Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference

Area of Science:

• Microbiology
• Molecular Biology
• Environmental Science

Background:

• Polymerase chain reaction (PCR) has evolved into powerful quantitative technologies like real-time PCR.
• Real-time PCR is crucial for nucleic acid detection and quantification in various settings, including community analysis.
• Absolute quantification in real-time PCR can be inaccurate due to DNA extraction losses or co-extracted inhibitors.

Purpose of the Study:

• To present an improved method for real-time PCR quantification using an artificial internal standard.
• To enable accurate estimation of total bacterial populations in diverse soil environments.
• To demonstrate the superiority of this novel relative quantification approach over absolute quantification.

Main Methods:

• An artificial internal standard was generated via in-situ mutagenesis from an E. coli template.
• The standard was added prior to nucleic acid extraction to account for losses and inhibition.
• Relative real-time PCR was employed, using the internal standard as a reference for quantification.

Main Results:

• The novel relative real-time PCR approach accurately estimated total bacterial populations across a wide range of soils.
• This method effectively compensated for DNA extraction inefficiencies and PCR inhibition.
• Comparison with ester-linked fatty acid analysis confirmed the improved accuracy over absolute quantification.

Conclusions:

• The developed internal standard and relative quantification method offer a robust solution for accurate bacterial enumeration in soil.
• This technique overcomes limitations of absolute quantification in real-time PCR for environmental samples.
• The findings have significant implications for microbial ecology and soil science research.