Rapid PCR-based monitoring of infectious enteroviruses in drinking water

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Abstract

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300-400l of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24-48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001 MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48 h against 5-16 d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

Original languageEnglish (US)
Pages (from-to)423-427
Number of pages5
JournalWater Science and Technology
Volume35
Issue number11-12
DOIs
StatePublished - 1997

Fingerprint

Cell culture
Potable water
virus
drinking water
Monitoring
monitoring
Viruses
water
freeze-thaw cycle
hepatitis
flocculation
integrated approach
resin
Water
assay
filter
enterovirus
detection
Flocculation
method

Keywords

  • Cell culture
  • Enteroviruses
  • Hepatitis A virus
  • ICC/PCR
  • Inhibition
  • Integrated cell culture/PCR
  • PCR

ASJC Scopus subject areas

  • Water Science and Technology

Cite this

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abstract = "Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300-400l of water were concentrated using charged filters followed by a modified 11, 1.5{\%} BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24-48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001 MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48 h against 5-16 d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.",
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AU - Pepper, Ian L

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AB - Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300-400l of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24-48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001 MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48 h against 5-16 d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

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