Detection of toxigenic Vibrio cholerae with new multiplex PCR

Abstract

Vibrio cholerae (V. cholerae) is the etiological agent of epidemic cholera. In recent years, cholera has become endemic in different regions of the world. Traditional culture and microscopic methods are considered the standard for the diagnosis of V. cholerae. However, these methods require days for confirmation. Therefore, molecular methods that may be used for the sensitive, accurate, and rapid analysis of V. cholerae are urgently needed. In the present investigation, a multiplex PCR assay was developed for the detection of virulence- and toxigenic-associated (VTA) genes (ctxA, tcpA and ompW). To evaluate PCR specificity, other bacteria from the Enterobacteriaceae family (Salmonella typhi, Shigella dysenteriae, and enterotoxigenic Escherichia coli) and Aeromonas hydrophila were examined. The assay sensitivity was evaluated using colony counting and genome dilution methods. Our results showed that this PCR-based method represents an ideal tool for the rapid detection of VTA genes due to its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used to determine the presence of VTA genes and can therefore distinguish V. cholerae bacteria from other Vibrio species and bacteria. This method can detect 10-100 CFU V. cholerae and 8.5-85 pg genomic DNA. This multiplex PCR method has higher sensitivity and specificity than other methods. The proposed test provides an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.