Optimization and Validation of Real Time PCR Assays for Absolute Quantification of toxigenic Vibrio cholerae and...

Quantitative real-time PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences and is more accurate compared to both traditional culture based techniques and 'end point' conventional PCR. Serial dilution of bacterial cell culture provides information on colony forming unit (CFU) counts. This is crucial for obtaining optimal standard curves representative of DNA concentration. This approach eliminates variation in the standard curves caused by loss of DNA by serial dilution of nucleic acid elute. In this study, an assay was developed to detect and quantify DNA by real-time PCR for two pathogenic species, Escherichia coli (E. coli) and Vibrio cholerae (V. cholerae). In order to generate a standard curve, total bacterial DNA was diluted in a 10-fold series and each sample was adjusted to an estimated cell count. The starting bacterial DNA concentration was 11ng/µL. An individual E. coli cell has approximately 5.16 femtograms of DNA. Therefore, 11 ng/µL of DNA would indicate 2.48×10⁷cells. Both SYBR Green and TaqMan assays were validated for uidA region in E. coli and ctxA region in V. cholerae, respectively and was based on previously published assays for this standard curve experiment. PCR efficiency for uidA gene and ctxA gene were obtained 103.8% and 99.21%, respectively. Analysis of Variance (ANOVA) and coefficient of variation (CV %) indicated that standard curve generated by genomic DNA dilution had higher repeatability. Although not statistically significant, low F ratios indicated that there was some variation in CT values when genomic DNA dilution was compared to dilution of cell suspension in media. Different water samples spiked with pure cultures of E. coli and V. cholerae were used as unknown samples. The standard curve constructed by the serial dilution of genomic DNA exhibited greater efficiency when compared to that of the standard curve obtained from serial dilution of cell suspension since in the former method DNA is not lost during extraction from culture dilutions.