荧光扫描法快速检测肽核酸分子信标标记的单增李斯特菌  

Molecular beacon based PNA-FISH method combined with fluorescence scanning for rapid detection of Listeria monocytogenes

作　　者：吴姗 张晓峰 帅江冰 李可 虞惠贞 金晨晨 

机构地区：[1]浙江省检验检疫科学技术研究院,浙江杭州310016

出　　处：《微生物学报》2016年第7期1105-1112,共8页Acta Microbiologica Sinica

基　　金：国家质量监督检验检疫总局科研项目(2013IK189;2015IK301);浙江省公益技术应用研究计划项目(2015C33042)~~

摘　　要：【目的】建立了单核细胞增多性李斯特菌(Listeria monocytogenes,单增李斯特菌)的肽核酸(Peptide nucleic acid,PNA)分子信标(Molecular beacon)的荧光扫描检测方法,以简化普通PNA原位荧光杂交(Fluorescence in situ hybridization,FISH)检测方法中显微镜观察步骤。【方法】在具有单增李斯特菌特异性的肽核酸探针的5′和3′分别标记报告荧光基团和淬灭基团形成分子信标PNA探针,利用FISH技术和荧光扫描技术对单增李斯特菌进行检测。【结果】用普通PNA探针进行荧光扫描检测时,以N1处理为空白对照,假阳性率11.4%,假阴性率为0;以N2处理为空白时,假阳性率降低至4.3%,但假阴性率上升为18.6%。用分子信标PNA探针进行检测时,用N1为空白对照时,假阳性率8.6%,假阴性率为1.4%;以N2处理作为空白时,假阳性率5.7%,假阴性率为1.4%。与普通探针比较,分子信标PNA探针能有效减少假阳性和假阴性的发生。2种普通PNA探针的杂交成功率分别为83.3%和95.2%;2种"分子信标化"的肽核酸探针的成功率分别为91.7%和90.5%,表明探针两端标记并不会降低与目标菌的杂交成功率。【结论】将液相PNA-FISH和荧光扫描技术结合,通过大通量快速的荧光扫描检测可大幅提高检测效率。同时将肽核酸探针分子信标化,有效的降低了荧光扫描结果的假阳性,并通过了N1和N2两种空白对照处理把假阴性控制在较低的范围。[Objective] To simplify the PNA-FISH（Peptide nucleic acid-fluorescence in situ hybridization） test,molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes.[Methods] The 5′ end and 3′ end of the L.monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively,to form a molecular beacon based PNA probe.[Results] When PNA probe used for fluorescence scanning and N1 treatment as the control,the false positive rate was 11.4%,and the false negative rate was 0;when N2 treatment as the control,the false positive rate decreased to 4.3%,but the false negative rate rose to 18.6%.When beacon based PNA probe used for fluorescence scanning,taken N1 treatment as blank control,the false positive rate was 8.6%,and the false negative rate was 1.4%;taken N2 treatment as blank control,the false positive rate was 5.7%,and the false negative rate was 1.4%.Compared with PNA probe,molecular beacon based PNA probe can effectively reduce false positives and false negatives.The success rates of hybridization of the two PNA probes were 83.3% and 95.2%respectively;and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively,which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria.[Conclusions] The combination of liquid phase PNA-FISH and fluorescence scanning method,can significantly improve the detection efficiency.

关 键 词：肽核酸荧光原位杂交 分子信标 荧光扫描 单增李斯特菌 

分 类 号：Q93[生物学—微生物学]