单 位：
河南师范大学生命科学学院;新乡学院生命科学技术学院;佛山市海天调味食品股份有限公司
关键词：
绿色魏斯氏菌;实时荧光定量PCR;SYBR GreenⅠ;rpoA基因
摘 要：
以rpoA基因为靶基因,建立绿色魏斯氏菌SYBR GreenⅠ实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)快速检测方法。针对rpoA基因设计特异性引物,建立绿色魏斯氏菌实时荧光定量PCR检测体系,通过特异性、灵敏度和重复性实验评价体系的检测效果,同时与常规PCR方法进行比较。结果表明:实时荧光定量PCR方法能够特异性检出绿色魏斯氏菌,对基因组DNA的检测灵敏度达到2.667×10~(-3) pg/μL,对纯培养物和模拟污染牛肉样品直接检测的灵敏度分别为30 CFU/mL和0.8 CFU/g;与常规PCR相比,实时荧光定量PCR检测的灵敏度是其1 000倍;不同浓度样品独立重复实验循环阈值的标准差均小于1,变异系数在0.02%～1.28%之间。本研究所建立的绿色魏斯氏菌实时荧光定量PCR检测方法具有特异性好、灵敏度高、重复性好的特点,能够进行准确的定量检测,是快速检测绿色魏斯氏菌的有效手段。
译 名：
Detection of Weissella viridescens by Quantitative Real-Time Polymerase Chain Reaction Assay
作 者：
ZHANG Yige;JIANG Xiaobing;YU Tao;SUN Liying;College of Life Sciences, Henan Normal University;College of Life Sciences and Technology,Xinxiang University;Foshan Haitian Flavouring and Food Co.Ltd.;
单 位：
ZHANG Yige%JIANG Xiaobing%YU Tao%SUN Liying%College of Life Sciences, Henan Normal University%College of Life Sciences and Technology,Xinxiang University%Foshan Haitian Flavouring and Food Co.Ltd.
关键词：
Weissella viridescens;;real-time polymerase chain reaction;;SYBR Green Ⅰ;;rpoA gene
摘 要：
A SYBR Green Ⅰ-based real-time polymerase chain reaction(PCR) assay for the detection of Weissella viridescens was established using rpo A as target gene in this study. Specific primers were designed targeting rpo A. The specificity, sensitivity, and repeatability of the real-time PCR method were evaluated and compared with those of conventional PCR. The real-time PCR method was found to be specific for W. viridescens. The limit of detection of this method was 2.667 × 10~(-3) pg/μL for genomic DNA from W. viridescens, and 30 CFU/m L and 0.8 CFU/g for pure bacterial culture and artificially inoculated beef, respectively. The sensitivity of real-time PCR was 1 000 times as high as that of conventional PCR. The results of independent repeated test on samples with different bacterial concentrations showed a standard deviation of less than 1 and coefficients of variation(CV) in the range of 0.02%–1.28% for cycle threshold(Ct).The real-time PCR method developed in this study can be considered to be a fast tool for detection of W. viridescens due to its good specificity, sensitivity, reproducibility, and accuracy.
