain very similar 16S rRNA genes; (3) It is difficult to generate a MSA results from a big number of input sequences, and it is almost impossible to generate MSA for many genomes.

Genome based LAMP primer designer uses a different strategy to avoid those problems. Firstly, GLAPD searches all candidate primers genome wide, then the candidate single primers are aligned with target and background genomes. The alignment information about positions and strands is used to check primers&#x; commonality and specificity. The distance between two single primers can be different in each target genome as long as the distance is within the allowed range. The sequences between primers can also be different in different target genomes as long as the primer regions are conserved among target genomes. In other words, GLAPD can design group-specific primers in variable genome regions with a higher success rate. This strategy can also be used in designing primers for other amplifications, like PCR.

The Parameter Configuration in GLAPD

Parameters and thresholds used in GLAPD were similar with PrimerExplorer V5. Good results were still achieved if GLAPD used loose thresholds. For example, when the 5&#x; stability of F1c or B1c was set to be bigger than -4 kcal/mol, the experimental results of the primers were still good. More experiments might be needed to decide better parameters or thresholds for a specific group of organisms.

GPU Version of GLAPD

In order to accelerate the LAMP primer design, a GPU version of GLAPD was developed. The GPU version was very promising to accelerate the primer design procedure in identifying candidate single primer regions (GPU version is three time faster than CPU version) and combining single primers to a primer set may be slower than CPU version in some scenarios (Supplementary Table S13). We are currently working on it to improve the performance of the GPU version.

Conclusion

Designing group-specific primers is a difficult task for amplifications like PCR, and it is even more challenging for LAMP due to the number of primers in LAMP primer set. Here we present a new LAMP primer designer, GLAPD, to design a LAMP primer set targeting on a group of genomes. Instead of using well-known gene regions, the whole genome could be used directly for primer design, which increased the success rate.

Genome based LAMP primer designer could be applied to design LAMP primers for the identification of any organisms without known regions as input. The results of GLAPD are similar to PrimerExplorer V5 when the same sequences are input. The effectiveness of GLAPD were validated in experiments. With GLAPD, the chance to successfully design a LAMP primer set to identify a group of organism is higher than before and it can be a good system to accelerating the application of LAMP technology in many fields such as food quarantine, epidemic disease surveillance and so on. GLAPD can be downloaded from http://cgm.sjtu.edu.cn/GLAPD/ or https://github.com/jiqingxiaoxi/GLAPD.git. Users can also learn and test GLAPD using the simple online version: http://cgm.sjtu.edu.cn/GLAPD/online/.

Data Availability Statement

All datasets generated for this study are included in the article/Supplementary Material.

Author Contributions

CW: conceptualization and design of the system. BJ and CW: system implementation. BJ and LM: system test. XLi, WL, CL, XLu, and Y-YL: experimental validation of designed LAMP primers. BJ, XLi, Y-YL, and CW: writing the manuscript. All authors have read and edited the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (), the National Basic Research Program of China (CB), the National High-Tech R&#x;D Program () (AA), and Cross-Institute Research Funding of Shanghai Jiao Tong University (YGZD01 and YGMS39).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We thank the High Performance Computing Center (HPCC) at the Shanghai Jiao Tong University for computing.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles//fmicb/full#supplementary-material

Footnotes

References

Borjesson, S., Gomez-Sanz, E., Ekstrom, K., Torres, C., and Gronlund, U. (). Staphylococcus pseudintermedius can be misdiagnosed as Staphylococcus aureus in humans with dog bite wounds. Eur. J. Clin. Microbiol. Infect. Dis. 34, &#x; doi: /sy

PubMed Abstract | CrossRef Full Text | Google Scholar

Chaumpluk, P., Plubcharoensook, P., and Prasongsuk, S. (). Rapid detection of aflatoxigenic Aspergillus sp. in herbal specimens by a simple, bendable, paper-based lab-on-a-chip. Biotechnol. J. 11, &#x; doi: /biot

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, C., Zhao, Q., Guo, J., Li, Y., and Chen, Q. (). Identification of Methicillin-resistant Staphylococcus aureus (MRSA) using simultaneous detection of meca, nuc, and femb by loop-mediated isothermal amplification (LAMP). Curr. Microbiol. 74, &#x; doi: /s

PubMed Abstract | CrossRef Full Text | Google Scholar

Curtis, K. A., Rudolph, D. L., Nejad, I., Singleton, J., Beddoe, A., Weigl, B., et al. (). Isothermal amplification using a chemical heating device for point-of-care detection of HIV PLoS One 7:e doi: /journal.pone

PubMed Abstract | CrossRef Full Text | Google Scholar

Demkin, V. V., Koshechkin, S. I., and Slesarev, A. (). A novel real-time PCR assay for highly specific detection and quantification of vaginal Lactobacilli. Mol. Cell Probes 32, 33&#x; doi: /j.mcp

PubMed Abstract | CrossRef Full Text | Google Scholar

Han, F., Wang, F., and Ge, B. (). Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification. Appl. Environ. Microbiol. 77, &#x; doi: /AEM

PubMed Abstract | CrossRef Full Text | Google Scholar

Hoegh, S. V., Skov, M. N., Boye, K., Worning, P., Jensen, T. G., and Kemp, M. (). Variations in the Staphylococcus aureus-specific nuc gene can potentially lead to misidentification of meticillin-susceptible and -resistant S. aureus. J. Med. Microbiol. 63, &#x; doi: /jmm

PubMed Abstract | CrossRef Full Text | Google Scholar

Huehn, S., Eichhorn, C., Urmersbach, S., Breidenbach, J., Bechlars, S., Bier, N., et al. (). Pathogenic vibrios in environmental, seafood and clinical sources in Germany. Int. J. Med. Microbiol. , &#x; doi: /j.ijmm

PubMed Abstract | CrossRef Full Text | Google Scholar

Kadariya, J., Smith, T. C., and Thapaliya, D. (). Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed. Res. Int. doi: //

PubMed Abstract | CrossRef Full Text | Google Scholar

Kalendar, R., Tselykh, T. V., Khassenov, B., and Ramanculov, E. M. (). Introduction on using the FastPCR software and the related java web tools for PCR and oligonucleotide assembly and analysis. Methods Mol. Biol. , 33&#x; doi: /_2

PubMed Abstract | CrossRef Full Text | Google Scholar

Karmakar, A., Dua, P., and Ghosh, C. (). Biochemical and molecular analysis of Staphylococcus aureus clinical isolates from hospitalized patients. Can. J. Infect. Dis. Med. Microbiol. doi: //

PubMed Abstract | CrossRef Full Text | Google Scholar

Kurosaki, Y., Grolla, A., Fukuma, A., Feldmann, H., and Yasuda, J. (). Development and evaluation of a simple assay for Marburg virus detection using a reverse transcription-loop-mediated isothermal amplification method. J. Clin. Microbiol. 48, &#x; doi: /JCM

PubMed Abstract | CrossRef Full Text | Google Scholar

Langmead, B., Trapnell, C., Pop, M., and Salzberg, S. L. (). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. R doi: /gbr25

PubMed Abstract | CrossRef Full Text | Google Scholar

Lim, J. Y., Yoon, J., and Hovde, C. J. (). A brief overview of Escherichia coli OH7 and its plasmid O J. Microbiol. Biotechnol. 20, 5&#x; doi: /jmb

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, J., Bai, L., Li, W., Han, H., Fu, P., Ma, X., et al. (). Trends of foodborne diseases in China: lessons from laboratory-based surveillance since Front. Med. 12, 48&#x; doi: /s

PubMed Abstract | CrossRef Full Text | Google Scholar

Mori, Y., and Notomi, T. (). Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 15, 62&#x; doi: /s

PubMed Abstract | CrossRef Full Text | Google Scholar

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., et al. (). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. E

Google Scholar

Okada, K., Chantaroj, S., Taniguchi, T., Suzuki, Y., Roobthaisong, A., Puiprom, O., et al. (). A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples. Diagn. Microbiol. Infect. Dis. 66, &#x; doi: /j.diagmicrobio

PubMed Abstract | CrossRef Full Text | Google Scholar

O&#x;Leary, N. A., Wright, M. W., Brister, J. R., Ciufo, S., Haddad, D., McVeigh, R., et al. (). Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D&#x;D doi: /nar/gkv

PubMed Abstract | CrossRef Full Text | Google Scholar

Parida, M., Sannarangaiah, S., Dash, P. K., Rao, P. V., and Morita, K. (). Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev. Med. Virol. 18, &#x; doi: /rmv

PubMed Abstract | CrossRef Full Text | Google Scholar

Paudyal, N., Anihouvi, V., Hounhouigan, J., Matsheka, M. I., Sekwati-Monang, B., Amoa-Awua, W., et al. (). Prevalence of foodborne pathogens in food from selected African countries - A meta-analysis. Int. J. Food Microbiol. , 35&#x; doi: /j.ijfoodmicro

PubMed Abstract | CrossRef Full Text | Google Scholar

Peng, H., Qin, L., Bi, Y., Wang, P., Zou, G., Li, J., et al. (). Rapid detection of the common avian leukosis virus subgroups by real-time loop-mediated isothermal amplification. Virol. J. doi: /s

PubMed Abstract | CrossRef Full Text | Google Scholar

Ran, G., Ren, L., Han, X., Liu, X., Li, Z., Pang, D., et al. (). Development of a rapid method for the visible detection of pork DNA in halal products by loop-mediated isothermal amplification. Food Anal. Methods 9, &#x; doi: /sz

CrossRef Full Text | Google Scholar

Tarr, P. I., Gordon, C. A., and Chandler, W. L. (). Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet , &#x; doi: /s(05)

PubMed Abstract | CrossRef Full Text | Google Scholar

Thompson, J. D., Higgins, D. G., and Gibson, T. J. (). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, &#x; doi: /nar/

PubMed Abstract | CrossRef Full Text | Google Scholar

Torres, C., Vitalis, E. A., Baker, B. R., Gardner, S. N., Torres, M. W., and Dzenitis, J. M. (). LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures. BMC Bioinformatics doi: /

PubMed Abstract | CrossRef Full Text | Google Scholar

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., et al. (). Primer3&#x;new capabilities and interfaces. Nucleic Acids Res. e doi: /nar/gks

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, X. R., Wu, L. F., Wang, Y., Ma, Y. Y., Chen, F. H., and Ou, H. L. (). Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification. Appl. Biochem. Biotechnol. , &#x; doi: /sx

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, Y., Li, D., Li, K., and Ye, C. (). Rapid and sensitive detection of Vibrio parahaemolyticus and Vibrio vulnificus by multiple endonuclease restriction real-time loop-mediated isothermal amplification technique. Molecules E doi: /molecules

PubMed Abstract | CrossRef Full Text | Google Scholar

Xu, W., Zhai, Z., Huang, K., Zhang, N., Yuan, Y., Shang, Y., et al. (). A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis. PLoS One 7:e doi: /journal.pone

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, L., Fu, S., Peng, X., Li, L., and Song, T. (). Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnol. Biotechnol. Equip. 28, &#x; doi: /

PubMed Abstract | CrossRef Full Text | Google Scholar

Yao, X., Li, P., Xu, J., Zhang, M., Ren, R., Liu, G., et al. (). Rapid and sensitive detection of Didymella bryoniae by visual loop-mediated isothermal amplification assay. Front. Microbiol. doi: /fmicb

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhao, X., Lin, C. W., Wang, J., and Oh, D. H. (). Advances in rapid detection methods for foodborne pathogens. J. Microbiol. Biotechnol. 24, &#x;

PubMed Abstract | Google Scholar

Keywords: LAMP, group-specific primer, whole genome based primer designer, foodborne pathogens, primer design

Citation: Jia B, Li X, Liu W, Lu C, Lu X, Ma L, Li Y-Y and Wei C () GLAPD: Whole Genome Based LAMP Primer Design for a Set of Target Genomes. Front. Microbiol. doi: /fmicb

Received: 17 June ; Accepted: 26 November ;
Published: 13 December

Reviewed by:

Debmalya Barh, Institute of Integrative Omics and Applied Biotechnology (IIOAB), India
Pallavi Singh, Northern Illinois University, United States

Copyright © Jia, Li, Liu, Lu, Lu, Ma, Li and Wei. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Yuan-Yuan Li, [email protected]; Chaochun Wei, [email protected]

&#x;These authors have contributed equally to this work and share first authorship

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Loop Mediated Isothermal Amplification (LAMP) Tutorial

References >> LAMP

LAMP - Loop Mediated Isothermal Amplification

"LAMP" stands for Loop-mediated Isothermal Amplification. This technology was developed by Notomi et al. It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.

Types of Primers used in LAMP

LAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions of the target gene. The four primers used are as follows:

1. Forward Inner Primer (FIP): The FIP consists of a F2 region at the 3'end and a F1c region at the 5'end. The F2 region is complementary to the F2c region of the template sequence. The F1c region is identical to the F1c region of the template sequence.

2. Forward Outer Primer (FOP): The FOP (also called F3 Primer) consists of a F3 region which is complementary to the F3c region of the template sequence. This primer is shorter in length and lower in concentration than FIP.

3. Backward Inner Primer (BIP): The BIP consists of a B2 region at the 3'end and a B1c region at the 5'end. The B2 region is complementary to the B2c region of the template sequence. The B1c region is identical to the B1c region of the template sequence.

4. Backward Outer Primer (BOP): The BOP (also called B3 Primer) consists of a B3 region which is complementary to the B3c region of the template sequence.

Stages in Loop-mediated Isothermal Amplification

1. F2 region of FIP hybridizes to F2c region of the target DNA and initiates complementary strand synthesis.


2. Outer primer F3 hybridizes to the F3c region of the target DNA and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5' end.


3. This single stranded DNA with a loop at the 5' end serves as a template for BIP. B2 hybridizes to B2c region of the template DNA. DNA synthesis is now initiated leading to the formation of a complementary strand and opening of the 5' end loop.


4. Now, the outer primer B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand. This results in the formation of a dumbbell shaped DNA.


5. The nucleotides are added to the 3' end of F1 by DNA polymerase, which extends and opens up the loop at the 5' end. The dumbbell shaped DNA now gets converted to a stem loop structure. This structure serves as an initiator for LAMP cycling, which is the second stage of the LAMP reaction.


6. To initiate LAMP cycling, the FIP hybridizes to the loop of the stem-loop DNA structure. Strand synthesis is initiated here. As the FIP hybridizes to the loop, the F1 strand is displaced and forms a new loop at the 3' end.


7. Now nucleotides are added to the 3' end of B1. The extension takes place displacing the FIP strand. This displaced strand again forms a dumbbell shaped DNA. Subsequent self-primed strand displacement DNA synthesis yields one complementary structure of the original stem loop DNA and one gap repaired stem loop DNA.


8. Both these products then serve as template for a BIP primed strand displacement reaction in the subsequent cycles. Thus, a LAMP target sequence is amplified 13 fold every half cycle.


The final products obtained are a mixture of stem loop DNA with various stem lengths and various cauliflower like structures with multiple loops. The structures are formed by annealing between alternatively inverted repeats of the target sequence in the same strand.

Lamp Detection

In a LAMP assay, the reaction takes place in a single tube containing buffer, target DNA, DNA polymerase and primers. The tube is incubated at 64°C in a regular laboratory water bath or heat block that helps in maintaining a constant temperature. The amplified product can be detected by naked eye as a white precipitate or a yellow-green color solution after addition of SYBR green to the reaction tube.

Advantages

1. Amplification of DNA takes place at an isothermal condition (63 to 65°C) with greater efficiency.

2. Thermal denaturation of double stranded DNA is not required.

3. LAMP helps in specific amplification as it designs 4 primers to recognize 6 distinct regions on the target gene.

4. LAMP is cost effective as it does not require special reagents or sophisticated equipment.

5. This technology can be used for the amplification of RNA templates in presence of reverse transcriptase.

6. LAMP assay takes less time for amplification and detection.

Applications

1. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases.

2. It helps in the identification of genus and species-specific parasites.

Efficient Primer Design for Loop-mediated Isothermal Amplification using LAMP Designer

LAMP Designer designs four primers along with two additional loop primers to identify six distinct regions. The software automatically interprets BLAST results and even avoids homologous regions on the sequence while designing primers to prevent non-specific amplification. The designed primers can also be BLAST searched to verify their specificity.

For details, please visit: http://www.premierbiosoft.com/isothermal/lamp.html


PREMIER Biosoft products
Sours: http://www.premierbiosoft.com/tech_notes/Loop-Mediated-Isothermal-Amplification.html

Primers lamp

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Loop Mediated Isothermal Amplification-Animation

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