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

Application of nested polymerase chain reaction combined with real-time polymerase chain reaction for diagnosis of Bordetella pertussis

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。

Objective To establish a highly sensitive and specific polymerase chain reaction （PCR） method for diagnosis of Bordetella pertussis （B. pertussis）. Methods Based on the sequence of B. per- tussis, an outer primer was designed for nested PCR （nPCR） by Primer 5.0 software, and the condition and procedure of amplification was improved. The PCR amplification was combined with real-time PCR （rPCR）, which was used to detect the IS481 target gene and to improve the sensitivity and specificity of diagnosing B. pertussis. This combined method was used to evaluate clinical specimens from 354 influen- za-like cases and 34 suspected clinical pertussis cases for DNA extraction. Results The sensitivity of the combined PCR method achieved the lowest detected limit of 0. 225 copies/l^L. Both B. pertussis and B. holmesii can test positive simultaneously, while other reference strains test negative, and then rPCR can detect B. holmesii. Using this combined PCR method, 17 of 354 nucleic acid samples from influenza-like cases, and 12 of 34 samples from clinical suspected pertussis cases were positive for B. pertussis, respectively, while 1 and 6 were positive when only using rPCR. All samples testing positive by rPCR were able to be identified by this combined method. Conclusions The combined nPCR and rPCR tech-nique, based on IS481 gene, can be used for diagnosis of B. Pertussis, with a high sensitivity and specificity.