A Rapid and Robust DNA Extraction Method for PCR-Based Diagnosis of V. cholerae

Abstract

Cholera, caused by _V. cholerae_, needs rapid diagnosis because of its threat for rapid spread. Recent molecular diagnosis by PCR assay is more advantageous than the conventional methods. The bottleneck in PCR diagnosis lies in the delay in template DNA extraction of _V. cholerae_, which is the basic requirement. This study describes a simple, less expensive, and rapid template DNA extraction method to lessen the total time for PCR diagnosis of _V. cholerae._ To obtain template DNA, our method involves boiling of _V. cholerae_ suspension in distilled water (takes 18-24 hours) instead of boiling the suspension in broth medium, which is a lengthy process (takes 72 hours). The validation of our template DNA was conducted using 40 _V. cholerae_ O1 strains that were confirmed previously, and its usefulness was checked on 20 clinical strains of _V. cholerae_ O1 isolated from acute diarrhea patients; results were compared with those of conventional template DNA. The analysis of our template DNA in simplex and quadruplex PCR assays revealed the presence of _ctxA_, _tcpA _(El Tor), _rfb _(O1), _ToxR_, _ctxB_, _zot_, _ace,rst _, and _OmpW _genes in _V. cholerae _O1. The comparison of the results of PCR assays using the template DNA from both sources showed equal results and matched correctly. The sensitivity of our template DNA was 1.5x103 CFU per assay. Our template DNA extraction method is simple, less expensive, and rapid, and it can be employed for early diagnosis of _V. cholerae_ during outbreaks, in research laboratories, and in hospital setups where infrastructures are available.