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

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.

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 described [2][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 malaria 6 and salmonella 7 , viral RNA rapid detection for HIV with reverse transcriptase LAMP (RT-LAMP) 8 , GM crop contamination 9 and in forensic science to specifically detect human DNA 10 .
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 primers 11 and STEM primers 12 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 primers 13 . 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 inhibitors 9,14,15 permitting less stringent DNA extraction procedures.
PCR has been developed to increase the specificity of the two primer amplification method. Real time PCR 16 developments have included amplicon specific TaqMan probes 17 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 beacons 18 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 reported 19 . 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, turbidity 20 from pyrophosphate precipitation, observations of LAMP ladder patterns with agarose gel electrophoresis 5 , quenched calcein 21 , hydroxy-naphthol blue 22 as well as a number of fluorescent probe based techniques 15,23,24 . 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 Green 26,27 and the SYTO family 28,29 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 turbidimetry 30 , colour change with pH sensitive dyes 31 and amplifying with the nucleic acid specific dye berberine 32 .
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 beacons 33 , 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 structures 34 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 fluorophores 35 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 guanine 36,37 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 LAMP 23 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 assay 19 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 probe 24 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 LAMP 42 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-ELISA 44 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.
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 120 minutes. All four replicates of samples with 500 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 0.4 to 0.8 micromolar JOE-FIP (Fig. 3A,E). 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 50000 template copies had an average Ct of 11.2 (SD 0.6) and for 50 copies an average Ct of 18.6 (SD 0.9), at a defined cycle threshold of −0.03. 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 50000 template copies assay had an average Ct of 11.7 (SD 0.4) and for 50 copies this figure was 17.2 (SD 1.4) at a cycle threshold of 0.4. The replicate assay with unlabelled FIP and SYTO dye had more rapid Ct values of 8.1 (SD 0.1) for the 50000 copies and 12.7 (SD 0.5) for the 50 copies at the cycle threshold of 0.4 (Fig. 4,3).
The linearity for the dilution range to a semi-log model with each method was high (R2 0.9799 to 0.9912), however it is apparent that the variation between replicates increases with decreasing concentration as previously observed 46 . 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 200 minutes to encourage amplification from non-specific primer interactions (Fig. 5,2A-C). www.nature.com/scientificreports www.nature.com/scientificreports/ Using SYTO9 for detection, five of the seventy two NTC replicates gave a positive signal between 150 and 200 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 200 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 470 and 510 nanometers separated from JOE excitation and emission at 520 and 555 nanometers. Assay times were extended to 180 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 100 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.
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 quantification 47,48 , 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).   www.nature.com/scientificreports www.nature.com/scientificreports/ 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 results 9 , 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 100 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 100 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 100 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. www.nature.com/scientificreports www.nature.com/scientificreports/ 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).
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 observed 35 . 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 10-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 www.nature.com/scientificreports www.nature.com/scientificreports/ 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 0.8 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 150 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 BART 25 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 programmes 49,50 . 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. www.nature.com/scientificreports www.nature.com/scientificreports/ Carrier DNA has previously been shown to enhance sensitivity, reduce variation between replicates, and increase reaction times and amplification frequency 9 when added at 100 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 100 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 100 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 495:520 and 544:576 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. The size of the linearised plasmid pART7 was assumed to be 4900 base pairs and 2.4 × 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 NK603, and the seed stock of Mon810 are made in accordance with Hardinge et al. 46 by adjusting transgenic copy numbers by 160/385. 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 100 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 optimised 9,46,52 . 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 quenching 35 . 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. 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), 300 micromolar each deoxynucleotide triphosphate (dNTP), 0.8 micromolar Betaine, 0.32 units per microlitre Bst polymerase v2.0 warm start (NEB), 0.5 micromolar SYTO9 Green, 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer, 0.2 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 www.nature.com/scientificreports www.nature.com/scientificreports/ degrees C. Results were analysed on RotorGene 6000 software v1.7 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 −0.05 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.

Methods
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 470 +/− 10 nanometers, detection 510 +/− 5 nanometers), JOE on the yellow channel (source 530 +/− 5 nanometers, detection 557 +/− 5 nanometers) and TAMRA with the orange channel (excitation at 585 +/− 5 nanometers, detection aat 610 +/− 5 nanometers) in the same reaction. Melt temperature analysis followed amplification between 60 and 95 degrees C. Results were analysed RotorGene 6000 software v1.7 and Microsoft Excel.