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

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

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.
TidsskriftTropical Biomedicine
Udgave nummer4
Sider (fra-til)641-651
Antal sider11
StatusUdgivet - 2016


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