Abstract
Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.
Highlights
Vibrio cholerae is the etiological pathogen of cholera, an acute watery diarrheal disease
We developed a multiplex real-time PCR assay on a BD MAX open system for detection and differentiation of V. cholerae and V. parahaemolyticus directly from human fecal specimens
The limit of detection (LoD) of BDM-VC for target genes of V. cholerae was 328– 655 CFU/ml and 195–780 CFU/ml when analyzed with stools spiked with N16961 (O1 serogroup) and ATCC51394 (O139 serogroup), respectively
Summary
Vibrio cholerae is the etiological pathogen of cholera, an acute watery diarrheal disease. Researchers have estimated that in endemic countries globally, there are approximately 1.3 billion people at risk for cholera, 1.3–4.0 million cholera cases occurring annually, and 21,000–143,000 deaths among these cases (Ali et al, 2015). The current seventh cholera pandemic continues to be a major public health threat for countries in Asia, Africa, and the Americas (WHO, 2018). Cholera toxin (CT) encoded by ctxA and ctxB is responsible for severe, cholera-like diseases in epidemic and sporadic forms (Holmgren, 1981; Wernick et al, 2010). In assessing the public health significance of an isolate of V. cholerae, the possession of the O1 or O139 antigen is a marker of epidemic or pandemic potential (Kaper et al, 1995), and the production of CT is one of the most important properties to be determined
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