巢式聚合酶链反应结合Real-time聚合酶链反应用于检测百日咳鲍特菌方法建立及应用

发布时间：2018-03-21 09:33

本文选题：巢式聚合酶链反应　切入点：Real-time聚合酶链反应　出处：《中国疫苗和免疫》2017年02期 　论文类型：期刊论文

【摘要】：目的建立一种具有高敏感性、高特异性的百日咳鲍特菌聚合酶链反应(Polymerase chain reaction,PCR)检测方法。方法以百日咳鲍特菌插入序列IS481为目的基因,通过Primer5.0软件设计巢式PCR(Nested PCR,nPCR)外引物,并优化扩增条件和程序,结合IS481目的基因的检测方法,建立nPCR结合Real-time PCR(rPCR)检测百日咳鲍特菌的方法,应用于354份流感样病例标本核酸检测和34份临床疑似百日咳病例标本核酸检测。结果该结合检测方法的最低检出限为0.225 copies/μL,能同时检测百日咳鲍特菌和霍氏鲍特菌,其他参考菌株均呈阴性,具有较好的特异性,进一步通过rPCR区分霍氏鲍特菌。采用该结合检测技术,354份流感样病例核酸标本中17份百日咳鲍特菌阳性,34份临床疑似百日咳病例核酸标本中12份百日咳鲍特菌阳性,而单独针对百日咳鲍特菌的rPCR方法分别仅检测出1份和6份阳性,rPCR检出的阳性标本,本方法均可检出。结论基于IS481基因的nPCR结合rPCR方法可用于百日咳鲍特菌的检测,具有较高的敏感性和特异性特点。
[Abstract]:Objective to establish a highly sensitive and specific polymerase chain reaction (PCR) method for detection of Bordetella pertussis by polymerase chain reaction (PCR). Methods the nested PCR(Nested PCRnPCRs were designed by Primer5.0 software, using the inserted sequence IS481 of Bordetella pertussis as the target gene. By optimizing the amplification conditions and procedures, combining with the method of detecting the target gene of IS481, the method of nPCR combined with Real-time PCRrPCRfor the detection of Bordetella pertussis was established. The method was used to detect nucleic acid in 354 influenza like cases and 34 clinical suspected pertussis cases. Results the minimum detection limit of the combined method was 0. 225 copies/ 渭 L, and it could be used to detect both Bordetella pertussis and Bordetella Hodgsoni at the same time. All other reference strains were negative and had good specificity. The combined detection technique was used to identify the nucleic acid samples of 354 influenza-like cases. Of the 17 nucleic acid samples positive for pertussis bacillus pertussis, 12 of 34 nucleic acid samples from clinical suspected pertussis cases were positive for Bordetella pertussis. However, only one and six positive samples were detected by rPCR alone for pertussis. Conclusion the nPCR combined with rPCR method based on IS481 gene can be used for the detection of Bordetella pertussis. It has high sensitivity and specificity.
【作者单位】：中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室;随州市疾病预防控制中心;随县疾病预防控制中心;随州市中心医院;
【分类号】：R440;R516.6

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