Virus Concentration and Taqman Real-time Polymerase Chain Reaction (qPCR) Quantification of Adenoviral DNA for Detection of Sewage and Farm Wastewater-Associated Adenoviral Markers
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Optimization and Validation of Virus Concentration Methods
Authors
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Asima Zehra
Guru Angad Dev Veterinary and Animal Sciences, GADVASU
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Simranpreet Kaur
Associate Professor, Centre for One Health, GADVASU, Punjab
Keywords:
Adenovirus, DNA extraction, Microbial Source Tracking, TaqMan qPCR assay, Virus ConcentrationAbstract
Library-independent techniques offer numerous advantages, particularly the utilization of Viral DNA as a promising microbial source tracking (MST) marker. However, the variability of genetic markers and their prevalence in populations from different geographical areas necessitate the prior characterization of MST markers' assay performance in each specific watershed before their implementation. Hence, the objective of this study was to evaluate a selected MST marker, namely adenoviruses, in fecal samples from known sources and optimize/validate virus concentration methods. Two rapid techniques for concentrating adenoviruses, including human adenoviruses (HAdV), bovine adenoviruses (BAdV), and porcine adenoviruses (PAdV), were compared in terms of their efficiency in virus recovery. In the direct extraction protocol (Protocol I), the viral genome was directly removed from the membrane, while in the adsorption-elution protocol (Protocol II), viruses were eluted using NaOH and concentrated through centrifugal ultrafiltration. No interference with the TaqMan qPCR assay was observed when processing river and tap water samples using Protocol I, and tap water samples using Protocol II. However, inhibition occurred in qPCR for river samples processed using Protocol II. The mean concentration of HAdVs (3.1 × 104 copy number) and PAdVs (2.6 × 103 copy number) was 10.47-fold and 9.51-fold higher when detected using Protocol I. All PCR markers exhibited high sensitivity and specificity, although none achieved 100% for both parameters. Despite the identification of some MST markers in hosts other than the expected ones, their abundance in the target group consistently exceeded that in non-target hosts, thus demonstrating their ability to differentiate between pollution sources.
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