A method for the detection and quantitation of PCR template in environmental samples by high performance liquid chromatography

Elizabeth M. Marlowe, Karen L. Josephson, Raina M. Miller, Ian L. Pepper

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

This study describes methodology for the detection and quantitation of PCR amplified DNA. Specifically we report the estimation of the prevalence of E. coli in marine waters and other environmental samples from Mamala Bay, Hawaii. High performance liquid chromatography (HPLC) was used to quantitate PCR products containing between 1 and 250 ng DNA. PCR was used to amplify E. coli DNA through the use of lamB primers. A standard curve was generated that related initial cell template concentrations to amplified product DNA concentrations within a template range of 520 to 5.2 x 107 cells. The standard curve was used to determine initial template concentrations of the lamB gene sequence present within 11 different environmental samples. Quantified PCR analyses were most useful when samples contained only low numbers of target organisms, and when environmental samples contained few PCR inhibitory substances, as for example in marine water samples. Quantitation of amplified DNA and comparison with culture data also suggested the presence of viable but nonculturable organisms in some environmental samples. Overall these data are unique in that they indicate the successful use of HPLC to quantitate PCR amplifications with concomitant estimation of PCR template within environmental samples.

Original languageEnglish (US)
Pages (from-to)45-53
Number of pages9
JournalJournal of Microbiological Methods
Volume28
Issue number1
DOIs
StatePublished - Jan 1 1997

Keywords

  • HPLC
  • PCR
  • bacteria
  • quantitation
  • seawater
  • sewage

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Fingerprint Dive into the research topics of 'A method for the detection and quantitation of PCR template in environmental samples by high performance liquid chromatography'. Together they form a unique fingerprint.

  • Cite this