DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method.
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.
Chen
Yen-Chih
YC
Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA. Electronic address: cheny@bucknell.edu.
Higgins
Matthew J
MJ
Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA.
Maas
Nicholas A
NA
Civil and Environmental Engineering, 235 Dana Engineering Building, Bucknell University, Lewisburg, PA 17837, USA.
Murthy
Sudhir N
SN
DC Water and Sewer Authority, Washington, DC 20032, USA.
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*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D007220:Industrial Waste; D008565:Membrane Proteins; D012722:Sewage; C121044:gadA protein, E coli; D005968:Glutamate Decarboxylase