食品科学 ›› 0, Vol. ›› Issue (): 203-206.

• 分析检测 • 上一篇    下一篇

副溶血弧菌的SYBR GreenⅠ实时定量PCR检测方法建立

张晓君,陈 丽,毕可然,秦 蕾,秦国民   

  1. 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
  • 收稿日期:2011-04-10 修回日期:2012-03-14 出版日期:2012-04-25 发布日期:2012-03-31
  • 通讯作者: 张晓君 E-mail:zxj9307@163.com
  • 基金资助:

    江苏省水产三项工程项目;江苏省自然科学基金项目

Development of SYBR Green-Based ⅠReal-time Quantitative PCR for Detection of Vibrio parahaemolyticus

  • Received:2011-04-10 Revised:2012-03-14 Online:2012-04-25 Published:2012-03-31
  • Contact: zhang xiaojun E-mail:zxj9307@163.com

摘要: 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 GreenⅠ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 GreenⅠ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2.06×108~2.06×103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。

关键词: 副溶血弧菌, gyrB基因, SYBR GreenⅠ, 实时定量聚合酶链式反应

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 amplification curves. Analysis of standard curves revealed excellent correlation between the number of copies (in the range of 2.06 × 108 to 2.06 × 103) and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R2 =0.992). It took only 4-5 h (from nucleic acid extraction to analysis of results) to detect samples by the method. Therefore, SYBR green-based I real-time PCR had the advantages of higher sensitivity and ease of operation over traditional methods and could be used for inspection  and quarantine of import and export commodities, food safety detection, and diagnostic studies and molecular epidemic survey of aquatic animal diseases caused by V. parahaemolyticus.

Key words: Vibrio parahaemolyticus, gyrB gene, SYBR green I, real-time PCR

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