Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of and on the BD MAX Platform.

and 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 and directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene , the cholera toxin (CT) coding gene , the O1 serogroup specific gene , and the O139 serogroup specific gene of . The other, BDM-VP, targets the gene and the toxin coding genes and of . 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 and 46-184 CFU/ml for , 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 , 100% for /, and lower (66.7%) for 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 and directly from human samples.