DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method

Yen Chih Chen, Matthew J. Higgins, Nicholas A. Maas, Sudhir N. Murthy

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.

Original languageEnglish (US)
Pages (from-to)3037-3044
Number of pages8
JournalWater Research
Volume40
Issue number16
DOIs
StatePublished - Sep 2006

All Science Journal Classification (ASJC) codes

  • Ecological Modeling
  • Water Science and Technology
  • Waste Management and Disposal
  • Pollution

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