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目的建立同时检测单增李斯特菌(Listeria monocytogenes,Lm)溶血素基因hlyA、内化素基因inlA和磷脂酶C基因plcB的TaqMan-MGB三重实时荧光PCR检测方法,并对101株单增李斯特菌的hlyA、inlA和plcB基因进行了检测,分析3个毒力基因在多位点序列分型(Multilocus sequence typing,MLST)聚类群组中的分布情况。方法根据单增李斯特菌毒力基因hlyA、inlA和plcB的保守序列设计引物和小沟结合物(minor groove binder,MGB)探针建立三重荧光PCR方法,对其特异性、灵敏度和可重复性进行了测试,并对分离的101株单增李斯特菌进行三重PCR检测。结果建立的三重实时荧光PCR方法特异于单增李斯特菌的检测,重复性良好,组内Ct值变异系数均小于1.5%,灵敏度可达100CFU/mL。对101株单增李斯特菌的hlyA、inlA和plcB的检出率分别为98%、75%和98%。在可被MLST分型的89株菌株中,groupI的30株菌株有20株均缺失inlA基因;groupII的57株菌株中有56株三个基因均检出;groupIII的菌株hlyA、inlA和plcB三个基因均未检出。结论本文建立的三重实时荧光PCR方法能准确、快速、稳定的检测单增李斯特菌,101株单增李斯特菌的hlyA、inlA、plcB三个毒力基因的检出情况与单增李斯特菌的MLST分型聚类有较一致的关系,对分析单增李斯特菌毒力的强弱有重要参考意义。
OBJECTIVE To establish a real-time TaqMan-MGB real-time PCR assay for the simultaneous detection of the hemolysin gene hlyA, the inilin gene and the phospholipase C gene plcB in Listeria monocytogenes (Listeria monocytogenes) The strains hlyA, inlA and plcB were tested for the distribution of the three virulence genes in the multilocus sequence typing (MLST) cluster. Methods Based on the conserved sequences of hlyA, inlA and plcB of Listeria monocytogenes, primers and minor groove binder (MGB) probes were designed and used to establish triplex fluorescence PCR. The specificity, sensitivity and repeatability The test was carried out, and 101 isolates of Listeria monocytogenes were tested by triplex PCR. Results The triplex real-time PCR method was specific for the detection of Listeria monocytogenes with good repeatability. The coefficients of variation of Ct values were less than 1.5% and the sensitivity was 100 CFU / mL. The detection rates of hlyA, inlA and plcB of 101 Listeria monocytogenes were 98%, 75% and 98% respectively. Of the 89 isolates that could be genotyped by MLST, 20 of the 30 isolates from groupI had the inlA gene deleted; 56 of the 57 isolates of group II were detected; and the strains hlyA, inlA and plcB of group III None of the genes were detected. Conclusion The triplex real-time fluorescence PCR method established in this paper can detect Listeria monocytogenes accurately, rapidly and stably. The detection of three virulence genes hlyA, inlA and plcB of 101 Listeria monocytogenes was significantly correlated with the increase of Listeria monocytogenes Mycobacterium tuberculosis MLST cluster has a more consistent relationship, which has important reference value for analyzing the virulence of Listeria monocytogenes.