DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive...

Area of Science:

• Environmental microbiology
• Molecular biology techniques
• Water and wastewater analysis

Background:

• Accurate enumeration of Escherichia coli (E. coli) is crucial for water and wastewater safety.
• Traditional cultivation methods may underestimate E. coli due to its viable but non-culturable state.

Purpose of the Study:

• Develop and validate a DNA extraction protocol for E. coli enumeration in digested biosolids.
• Establish a quantitative polymerase chain reaction (PCR) method for reliable E. coli detection.

Main Methods:

• A solvent-based DNA extraction protocol was optimized for efficient cell lysis.
• A competitive touchdown PCR (cPCR) method was developed and compared to real-time PCR (rt-PCR) and cultivation.

Main Results:

• The developed DNA extraction protocol recovered 78-84% of spiked DNA, outperforming a commercial kit (28-42%).
• The cPCR method demonstrated comparable sensitivity to rt-PCR and cultivation, with a detection limit of 50,000-500,000 E. coli per gram dry solids.
• The cPCR method is suitable for monitoring Class B and "conventional" biosolids.

Conclusions:

• The developed DNA extraction and cPCR protocol offer a reliable, culture-independent method for E. coli enumeration in biosolids.
• This approach provides a cost-effective alternative to rt-PCR for water safety assessments.