Removal of PCR inhibiting substances in sewage sludge amended soil

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Current methods for the detection of enteric viruses in soil involve elution of viruses from soil colloids using beef extract or other proteins. These eluates are then assayed in cell culture and observed daily for cytopathic effects (CPE). While this method is suitable for detection of enteric viruses by cell culture, these eluates contain humic acids and heavy metals that interfere with polymerase chain reaction (PCR) detection. Using beef extract eluates prepared from sludge amended soil, 10 different methods of eluate purification were evaluated for their ability to remove PCR inhibition and maximize sensitivity. The treatment method providing the greatest sensitivity of poliovirus detection by PCR was the combination of Sephadex G-50 and Chelex-100. Using this method 2 plaque forming units (PFU) could be detected after reverse transcription and 30 cycles of PCR. Thirty (30) cycles of seminested PCR were performed on these samples to verify nucleic acid sequences and increase sensitivity after the first 30 cycles of PCR. Using seminested PCR sensitivity of detection using the Sephadex G-50 and Chelex-100 treatment method to 0.2 PFU. In addition to providing excellent sensitivity for viruses in sludge amended soils, this treatment method is relatively simple compared to other methods.

Original languageEnglish (US)
Pages (from-to)311-315
Number of pages5
JournalWater Science and Technology
Volume31
Issue number5-6
DOIs
StatePublished - 1995

Fingerprint

Polymerase chain reaction
Sewage sludge
polymerase chain reaction
Soils
Viruses
virus
soil
Beef
Cell culture
Nucleic acid sequences
sludge
Transcription
removal
sewage sludge
method
Colloids
nucleic acid
Heavy metals
colloid
Purification

Keywords

  • Enteric viruses
  • Land application
  • RT-PCR
  • Seminested PCR
  • Sewage sludge

ASJC Scopus subject areas

  • Water Science and Technology

Cite this

Removal of PCR inhibiting substances in sewage sludge amended soil. / Straub, T. M.; Pepper, Ian L; Gerba, Charles P.

In: Water Science and Technology, Vol. 31, No. 5-6, 1995, p. 311-315.

Research output: Contribution to journalArticle

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AB - Current methods for the detection of enteric viruses in soil involve elution of viruses from soil colloids using beef extract or other proteins. These eluates are then assayed in cell culture and observed daily for cytopathic effects (CPE). While this method is suitable for detection of enteric viruses by cell culture, these eluates contain humic acids and heavy metals that interfere with polymerase chain reaction (PCR) detection. Using beef extract eluates prepared from sludge amended soil, 10 different methods of eluate purification were evaluated for their ability to remove PCR inhibition and maximize sensitivity. The treatment method providing the greatest sensitivity of poliovirus detection by PCR was the combination of Sephadex G-50 and Chelex-100. Using this method 2 plaque forming units (PFU) could be detected after reverse transcription and 30 cycles of PCR. Thirty (30) cycles of seminested PCR were performed on these samples to verify nucleic acid sequences and increase sensitivity after the first 30 cycles of PCR. Using seminested PCR sensitivity of detection using the Sephadex G-50 and Chelex-100 treatment method to 0.2 PFU. In addition to providing excellent sensitivity for viruses in sludge amended soils, this treatment method is relatively simple compared to other methods.

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