Specific Primer Design for Detection and Quantification of Entomopathogenic Fungi Metarhizium anisopliae using Quantitative PCR (qPCR) in Soil and Cocoon Samples

<p>The information on the abundance and dynamics of entomopathogenic fungus <em>Metarhizium anisopliae</em> on the soil is limited by the existence of insect hosts. In this study, to detect and quantify specifically for <em>M. anisopliae</em> from extract of soil DNA, a culture-independent approach based on DNA and employing quantitative Polymerase Chain Reaction (qPCR) was developed. Primer pairs were designed and tested for their specificity to get a specific primer pair. The best primer pair was determined to be MA6071F/MA6218R. Two standard curves were created using 10 concentration levels (10<sup>1</sup> to 10<sup>10</sup>) by qPCR. Standard curves for genomic DNA showed a strong relationship and good fitting (R<sup>2</sup> > 0.980). Six levels were obtained to generate standard genomic DNA (<em>R<sup>2</sup></em>= 0.98, E = 1.05). Eight levels (<em>R<sup>2</sup></em>= 0.9854, E = 0.91) were created for standard soil DNA. By qPCR, <em>M. anisopliae</em> was not found in all soil samples, possibly due to the samples' low fungal density. However, 13 dead cocoon samples out of 80 showed positive for <em>M. anisopliae</em>. To successsfully detect and quantify <em>M. anisopliae</em> in soil, the method of DNA extraction and soil sampling should be enhanced.</p>