Computational protocol: Regional Differences of Leptospirosis in Sri Lanka: Observations from a Flood-Associated Outbreak in 2011

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Protocol publication

[…] All qPCR positive samples were amplified using a previously published nested PCR protocol . This single-tube nested PCR was used to amplify a region of the 16S ribosomal DNA gene specific for pathogenic and intermediate Leptospira spp. The PCR primers were rrs- outer-F (5-CTCAGAACTAACGCTGGCGGCGCG-3′), rrs- outer-R(5′-GGTTCGTTACTGAGGGTTAAAACCCCC-3′), rrs-inner-F (5′-CTGGCGGCGCGTCTTA-3′), and rrs-inner-R (5′-GTTTTCACACCTGACTTACA-3′). PCR products were purified using the USB ExoSAP-IT PCR product cleanup kit (Affymetrics) according to manufacturer's instructions. At least two forward and two reverse sequencing reads were obtained for all samples; sequences were assembled and aligned using Geneious software then the alignments trimmed to yield a 443 bp region for phylogenetic analysis [positions 89–531 of the rrs gene of L. interrogans serovar Lai strain 56601 (NC_004342) for phylogenetic analysis. For species identification, maximum likelihood trees with support from bootstrap 500 replicates were created in MEGA5. […]

Pipeline specifications

Software tools Geneious, MEGA
Application Phylogenetics
Organisms Leptospira interrogans, Leptospira kirschneri
Diseases Communicable Diseases, Leptospirosis, Myocarditis, Oculocerebrorenal Syndrome, Thrombocytopenia, Acute Kidney Injury