Optimization and Comparative Evaluation of SimpliAmp and Bio-Rad Thermal Cyclers for PCR-Based Detection of Vibrio cholerae

Cholera continues to pose a significant public health risk in low- and middle-income countries (LMICs), necessitating quick, cost-effective, and dependable diagnostics for effective outbreak surveillance and management. In this study, we optimized and tested two PCR-based assays targeting a uniplex assay with the ompW gene, and a multiplex assay with ctxA, O1-rfb, and O139-rfb genes of Vibrio cholerae (V. cholerae) on two thermal cycler platforms with different cost: the Bio-Rad C1000 Touch, which is more expensive, and the SimpliAmp, which is cheaper. Cycling conditions, annealing temperatures, and primer concentrations were systematically optimized to improve efficiency and reagent use. We tested our assay sensitivity by serially diluting gBlock DNA from 2000 to 50 copies. Quo data software was used to estimate the limit of detection and Fishers exact tests with Holm-Bonferroni correction to assess any differences (p<0.05) across all dilution levels on both platforms. The ompW assay achieved identical performance on both platforms, with 100% detection down to 250 copies and an LOD of 523 copies (95% CI: 347- 786; p = 1.000). For the multiplex assay, O1-rfb showed slightly higher sensitivity on SimpliAmp (LOD= 509 copies, 95% CI: 339-766) than on Bio-Rad (LOD = 333 copies, 95% CI: 255-688; p = 0.217). The ctxA assay had an LOD of 684 copies (95% CI: 501-1323) on SimpliAmp and 974 copies (95% CI: 714-1784) on Bio-Rad (p = 0.089). For O139-rfb, SimpliAmp and Bio-Rad achieved LOD values of 639 (95% CI: 490-1251) and 1060 copies (95% CI: 719-1562), respectively (p = 0.179). In general, the differences in performance between platforms were not statistically significant. These results highlight the potential of SimpliAmp as a reliable, low-cost alternative for cholera diagnostics in LMICs and low-resource settings, supporting broader access to molecular tools for outbreak investigation and public health surveillance.