Impact of DNA extraction efficiency on the sensitivity of PCR-based plant disease diagnosis and pathogen quantification

PCR and qPCR are important methods for plant disease diagnosis and quantification of pathogen populations in host tissues or soil. For most plant diseases, DNA extracted from infected tissue or soil is a prerequisite for PCR or qPCR. The efficiency of DNA extraction should have a direct impact on the sensitivity of PCR and qPCR systems. In this study, plant pathogen DNA extraction efficiencies were evaluated based on three plant disease systems and three DNA extraction methods. DNA of bacterial (Pectobacterium atrosepticum), protist (Plasmodiophora brassicae) and fungal (Botrytis cinerea) pathogens was extracted and aliquots were prepared. Aliquots of the DNA were mixed with healthy tissues of the corresponding host plant and DNA was extracted again from the mixture. The resultant mixed DNA, as well as the original pathogen DNA, were analysed by qPCR to assess the quantities of the pathogen DNA. Extraction efficiency was calculated based on the qPCR Cq values of the mixed DNA and the original pathogen DNA for each extraction method on each pathogen. Our results indicated the pathogen DNA extraction efficiencies were generally less than, or near, 50%. This result called attention to the importance of: 1) including the DNA extraction efficiency in the evaluation and announcement of new qPCR diagnostic systems, and 2) developing and using high efficient DNA extraction methods in plant disease diagnosis and pathogen quantification.