| 田国忠,邵祝军,张砺,李晓静,朱兵清,杨亚静,徐丽,高源,王晓蕾.多重聚合酶链反应检测流感嗜血杆菌[J].Chinese journal of Epidemiology,2008,29(8):806-809 |
| 多重聚合酶链反应检测流感嗜血杆菌 |
| Detection of Haemophilus influenzae by multiplex polymerase chain reaction method |
| Received:December 26, 2007 Revised:June 26, 2012 |
| DOI： |
| KeyWord: 流感嗜血杆菌 多重聚合酶链反应 检测 |
| English Key Word: Haemophilus influenzae Muhiplex-polymerase chain reaction Detection |
| FundProject:国家自然科学基金资助项目(30570080);北京市自然科学基金资助项目(052020);北京市科委新星计划资助项目(2004B34) |
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| Abstract: |
| 目的<\b>建立检测流感嗜血杆菌的多重聚合酶链反应(M—PCR)方法<\b>。方法<\b> 合成扩增流感嗜血杆菌编码P6外膜蛋白基因的引物(Hi)，鉴定流感嗜血杆菌种特异性；合成扩增流感嗜血杆菌编码荚膜基因的引物(Hi．cap)，鉴定菌株是否具有荚膜；设计并合成6对扩增流感嗜血杆菌不同血清型(荚膜型)编码基因的引物(Hia～Hif)，鉴定菌株的血清型，以其他呼吸道常见病原体菌株做对照，应用M—PCR方法<\b>对200株临床分离的疑似流感嗜血杆菌菌株进行鉴定和血清分型。结果<\b>M—PCR方法<\b>检测流感嗜血杆菌具有高度的敏感性和特异性。对照菌株应用种(Hi)、荚膜(Hi．cap)和荚膜型(Hia～Hif)特异引物没有扩增出DNA片段。M—PCR方法<\b>检测DNA的最低检测量为0．935Pg。对临床分离的200株疑似流感嗜血杆菌鉴定，189株为流感嗜血杆菌，与API@NH鉴定结果<\b>一致。其中1株具有荚膜，为f血清型，与玻片凝集法鉴定结果<\b>一致。结论<\b>M—PCR方法<\b>检测流感嗜血杆菌具有较高的敏感性和特异性，可用于流感嗜血杆菌感染病例标本的快速检测和病例诊断。 |
| English Abstract: |
| Objective To develop a rapid method for detecting Haemophilus influenzae by multiplex polymerase chain reaction (M-PCR). Methods Primers (Hi) were designed for amplification of p6 gene coding P6 protein of Haemophilus influenzae, which was used to identify Haemophilus influenzae species. Primers (Hi-cap) were designed for amplification of bexA gene which coding capsular polysaccharide (cap) synthesis was used for detecting whether Haemophilus influenzae isolates possess bexA gene relating to cap synthesis. Twelve primers (Hia-Hif) were designed for amplification of cap synthesis gene to identify the cap-type of Haemophilus influenzae. Other relative enteric pathogenic bacteria were amplified by M-PCR to serve as controls. 200 strains isolated from patients were identified.Results from M-PCR were compared to two methods including V and X factors grow requirement test and standard slide agglutination serotyping (SAST). Results The results indicated that the M-PCR assay was high specificity and sensitivity and might be valuable for differential diagnosis of Haemophilus influenzae.The sensitivity of detection was 0. 935 pg. 189 strains out of the 200 belonged to Haemophilus influenzae isolates, and one isolate was cap-type f. An agreement results were seen among the V and X factors grow requirement test, SAST and M-PCR methods. Conclusion M-PCR method showed satisfactory sensitivity, specificity and stability for detecting and identifying Haemophilus influenzae ,and could be used in clinic diagnosis, surveillance and rapid diagnosis for plague of Haemophilus influenzae. |
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