Detection of leptospiral DNA by PCR.
An EcoRI fragment (1.2 kb) which is highly conserved among Leptospira interrogans isolated in Korea was cloned into pBluescript vector from L. interrogans serovar lai WH20. The EcoRI fragment was sequenced, and a pair of primers (LP1 and LP2) was designed for PCR assay. PCR amplification of target DNA obtained from cultured L. interrogans showed that 274 bp could be detected when as little as 100 fg of leptospiral genomic DNA was used in the reaction mixture. No amplification of DNA was detected from DNA of Leptospira biflexa serovars patoc and sau paulo, Borrelia burgdorferi, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium. Amplification of 274-bp target DNA could be detected in DNA samples purified from 500 microliters of blood collected from experimentally infected gerbils 2 days after infection, while antibodies to L. interrogans could be detected by the microscopic agglutination test 7 days after infection. The specificity and high sensitivity of the test provided valuable tools for the early diagnosis of leptospirosis.
Kee
S H
SH
Department of Microbiology, Seoul National University College of Medicine, Republic of Korea.
Kim
I S
IS
Choi
M S
MS
Chang
W H
WH
D000372:Agglutination Tests; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005849:Gerbillinae; D006801:Humans; D007921:Leptospira interrogans / Q000235:genetics* / Q000302:isolation & purification; D007922:Leptospirosis / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial