A simple and rapid nested polymerase chain reaction-restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp.
A rapid and specific nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to detect and differentiate pathogenic and nonpathogenic Leptospira spp. Leptospiral genomic DNA was extracted from suspected human sera using an improved method of standard phenol-chloroform, and specific primers have been used to amplify 16S ribosomal RNA from all pathogenic and nonpathogenic Leptospira spp. The PCR products of all nonpathogenic species were digested with ApoI enzyme, but not pathogenic. To evaluate this assay, we analyzed 283 serum samples collected from suspected patients with leptospirosis. Nested PCR assay confirmed that 42 (14.8%) of 283 samples harbored Leptospira infection, and RFLP assay confirmed 38 (90.5%) of 42 and 4 (9.5%) of 42 positive cases had pathogenic and nonpathogenic Leptospira spp., respectively. Based on sequencing results, Leptospira interrogans, Leptospira kirschneri, and Leptospira wolffii and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected among analyzed samples. The nested PCR-RFLP assay developed in this study fulfills this requirement in the early stage of infection.
Djadid
Navid Dinparast
ND
Biotechnology Research Center, Pasteur Institute of Iran, Tehran.
Ganji
Zahra Faghanzadeh
ZF
Gouya
Mohammad Mehdi
MM
Rezvani
Mahmood
M
Zakeri
Sedigheh
S
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005260:Female; D006801:Humans; D007919:Leptospira / Q000145:classification* / Q000235:genetics* / Q000472:pathogenicity; D007922:Leptospirosis / Q000175:diagnosis* / Q000382:microbiology; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D044967:Serum / Q000382:microbiology
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C075297:endodeoxyribonuclease ApoI; D015252:Deoxyribonucleases, Type II Site-Specific