重组酶聚合酶扩增-侧流层析技术检测乳制品中单核细胞增生李斯特氏菌

Detection of Listeria monocytogenes in dairy products by recombinase polymerase amplification-lateral flow technology

中文关键词:  单核细胞增生李斯特氏菌  重组酶聚合酶扩增技术  侧流层析  快速检测

英文关键词:Listeria monocytogenesi  recombinase polymerase amplification technology  lateral flow  rapid detection

中文摘要:

      目的 以特异性基因16S rRNA和溶血素编码基因hly为靶序列, 建立重组酶聚合酶扩增-侧流层析技术(recombinase polymerase amplification-lateral flow, RPA-LF)检测乳制品中单核细胞增生李斯特氏菌的方法。方法 根据目标序列保守片段设计适用于RPA-LF的引物和探针, 筛选最佳引物探针组合, 建立相关检测方法并对其反应条件进行优化。结果 RPA-LF优化条件后, 总反应时间仅为20 min, 即可用肉眼直观观察到检测结果。该方法与8种常见的食源性病原微生物无交叉反应, 具有较好的特异性。能够区分李斯特氏菌同属菌, 表明该方法对李斯特氏菌同属菌具有抗干扰性。最低检出限可达到82 fg/μL, 菌含量约为1.8×102 CFU/mL, 表明该方法具有较高的灵敏度。此外, 用该方法进行常规样品检测可获得与传统微生物方法一致的检测结果。结论 本研究中建立的应用于单核细胞增生李斯特氏菌溶血素基因的RPA-LF检测方法, 具有仪器依赖性低、操作简便、灵敏度高、特异性好, 结果直观可视等优点, 为该菌现场的快速检测和筛选诊断提供了一条新的思路。

英文摘要:

      Objective To establish a recombinase polymerase amplification-lateral flow (RPA-LF) detection method for the detection of Listeria monocytogenes in dairy products, targeting the species-specific 16S rRNA gene and the hemolysin-encoding gene hly. Methods Primers and probes suitable for RPA-LF were designed based on the conserved fragments of the target sequence, and the optimal primer-probe combination was selected, the corresponding detection method was established, and its reaction conditions were optimized. Results Under optimized conditions, the total reaction time of the RPA-LF assay was only 20 min, and the results could be observed visually by the naked eye. The method showed no cross-reactivity with 8 kinds of common foodborne pathogenic microorganisms, demonstrating good specificity. It could also distinguish Listeria monocytogenes from other Listeria species, indicating good anti-interference ability against bacteria from the same genus. The limit of detection was as low as 82 fg/μL, corresponding to approximately 1.8×102 CFU/mL, demonstrating high sensitivity. Furthermore, when used for routine sample detection, the method yielded results consistent with those obtained by traditional microbiological methods. Conclusion The RPA-LF method targeting the hemolysin gene of Listeria monocytogenes established in this study has several advantages, including low instrument dependency, simple operation, high sensitivity, good specificity and direct visual readout. It provides a new approach for the rapid on-site detection and screening diagnosis of this pathogen.