Sensitive detection of human adenovirus from small volume of primary wastewater samples by quantitative PCR

• Published in: Journal of virological methods Vol. 187; no. 2; pp. 395 - 400
• Main Authors: Sidhu, Jatinder P.S., Ahmed, Warish, Toze, Simon
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01-02-2013; Elsevier
• Subjects: Adenovirus; Adenoviruses, Human; assays; Biological and medical sciences; Blood; Centrifugation - methods; DNA; DNA viruses; DNA, Viral - genetics; DNA, Viral - isolation & purification; filters; Filtration - methods; Fundamental and applied biological sciences. Psychology; humans; Microbiology; PCR inhibitors; Quantitative PCR; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Techniques used in virology; Virology; Virology - methods; Virus concentration; viruses; Waste Water - virology; Wastewater; Virus concentration; Wastewater; Adenovirus; PCR inhibitors; Quantitative PCR; Adenoviridae; Method; Waste water; Virus; Polymerase chain reaction; Human adenovirus; Mastadenovirus; Detection; Quantitative analysis
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.11.002

► Efficient method for sensitive and reproducible quantification of adenovirus. ► Ten millilitre wastewater concentrated and purified with centrifugal filters. ► Sensitivity of qPCR depends upon nucleic acid extraction method used. ► Method applicable for the qPCR detection of other DNA virus in wastewater.
An accurate quantitative detection of enteric viruses from the primary wastewater requires, sample concentration followed by extraction of nucleic acid with high purity. A highly efficient and sensitive method was developed for the concentration and quantitative detection of human adenovirus (HAdv) from wastewater samples. The two-step method which combines concentration of virus from 10mL sample with centrifugal filters followed by extraction and purification of DNA with commercially available nucleic acid extraction kit resulted in high purity DNA for downstream quantitative PCR (qPCR). The results obtained on analytical sensitivities of five commercial nucleic acid extraction kits show that they differ in their ability for DNA yield and purity. Nevertheless, despite variable analytical sensitivities extracted nucleic acid was found to be relatively PCR inhibition free. The genomic copy numbers of HAdv detected from the same concentrated wastewater sample were significantly higher (P<0.01) when Qiagen Blood and Tissue kit (1.54×106L−1) was used as compared to Mo-Bio PowerSoil kit (5.30×105L−1) which suggests that the nucleic acid extraction kit can influence the sensitivity of qPCR assays. The method developed in this study is simple, rapid, sensitive, and can be applicable for the qPCR detection of adenovirus and other DNA virus in wastewater.