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目的:建立二重PCR方法特异检测食品中的单核细胞增生李斯特菌(Listeria monocytogenes,LM)。方法:以编码内化素B的inlB基因和以编码LM溶血素O的hly基因为靶基因,设计筛选引物,建立二重PCR体系。对5株LM和35株非LM进行特异性检测。梯度稀释LM基因组DNA,以不同稀释度DNA作PCR扩增。在黑椒烤鸡样品中以不同菌量人工污染,增菌培养10 h,提取DNA进行PCR扩增。应用该方法对实际样品进行检测。结果:以inlB、hly为靶基因的2对引物对LM的检出有很好的特异性。PCR检测的灵敏度在DNA水平上达到0.8769 ng。人工污染样品,当起始污染量为2.7×102cfu/g时,37℃增菌培养10 h即可检出。一共检测了55份食品样品,检出1份阳性样品,与传统方法一致。结论:建立了适用于食品中LM检测的特异二重PCR方法。
Objective: To establish a dual PCR method for the specific detection of Listeria monocytogenes (LM) in food. Methods: InlB gene encoding endonuclease B and hly gene encoding LM hemolysin O were selected as target genes. Primers were designed and screened to establish a duplex PCR system. Five LM and 35 non-LM were tested for specificity. The LM genomic DNA was diluted in a gradient and amplified with different dilutions of DNA. Artificially polluted black pepper roast chicken with different amount of bacteria, cultured for 10 h, DNA was extracted for PCR amplification. Use this method to test the actual sample. Results: Two pairs of primers targeting inlB and hly had good specificity for the detection of LM. The sensitivity of PCR detection reached 0.8769 ng at the DNA level. Artificially contaminated samples, when the initial contamination of 2.7 × 102cfu / g, 37 ℃ enrichment culture can be detected 10 h. A total of 55 food samples were tested and 1 positive sample was detected, consistent with the traditional method. Conclusion: A specific duplex PCR method suitable for LM detection in food was established.