16S ribosomal RNA 유전자 증폭에 의한 leptospira interrogans 검출

Detection of leptospira interrogans by amplification of 16S ribosomal RNA gene

Two sets of primers(LIF and LIR, LHF and LHR) for PCR assay were designed to detect the species of Leptospira(L.) interrogans and L. interrogans serovar hardjo based on nucleotide sequences of the 16S ribosomal RNA genes, respectively. The optimal annealing temperature was about 55℃ for primers LIF and LIR,and was 53℃ for primers LHF and LHR. The 460 base pair DNA fragment was amplified with the primers LIF and LIR in all of the serovar of L. interrogans tested. No amplified DNA was detected from L. bifelexa and Leptonema illini. The 384 base pair DNA fragment produced by the primers LHF and LHR was only amplified in Leptospira interrogans serovar hardjo. None of the other serovar of L. interrogans, L. bifelexa and Leptonema illini was detected the DNA with the PCR assay. As little as 100pg of the leptospiral genomic DNA could be detected using the PCR assay. The amplified DNA by PCR assay could be detected in liver samples colleted from experimentally infected guinea pigs at 2 days after infection, while the L. interrogans serovar hardjo-bovis could be isolated at 3 days after infection. This study suggested that the developed PCR assay is a rapid and specific method for the early diagnosis of leptospirosis.