绿色魏斯氏菌实时荧光定量聚合酶链式反应检测方法的建立

以rpoA基因为靶基因，建立绿色魏斯氏菌SYBR Green Ⅰ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。

Detection of Weissella viridescens by Quantitative Real-Time Polymerase Chain Reaction Assay

A SYBR Green Ⅰ-based real-time polymerase chain reaction (PCR) assay for the detection of Weissella viridescens was established using rpoA as target gene in this study. Specific primers were designed targeting rpoA. 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/mL 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.