Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction

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20 Citations (Scopus)

Abstract

This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 1 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 × 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/l tap water).

Original languageEnglish (US)
Pages (from-to)295-302
Number of pages8
JournalJournal of Virological Methods
Volume55
Issue number3
DOIs
StatePublished - 1995

Fingerprint

Poliovirus
Reverse Transcriptase Polymerase Chain Reaction
Viruses
Water
Enterovirus
Viral RNA
Chloroform
Phenol
Polymerase Chain Reaction
Flocculation
Water Purification
Hot Temperature
Chelex 100
sephadex

Keywords

  • Concentration
  • Coxsackievirus
  • Poliovirus
  • Reverse transcriptase-polymerase chain reaction (RT-PCR)
  • Water

ASJC Scopus subject areas

  • Virology

Cite this

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title = "Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction",
abstract = "This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 1 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 × 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99{\%} of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/l tap water).",
keywords = "Concentration, Coxsackievirus, Poliovirus, Reverse transcriptase-polymerase chain reaction (RT-PCR), Water",
author = "Ma, {Ju Fang} and Gerba, {Charles P} and Pepper, {Ian L}",
year = "1995",
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language = "English (US)",
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AU - Ma, Ju Fang

AU - Gerba, Charles P

AU - Pepper, Ian L

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N2 - This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 1 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 × 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/l tap water).

AB - This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 1 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 × 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 μl (0.2 PFU/l tap water).

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