title: DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method.
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.
authors.affiliation: Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA. Electronic address: cheny@bucknell.edu.
authors.forename: Yen-Chih
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Higgins
authors.affiliation: Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Maas
authors.affiliation: DC Water and Sewer Authority, Washington, DC 20032, USA.
authors.forename: Sudhir N
authors.initials: SN
authors.lastname: Murthy
mesh_terms: D000693:Anaerobiosis; D015169:Colony Count, Microbial / Q000191:economics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics; D007220:Industrial Waste / Q000191:economics; D008565:Membrane Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012722:Sewage / Q000737:chemistry / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D007220:Industrial Waste; D008565:Membrane Proteins; D012722:Sewage; C121044:gadA protein, E coli; D005968:Glutamate Decarboxylase