Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Method for Penicillium sclerotiorum
SONG Jia-Yi1, ZHANG Hai-Xia2, QI Shi1, ZHANG Ji-Li3, CHEN Dong-Hao1, WEI Jian-Yu3, LI Ping1, CHEN Ze-Peng4, YAN Jian1,*
1 College of Resources and Environment, South China Agricultural University, Guangzhou 510642, China; 2 Guangdong Tobacco Shaoguan City Co., Ltd, Shaoguan 512026, China; 3 China Tobacco Guangxi Industrial Co., Ltd, Nanning 530001, China; 4 China Tobacco Corporation Guangdong Province Company, Guangzhou 510642, China
Abstract：Penicillium sclerotiorum is an endophytic fungus in the genus Penicillium with high biological activity that produces secondary metabolites with certain biological control potentials and has been widely used in medicine, agriculture, and other fields. This study utilized P. sclerotiorum obtained from Portulaca oleracea, and developed primers for its specific conserved sequence. The target segment was inserted into the pBM73-T vector by molecular cloning to create a recombinant plasmid as a positive control. A real-time PCR method for P. sclerotiorum was developed using the SYBR GreenⅠdye. PCR amplification revealed that only P. sclerotiorum exhibited bands, and there were no melting curves or amplification curves for P. chrysogenum and P. polonicum. The findings demonstrated a good linear relationship between the cycling threshold (Ct) and the template concentration between 2.35×103 and 2.35×1010 copies/μL in the standard curve created using recombinant plasmid as the standard. The obtained standard curve was y=-3.211x+39.42 with a correlation coefficient R2=0.999 3. The findings from the soil detection showed that the method's ability to accurately count endophytic P. sclerotiorum. The method established in this study had good specificity, reproducibility and practicability, which could be used to investigate the colonization of P. sclerotiorum in plants and soil and quantify the number in different tissues of plants. This study provides some data and theoretical support for the study of the promotion and resistance mechanism of P. sclerotiorum.
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