摘要
【目的】建立同时检测副溶血性弧菌gyrase、tdh、trh基因的三重PCR快速检测方法。【方法】将已报道的这3种基因的引物加入一个PCR反应管中,对引物浓度和退火温度进行优化,找到最佳引物比例和扩增条件。通过特异性验证、灵敏度验证以及方法间对比进行方法确认,其PCR产物使用全自动毛细管电泳分析系统进行分析。【结果】仅在91、269、485 bp处分别出现预期DNA扩增条带;纯培养条件下,扩增gyrase、tdh、trh的菌浓度检测限分别为6.6×101、6.6×102和6.6×101 CFU/mL;本底干扰物存在时,扩增gyrase、tdh、trh的菌浓度检测限分别为6.6×103、6.6×104和6.6×103 CFU/mL;模板DNA浓度检测限为1.36μg/L。检测进境海产品时,检测结果和FDA 2004标准结果一致,且更易辨认和判断。【结论】此检测方法的成功建立,为副溶血性弧菌及携带tdh和/或trh基因的致病性副溶血性弧菌的检测提供了一种准确、高效、便捷的分子技术手段。
[Objective] This study aims to establish a rapid specific multiplex-PCR detection of Vibrio parahaemolyticus by targeting gyrase, tdh, trh genes simultaneously. [Methods] Three pairs of reported primers were mixed in a single PCR tube. Through optimizing the concentration of primers and anneal temperature, the best reaction condition and primers ratio were determined. This method was validated by specificity test, sensitivity test and comparison test. The PCR-amplified products were analyzed by automatic capillary electrophoresis device. [Results] The predicted DNA amplified bands exhibited at sequences of 91,269 and 485 bp, indicating high specificity. Assay on sensitivity of cell concentration in pure culture condition showed that the detection limit of amplified gyrase, tdh and trh genes were 6.6×10^1, 6.6×10^2 and 6.6×10^1 CFU/mL respectively. When background interference existed, the detection limit of cell concentration of amplified gyrase, tdh and trh genes were 6.6×10^3, 6.6×10^4 and 6.6×10^3 CFU/mL respectively. The sensitivity experiment for testing genomic DNA template concentration showed that the detection limit was 1.36 μg/L. This method was applied to the test for imported seafood, the results matched the FDA 2004 standard. The amplified bands can be distinguished from each other more easily, comparing with those of FDA 2004 standard. [Conclusion] The multiplex-PCR method was successfully established. It is accurate, rapid and convenient. It can be applied to detect Vibrio parahaemolyticus or pathogenic Vibrio parahaemolyticus with tdh gene or trh gene.
出处
《微生物学通报》
CAS
CSCD
北大核心
2014年第4期764-775,共12页
Microbiology China
基金
国家认证认可监督管理委员会行业标准专项项目(No.2010B152)
