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目的:建立二重PCR方法快速检测食品中的志贺菌(Shigella spp.)。方法:以侵袭性质粒抗原H基因ipaH和铁载体基因iuc为靶基因,筛选引物,建立二重PCR体系。对3株志贺菌和28株非志贺菌进行特异性检测。梯度稀释志贺菌基因组DNA,以不同稀释度DNA作PCR扩增。在孜然牛肉中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增。应用该方法检测实际样品。结果:以ipaH、iuc为靶基因的2对引物对志贺菌的检出有很好的特异性。PCR检测的灵敏度在DNA水平上达到78.73 pg。人工污染样品,当起始污染量为0.56 cfu/g时,37℃增菌培养10 h即可检出。一共检测了18份样品,未检出志贺菌,与传统方法一致。利用该方法检测了一份盲样冻干样品,检出志贺菌,与实际结果相符。结论:建立了适用于食品中志贺菌检测的特异灵敏二重PCR方法。
Objective: To establish a dual PCR method for the rapid detection of Shigella spp. In food. Methods: Using the ipaH gene of invasive plasmid antigen and iuc gene as the target gene, the primers were screened and a duplex PCR system was established. Three strains of Shigella and 28 strains of non-Shigella were tested for specificity. Gradient dilution of Shigella genomic DNA with different dilutions of DNA for PCR amplification. In cumin beef artificial contamination of different bacteria, different enrichment time training, DNA extraction for PCR amplification. Apply this method to test actual samples. Results: Two pairs of primers with ipaH and iuc as target genes showed good specificity for the detection of Shigella. The sensitivity of PCR detection reached 78.73 pg at the DNA level. Artificially contaminated samples, when the initial pollution level of 0.56 cfu / g, 37 ℃ enrichment culture can be detected 10 h. A total of 18 samples were tested, Shigella was not detected, in line with the traditional method. Using this method, a blind sample of lyophilized samples was detected and Shigella was detected, consistent with the actual results. Conclusion: The specific sensitive duplex PCR method for detecting Shigella in food was established.