本文選題：巢式聚合酶鏈反應(yīng)　切入點(diǎn)：Real-time聚合酶鏈反應(yīng)　出處：《中國(guó)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(yīng)(Polymerase chain reaction,PCR)檢測(cè)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(shè)計(jì)巢式PCR(Nested PCR,nPCR)外引物,并優(yōu)化擴(kuò)增條件和程序,結(jié)合IS481目的基因的檢測(cè)方法,建立nPCR結(jié)合Real-time PCR(rPCR)檢測(cè)百日咳鮑特菌的方法,應(yīng)用于354份流感樣病例標(biāo)本核酸檢測(cè)和34份臨床疑似百日咳病例標(biāo)本核酸檢測(cè)。結(jié)果該結(jié)合檢測(cè)方法的最低檢出限為0.225 copies/μL,能同時(shí)檢測(cè)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(jìn)一步通過rPCR區(qū)分霍氏鮑特菌。采用該結(jié)合檢測(cè)技術(shù),354份流感樣病例核酸標(biāo)本中17份百日咳鮑特菌陽(yáng)性,34份臨床疑似百日咳病例核酸標(biāo)本中12份百日咳鮑特菌陽(yáng)性,而單獨(dú)針對(duì)百日咳鮑特菌的rPCR方法分別僅檢測(cè)出1份和6份陽(yáng)性,rPCR檢出的陽(yáng)性標(biāo)本,本方法均可檢出。結(jié)論基于IS481基因的nPCR結(jié)合rPCR方法可用于百日咳鮑特菌的檢測(cè),具有較高的敏感性和特異性特點(diǎn)。
[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(guó)疾病預(yù)防控制中心傳染病預(yù)防控制所傳染病預(yù)防控制國(guó)家重點(diǎn)實(shí)驗(yàn)室;隨州市疾病預(yù)防控制中心;隨縣疾病預(yù)防控制中心;隨州市中心醫(yī)院;
【分類號(hào)】：R440;R516.6

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