可视化跨越式滚环扩增技术检测食品中单核细胞增生李斯特氏菌

Detection of Listeria monocytogenes in Foods by Visual Saltatory Rolling Circle Amplification

建立一种新型的单核细胞增生李斯特氏菌检测方法,即通过可视化跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)技术检测单核细胞增生李斯特氏菌。根据单核细胞增生李斯特氏菌hlyA基因序列设计引物,进行特异性验证,对灵敏度和人工污染样品的检出限进行测定。通过检测70种实际食品样品对SRCA方法的敏感性、特异性和符合率进行评价。结果表明:所有单核细胞增生李斯特氏菌呈阳性结果,非单核细胞增生李斯特氏菌呈阴性结果,说明该方法特异性良好。SRCA方法的灵敏度为8.9×10^(0)fg/μL,是传统聚合酶链式反应(polymerase chain reaction,PCR)方法的1000倍,检出限为2.8×10^(0)CFU/g,是传统PCR方法的1/1000。在实际样品的检测中,SRCA方法与GB 4789.30—2016《食品微生物学检验单核细胞增生李斯特氏菌》方法进行比较,其敏感性、特异性和符合率分别为100%、97.01%和97.14%。可视化SRCA技术具有操作简便、设备简单、特异性强、灵敏度高、检出限低、检测成本低等优点,适合在基层单位和中小型食品企业中推广应用。

In this study,a novel method for the detection of Listeria monocysteia was established using visual saltatory rolling circle amplification(SRCA).According to the hlyA gene sequence of L.monocytogenes,primers were designed.The specificity,sensitivity and detection limit for artificial contaminated samples were determined.In addition,the sensitivity,specificity and accuracy of SRCA were evaluated by applying it to detect 70 actual food samples.Results showed that the developed method gave positive results for all L.monocytogenes strains and negative results for non L.monocytogenes strains,indicating good specificity of the assay.The sensitivity of the SRCA method was 8.9×10^(0) fg/μL,and the detection limit was 2.8×10^(0) CFU/g,which were 1000-fold better than those of polymerase chain reaction(PCR).Compared with the national standard method GB 4789.30-2016,the relative sensitivity,specificity and accuracy of SRCA were 100.00%,97.01%and 97.14%,respectively.In summary,this method has the advantages of easy operation,simple equipment,strong specificity,high sensitivity,low detection limit and low cost,and can be widely used in grass-root inspection and testing institutions and small-and medium-sized food companies.