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目的建立基于流感嗜血杆菌(Haemophilus influenzae,Hi)外膜蛋白P6编码基因omp6和荚膜编码基因bex A的双重PCR检测方法,并确定其检测限、灵敏度和特异度。方法根据Gen Bank公布流感嗜血杆菌的omp6和bex A基因核苷酸序列设计引物,用Hi标准菌株确定其检测限,双重PCR检测289例鼻窦炎患者鼻咽部分泌物(NPS)确定方法的灵敏度与特异度。结果 289例NPS培养阳性率为50.5%,14株为荚膜型;双重PCR检测omp6的阳性率为54.7%,bex A阳性16株。双重PCR与细菌培养及血清凝集试验符合率分别为95.8%和99.3%。检测时模板的下限为3.8 pg/μl。双重PCR检测omp6和bex A基因的灵敏度均为100.0%,特异度分别为91.6%和99.3%。结论建立的双重PCR是一种快速、特异和敏感的Hi感染的检测和分型方法,可代替细菌培养和血清凝集试验用于临床标本诊断。
Objective To establish a dual PCR method based on the gene omp6 encoding P6 and the gene encoding bex A encoding the outer membrane protein of Haemophilus influenzae (Hi), and to determine its detection limit, sensitivity and specificity. Methods Primers were designed according to nucleotide sequences of omp6 and bex A gene of Haemophilus influenzae from Gen Bank. The detection limit was determined by Hi standard strains. Nasopharyngeal secretion (NPS) was determined by double PCR in 289 patients with sinusitis Sensitivity and specificity. Results The positive rate of NPS culture in 289 cases was 50.5% and 14 strains were capsular type. The positive rate of omp6 was 54.7% by double-PCR and 16 bex-A positive. The coincidence rates of double PCR with bacterial culture and serum agglutination test were 95.8% and 99.3% respectively. The lower limit of the template when testing is 3.8 pg / μl. Double PCR showed that the sensitivity of omp6 and bex A genes were 100.0% and 91.6% and 99.3% respectively. Conclusion The established dual PCR is a rapid, specific and sensitive method for the detection and typing of Hi infection, which can replace the bacterial culture and serum agglutination test for clinical specimen diagnosis.