A simple and rapid nested polymerase chain reaction-restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp.

作者：Djadid Navid Dinparast; Ganji Zahra Faghanzadeh; Gouya Mohammad Mehdi; Rezvani Mahmood; Zakeri Sedigheh^*

来源：Diagnostic Microbiology and Infectious Disease, 2009, 63(3): 251-256.

DOI：10.1016/j.diagmicrobio.2008.10.017

• Diagnosis
• Leptospira
• Nonpathogen
• Pathogen
• PCR/RFLP
• 16S RNA

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 Apol 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, and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected Leptospira kirschneri, and Leptospira wolffii among analyzed samples. The nested PCR-PFLP assay developed in this study fulfills this requirement in the early stage of infection.

• 出版日期2009-3