CAJ | 학술논문

目的：建立同时检测单增李斯特菌(Listeria monocytogenes)及其3种毒力因子的多重荧光PCR快速检测方法,并应用于日常食品的检测。方法根据单增李斯特菌溶血素基因 hlyA、内化素基因 inlA 和表面蛋白actA 基因的保守序列,分别设计合成特异性引物和探针,优化多重荧光 PCR 反应体系。对该方法的特异性、敏感性和重复性进行评估。结果该法特异性强、敏感性高,对单增李斯特菌纯培养物的最低检出限410 cfu/mL；重复性好,变异系数均小于2%。对84份食品检测结果与传统国标法相符,共检出单增李斯特菌4份,检出率为4.76%。多重荧光PCR检测方法耗时1 h,比传统方法节约2~5 d。4株单增李斯特菌分离株中2株同时含有inlA、actA、hlyA 3种毒力基因,另2株为毒力基因actA缺失株,提示目前流行株并非同一来源。结论本研究建立的多重实时荧光 PCR方法能同时对单增李斯特菌及其3种毒力因子进行快速检测,且灵敏度高、特异性好,为食源性疾病的病原学检测提供了快速可靠的方法。
목적：건립동시검측단증리사특균(Listeria monocytogenes)급기3충독력인자적다중형광PCR쾌속검측방법,병응용우일상식품적검측。방법근거단증리사특균용혈소기인 hlyA、내화소기인 inlA 화표면단백actA 기인적보수서렬,분별설계합성특이성인물화탐침,우화다중형광 PCR 반응체계。대해방법적특이성、민감성화중복성진행평고。결과해법특이성강、민감성고,대단증리사특균순배양물적최저검출한410 cfu/mL；중복성호,변이계수균소우2%。대84빈식품검측결과여전통국표법상부,공검출단증리사특균4빈,검출솔위4.76%。다중형광PCR검측방법모시1 h,비전통방법절약2~5 d。4주단증리사특균분리주중2주동시함유inlA、actA、hlyA 3충독력기인,령2주위독력기인actA결실주,제시목전류행주병비동일래원。결론본연구건립적다중실시형광 PCR방법능동시대단증리사특균급기3충독력인자진행쾌속검측,차령민도고、특이성호,위식원성질병적병원학검측제공료쾌속가고적방법。
Objective To establish a multiplex real-time PCR method for the determination of Listeria monocytogenes and 3 toxic genes, and apply for the detection of food. Methods The specific primers and probes were designed in the conserved region of the hlyA gene, inlA gene and actA gene. The reaction conditions were optimized, and the sensitivity, specificity, and stability of the assay were evaluated. Results The results showed high specificity for the detection of L. monocytogenes without any evident cross-reaction with other pathogens such as E.coli, S.aureus, S.typhi, C.freumdii, P.mirabillis and E.tarda. The detection limit was 410 cfu/mL in pure culture. It demonstrated the high reproducibility with a coefficient of variation (CV) less than 2%. Investigation of 84 samples showed that the real-time PCR method was fully compatible with the standard method and 4 positive results were detected. The multiplex real-time PCR method provided results more rapidly and can be performed in 2 working days compared to up to 7 d for the standard method. Two isolates have 3 toxic genes of hlyA, inlA and actA, and the other two were lack of actA gene. The results suggested that the pandemic strains of L. monocytogenes currently did not come from the same source. Conclusion The method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food. It has a high sensitivity and specificity, and provides fast and reliable method for the detection of foodborne diseases.