Real-time quantitative PCR

To detect and quantify M. ulcerans DNA in environmental samples, we performed two TaqMan qPCR runs; one targetting the insertion sequence IS2404 and one targetting the ketoreductase B (KR) domain of the mycolactone polyketide synthase gene that is specifically found in the virulence plasmid of M. ulcerans strains. To amplify IS2404 genetic marker, we used the following primer and probes: IS2404 forward primer 5’-ATTGGTGCCGATCGAGTTG-3’, IS2404 reverse primer 5’-TCGCTTTGGCGCGTAAA-3’ and IS2404 probe FAM-CACCACGCAGCATTCTTGCCGT-BHQ1 [21]. For KR amplification we used KR forward primer 5’-TCACGGCCTGCGATATCA-3’, KR reverse primer 5’-TTGTGTGGGCACTGAATTGAC-3’, and KR probe FAM-ACCCCGAAGCACTG-MGBNFQ [19]. The qPCR reaction consisted of 1X TaqMan Gene Expression Master Mix (LifeTechnologies), 0.3 μM (final concentration) of each primer, 0.1 μM (final concentration) of the probe, 5 μl of DNA and water adjusted to a final volume per reaction of 25 μl. For KR we followed the same protocol except that we used the probe at a final concentration of 0.25 μM. An internal positive control (IPC) was added in each IS2404 reaction in order to test for the presence of PCR inhibitors in the environmental samples. In each qPCR plate, a positive (M. ulcerans DNA at a concentration of 10⁵ bacteria/mL) and negative (DNA replaced by water) controls were included. The positive control for M. ulcerans consisted of genomic DNA purified from a cultured strain from French Guiana (strain 1G897) and provided by Laurent Marsollier (ATOMYCA, Université d’Angers). This positive control was also used in our study to run standard curves based on serial dilutions of purified DNA from 10⁵ to 10⁰ bacteria/mL (in triplicates). Standard curves allowed us to determine a threshold value above which we considered our samples as negative (CT-values > 38). The assays were run in duplicates on an Applied Biosystems 7300 Real Time PCR system, with the following program: one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec and at 60°C for 1 min. Only samples with cycle threshold values < 38 for both IS2404 and KR markers in 1 out of 2 replicates were considered as positives. In all assays the negative controls remained negative.

To date, 22 species of Leptospira have been described and arranged into 3 groups based on their pathogenicity; pathogenic species (L. interrogans, L. kirschneri, L. borgpetersenii, L. mayottensis, L. santarosai, L. noguchii, L. weilii, L. alexanderi, L. kmetyi, L. alstonii), intermediate species of unclear or low pathogenicity (L. broomii, L. fainei, L. inadai, L. licerasiae, L. wolffii), and saprophytic species which are free-living cells in water and soil and are not infectious (L. biflexa, L. idonii, L. meyeri, L. terpstrae, L. vanthielli, L. wolbachii, L. yanagawae) [22]. Whilst pathogenic and intermediate Leptospira species are infectious for humans or animals [22], most diagnotic PCR tools only detect Leptospira from the pathogenic cluster and fail to detect intermediate species [23]. Among these tools the TaqMan qPCR assay targetting the lipL32 gene is commonly used to detect pathogenic Leptospira [5,23,24] since it encodes outer membrane proteins and virulence factors found in pathogenic species [22]. This qPCR assay has been optimized for both sensitivity and specificity, allowing thus to detect and characterize Leptospira sp. in low number or in samples that contain high concentrations of non-Leptospira DNA [22,23]. However since this target gene is highly conserved among Leptospira species it does not allow the discrimination between species. Therefore, to detect the presence of Leptospira sp. DNA we performed a qPCR targetting the lipL32 gene. To do so, we used forward primer LipL32-45F 5’-AAGCATTACCGCTTGTGGTG-3’, reverse primer LipL32-Rb 5’-GAACTCCCATTTCAGCGAT-3’ and the probe LipL32-189P FAM-AAAGCCAGGACAAGCGCCG-BHQ1 [23]. The qPCR reaction consisted of 1X TaqMan Gene Expression Master Mix (LifeTechnologies), 0.7 μM (final concentration) of each primer, 0.15 μM (final concentration) of the probe, 5 μl of DNA and water adjusted to a final volume per reaction of 25 μl. In each qPCR plate, a positive (L. santarosai DNA at a concentration of 10² bacteria/mL) and negative (DNA replaced by water) controls were included. The positive control for Leptospira sp. consisted of genomic DNA of the strain L. santarosai that was provided by the Pasteur Institute in Paris (Pascale Bourhy, Centre National de Référence de la Leptospirose). Standard curves were run with serial dilutions of genomic DNA from L. santarosai from 10⁵ to 10⁰ bacteria/mL (in triplicates). The assays were run on an Applied Biosystems 7300 Real Time PCR system, with the following program: one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec and at 60°C for 1 min. Only samples with cycle threshold values < 40 were considered as positives. In all assays the negative controls remained negative.