基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立  

Establishment of a fluorescence quantitative PCR assay in detection of Vibrio parahaemolyticus based on internal reference

作　　者：王建昌[1] 王金凤[1] 李静[1] 孙晓霞[1] 陈瑞春[1] 

机构地区：[1]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051

出　　处：《中国食品卫生杂志》2015年第4期408-413,共6页Chinese Journal of Food Hygiene

基　　金：质检公益性行业科研专项(201210128;201310126)

摘　　要：目的建立基于内参的副溶血性弧菌实时荧光定量PCR方法,快速检测样品中的副溶血性弧菌。方法根据Gen Bank已公布的副溶血性弧菌基因组序列,筛选特异性靶基因,设计特异性引物探针,优化反应体系,并在体系中加入内参（IAC）,通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应。按照5~50 cfu/25 g的细菌量人工污染样品,以评价所建立反应的体系。结果以副溶血性弧菌基因组DNA为模板,最低检测限为1 pg/μl;以10倍梯度稀释的菌液经水煮法提取的DNA为模板,最低检测限为4×102cfu/ml;以含有gyr B的质粒为模板,最低检测极限可以达到100 copies/μl;建立gyr B和gyr B-IAC标准曲线,Ct值与模板拷贝数均呈良好线性关系（r2=0.999）;人工污染初始菌量为7 cfu/25 g时,样品中副溶血性弧菌增菌6 h即可检出。结论本研究所建立的gyr B-IAC实时荧光定量PCR方法,既能有效检测食品中副溶血性弧菌,又能实时监测PCR反应过程,有效防止＂假阴性＂的发生,结果可靠,有利于实现海产品中副溶血性弧菌实时荧光定量PCR检测方法的标准化。Objective Based on Vibrio parahaemolyticus real-time fluorescent quantitative PCR method, rapid detection method of Vibrio parahaemolyticus was developed. Methods According to the Vibrio parahaemolyticus gene sequences published in GenBank, specific target genes were selected for primers and probes design, and the reaction system was optimized. An internal amplification control （IAC） was added to the faction system. This IAC was detected by TaqMan probes labeled with different fluorophore. The samples were artificially contaminated in 5-50 cfu/25 g, and were used to evaluate the performance of the reaction system. Results The assay could be used reliably for detection of Vibrio parahaemolyticus with the sensitivity of 1 pg/μl. For the 10-fold dilutions bacteria DNA extracted by cooking water, the lowest detection limit was 4 ×102 cfu/ml; and for the plasmid with gyrB, the lowest detection limit could reach 100 eopies/μl. The standard curves of gyrB and gyrB-IAC were established, which the quantification was linear between Ct and template copy number （r2 = 0. 999）. When the initial sample amount of artificially contaminated bacteria was 7 cfu/25 g seafood, the Vibrio parahaemolyticus could be detected after 6 hours culture. Conclusion The gyrB-IAC fluorescence quantitative PCR assay was developed. It could not only be applied for detection of Vibrio parahaemolyticus in food, but also monitor the PCR reaction system to prevent ＇false negatives＇. Therefore, the gyrB-IAC fluorescence quantitative PCR assay further ensures the reliability of the results and is helpful to standardize quantitative PCR method for Vibrio parahaemolyticus in seafood.

关 键 词：副溶血性弧菌 GYRB 实时荧光定量PCR IAC 食源性致病菌 食品安全 

分 类 号：R155.5[医药卫生—营养与食品卫生学]