基于内参的志贺氏菌实时荧光定量PCR快速检测

Establishment of Fluorescence Quantitative PCR Assay in Detection of Shigella Based on Internal Reference

根据Genbank已公布的志贺氏菌基因组序列,筛选特异性靶基因ipa H,设计特异性引物和探针,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反应过程。按照人工污染样品,以评价所建立反应体系的性能。以志贺氏菌基因组DNA为模板,最低检测限为1 pg/u L;以10倍梯度稀释的菌液用水煮法提取DNA为模板,最低检测限为9×103CFU/m L;以含有ipa H的质粒为模板,最低检测限可以达到103考贝/u L;建立ipa H和ipa H-IAC标准曲线,Ct值与模板拷贝数均呈良好的线性关系(R2=0.999);人工污染初始菌量为20 g中10 CFU的羊肉时,志贺氏菌在增菌6 h后即可检出(水洗加试剂盒法)。作者建立的ipa H-IAC实时荧光定量PCR方法,既能有效检测食品中志贺氏菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,进一步保证了结果的可靠性,有利于实现样品中志贺氏菌实时荧光定量PCR检测方法的标准化。

According to Shigella gene sequences published by Genbank,we selected specific target genes,designed specific primers and probes and optimized the reaction system. An internal amplification control （IAC） was added to reaction system. This IAC was detected by TaqMan probes labeled with different fluorophore. 5-50 CFU/25 g artificially contaminated sample was added to evaluate the performance of reaction system. The assay was could be used reliably for detection of ShigeUa with a sensitivity of lpg/uL. For the 10 -fold dilutions bacteria DNA extracted by cooking water,the lowest detection limit was 9 ×10^3 cfu/mL. But for the plasmid with ipaH,the lowest detection limit can reach 103 copies/uL. The standard curve of ipaH and ipaH-IAC was established, and the quantification was linear between Ct and template copy number （R2=0.999）. While the initial sample amount of artificially contaminated bacteria was 10 CFU/25 g mutton,the Shigella could be detected aider 6 hours culture. The ipaH-IAC fluorescence quantitative PCR assay was developed. It could not only be applied for detection of Shigella in food ,but also monitor the PCR reaction system and prevent the false negatives. Therefore, the ipaH-IAC fluorescence quantitative PCR assay further ensures the reliability of the results and is conducive to the realization of standardized quantitative PCR method for Shigella in samples.