New Process Control to Evaluate DNA Recovery Efficiency in Environmental Samples.

Quantitative real-time polymerase chain reaction (qPCR) is frequently used to measure target genes (e.g., pathogens) present in environmental samples (e.g., water, soil, and sediment). For accurate and reliable qPCR process, it is important to extract high-quality DNA from environmental samples because humic and other substances can inhibit PCR amplification. In addition, some portion of DNA can be lost during DNA extraction process. However, DNA recovery efficiency has not been evaluated each time when processing soil and other environmental samples. In this study, we created a process control strain and a qPCR assay to evaluate DNA recovery efficiency in environmental samples. Strain NH8B-1D2, a mutant strain that is not present in natural environments, was created by inserting kanamycin-resistance gene to one of the 23S rRNA gene of Pseudogulbenkiania sp. NH8B, a soil denitrifier belonging to the class Betaproteobactera. To specifically detect and quantify this strain NH8B-1D2, we designed a new TaqMan qPCR assay that produced 61-bp fragment. We could evaluate the DNA recovery efficiency in environmental samples by comparing the quantity of NH8B-1D2 cells spiked to the environmental samples prior to DNA extraction and the quantity of NH8B-1D2 cells measured by qPCR. To validate this approach, we co-spiked NH8B-1D2 and one of the four pathogens (E. coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, or Listeria monocytogenes) to pond water samples at various concentrations (10 to 10⁷ cells/L). Similar recovery efficiencies were obtained between NH8B-1D2 and the target pathogens, suggesting that the process control developed in this study (strain NH8B-1D2) can be used to evaluate and normalize the loss of the target pathogen genes during DNA extraction. Our process control approach can be used to analyze and evaluate data obtained by advanced techniques such as microfluidic qPCR, in which multiple genes can be quantified simultaneously, and next-generation sequencing.