实时荧光PCR法快速检测耐热霉菌费氏新萨托菌

Real-time PCR assays for rapid detection of heat-resistant mould-Neosartorya fescheri

费氏新萨托菌是果蔬汁中易污染的耐热霉菌之一,根据Genbank公布的beta-tubulin基因序列,设计引物和探针,对实时荧光PCR的扩增反应体系进行优化。结果表明:1.2μL ROX染料(10μΜ)、2.5μL MgCl2(25mM)、0.2μLDNA聚合酶(5U/μL)、2.0μL dNTP混合物(2.5 mM)、2.5μL 10×PCR反应缓冲液为最佳反应体系;考察实时荧光PCR方法的灵敏度和特异性,基因组DNA的灵敏度为8.6×10-4 ng/反应体系,检测特异性好。该方法可实现耐热霉菌费氏新萨托菌的快速检测。

According to the sequence of beta tubulin oi Neosartorya fescheri published in Genbank, one pairs of primers and one Taqman probe were designed. Factors affecting real time PCR were opti- mized. 2 μLROX dye （10 μM）, 2.5 μL MgCl2（25 mM）, 0.2 μL DNApolymerase（5 U//μL）, 2. 0 μL dNTP mixture（2. 5 mM）, 2.5 μL 10 × PCR buffer were optimal conditions. The specificity, sensitivity and reproducibility of real-time PCR were also analyzed. The detection limit of genomic DNA in the real-time PCR assay was 8.6 × 10^-4 ng/reaction system. The established method provides a better choice for rapid detection of Neosartorya fescheri.