牛乳中白色念珠菌和近平滑念珠菌双重定量PCR检测方法的建立

Establishment of a Dual Real-time Quantitative PCR Method for Detection of Candida albicans and Candida parapsilosis in Raw Milk

为建立生牛乳中白色念珠菌和近平滑念珠菌的快速检测方法,根据GenBank已公布的基因序列分别设计2种念珠菌的种特异性引物,采用SYBR GreenⅠ荧光定量PCR方法检测目标菌的基因组DNA,并进行特异性、敏感性和重复性试验,用该方法对含有目标菌的生牛乳样本进行检测。结果表明,该方法特异性强,与对照菌株光滑念珠菌、克柔念珠菌、热带念珠菌和烟曲霉等病原真菌无交叉反应;灵敏度高,对白色念珠菌和近平滑念珠菌的最低检出量均为10 CFU/mL;重复性好,T m值和Ct值变异系数分别在0.1%和2.5%之内。该方法操作简便、耗时短,2 h^3 h即可完成整个检测过程。建立的方法能用于生牛乳中白色念珠菌和近平滑念珠菌的一次性快速、准确和定量检测。

In order to establish a rapid detection method for Candida albicans and Candida parapsilosis in raw milk,we designed the specific primers for the two species respectively according to the published gene sequences in GenBank,employed real-time quantitative PCR for detecting the genomic DNA of the two target Candida strains,and conducted tests of primer specificity,sensitivity and repeatability.Then the method was used to detect the raw milk with the two target strains.Results showed that the method had strong specificity,with no cross-reaction to the control including Candida glabrata,Candida krusei,Candida tropicalis and Aspergillus fumigatus;high sensitivity,with the minimum of 10 CFU/mL for both Candida albicans and Candida parapsilosis;and good repeatability with T m value and Ct value coefficients of variation within 0.1%and 2.5%,respectively.In addition,It was convenient and time-saving,taking a period of 2-3 hours to complete the whole test.This study indicated that the method could be applied to quick,accurate and quantitative detection of Candida albicans and Candida parapsilosis in raw milk at one time.