副溶血弧菌的sybrgreenⅰ实时定量pcr检测方法建立-食品科学.pdfVIP

※分析检测 食品科学 2012, Vol. 33, No. 08 203 SYBR Green ⅠPCR 张晓君，陈 丽，毕可然，秦 蕾，秦国民 (淮海工学院海洋学院，江苏省海洋生物技术重点建设实验室，江苏 连云港 222005) 基于副溶血弧菌gyrB 基因保守序列设计 1 对特异性引物，建立 SYBR Green Ⅰ实时定量聚合酶链式反应 (polymerase chain reaction，PCR)检测副溶血弧菌的方法。SYBR Green Ⅰ实时定量PCR 的Tm 为90 ℃，扩增产物的 熔解曲线只出现 1 个单特异峰，无引物二聚体，表明该引物具有较好的特异性；所制作的实时定量 PCR 扩增标准 曲线在2.06 ×108 3 ～2.06 ×10 拷贝数之间有较好的线性关系，相关系数为 0.992，能对副溶血弧进行准确的定量分 析。该方法检测时间从核酸抽提到结果分析仅需 4 ～5h ，且较传统方法敏感、操作简单，可用于针对副溶血弧菌 的进出口检验检疫、食品安全检测及该菌引起的水产动物疾病的诊断与分子流行病学调查。 副溶血弧菌；gyrB 基因；SYBR Green Ⅰ；实时定量聚合酶链式反应 Development of SYBR Green-Based ⅠReal-time Quantitative PCR for Detection of Vibrio parahaemolyticus ZHANG Xiao-jun ，CHEN Li ，BI Ke-ran ，QIN Lei ，QIN Guo-min (College of Ocean, Key Laboratory of Oceanic Biotechnology of Jiangsu, Huaihai Institute of Technology, Lianyungang 222005, China) Abstract： The gyrB gene, which encodes the B subunit protein of DNA gyrase, is a single copy gene and has conserved regions for PCR primers. A pair of specific primers target to the gyrB gene of V. parahaemolyticus was designed, and a SYBR green I- based real-time PCR for V. parahaemolyticus detection was established. The PCR primers could amplify 285-bp gene fragment from chromosomal DNA of V. parahaemolyticus, and no positive reaction was detected in 8 other pathogenic bacteria using conventional PCR. In addition, the results of melting curve analysis showed only a specific peak with a melting temperature (Tm) of 90 ℃, and no primer-dimers peak was observed. These findings indicated that the PCR primers had high specificity. Both geometric growth and plateau phases were observed in PCR amplificatio