Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers

Ayano Kobayashi, Daisuke Sano, Asami Taniuchi, Satoshi Ishii, Satoshi Okabe

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.

Original languageEnglish (US)
Pages (from-to)9165-9173
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume97
Issue number20
DOIs
StatePublished - Oct 2013

Bibliographical note

Funding Information:
Acknowledgments This work was funded by Core Research for Evolutionary Science and Technology (CREST) from Japan Science and Technology Agency (JST) and grant-in-aid for Scientific Research A (23246094) from Japan Society for the Promotion of Science (JSPS).

Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.

Keywords

  • Bacteroides-Prevotella 16S rRNA genetic markers
  • Fecal contamination
  • Microbial source tracking
  • Propidium monoazide
  • Quantitative PCR assay
  • Sample process control

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