本文選題：巢式聚合酶鏈反應　切入點：Real-time聚合酶鏈反應　出處：《中國疫苗和免疫》2017年02期 　論文類型：期刊論文

【摘要】：目的建立一種具有高敏感性、高特異性的百日咳鮑特菌聚合酶鏈反應(Polymerase chain reaction,PCR)檢測方法。方法以百日咳鮑特菌插入序列IS481為目的基因,通過Primer5.0軟件設計巢式PCR(Nested PCR,nPCR)外引物,并優(yōu)化擴增條件和程序,結合IS481目的基因的檢測方法,建立nPCR結合Real-time PCR(rPCR)檢測百日咳鮑特菌的方法,應用于354份流感樣病例標本核酸檢測和34份臨床疑似百日咳病例標本核酸檢測。結果該結合檢測方法的最低檢出限為0.225 copies/μL,能同時檢測百日咳鮑特菌和霍氏鮑特菌,其他參考菌株均呈陰性,具有較好的特異性,進一步通過rPCR區(qū)分霍氏鮑特菌。采用該結合檢測技術,354份流感樣病例核酸標本中17份百日咳鮑特菌陽性,34份臨床疑似百日咳病例核酸標本中12份百日咳鮑特菌陽性,而單獨針對百日咳鮑特菌的rPCR方法分別僅檢測出1份和6份陽性,rPCR檢出的陽性標本,本方法均可檢出。結論基于IS481基因的nPCR結合rPCR方法可用于百日咳鮑特菌的檢測,具有較高的敏感性和特異性特點。
[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.
【作者單位】： 中國疾病預防控制中心傳染病預防控制所傳染病預防控制國家重點實驗室;隨州市疾病預防控制中心;隨縣疾病預防控制中心;隨州市中心醫(yī)院;
【分類號】：R440;R516.6

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