Matar G M, Chahwan R, Fuleihan N, Uwaydah M, Hadi U
Departments of Microbiology and Immunology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
Clin Diagn Lab Immunol. 2001 Mar;8(2):221-4. doi: 10.1128/CDLI.8.2.221-224.2001.
We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.
我们开发并评估了一种基于聚合酶链反应-限制性内切酶分析的方法，用于检测和分析接受扁桃体切除术和腺样体切除术的儿科患者中流感嗜血杆菌和副流感嗜血杆菌的tonB基因。从同一患者的多个部位，包括腺样体和扁桃体表面以及扁桃体核心，在巧克力琼脂上进行培养，并使用标准程序和API NH试剂盒进行鉴定。共从20名患者的不同部位分离出55株流感嗜血杆菌，从12名患者的不同部位分离出32株副流感嗜血杆菌。通过PureGene试剂盒从美国典型培养物保藏中心菌株和测试分离株中提取DNA。我们设计了两条引物，G1（21个碱基）和G2（23个碱基），用于通过聚合酶链反应扩增由一个813碱基对片段组成的tonB基因。使用引物T1（23个碱基）和T2（24个碱基）进行巢式聚合酶链反应，该引物位于该基因内部约257碱基对序列的两侧，并使用XhoI和BglII进行限制性内切酶消化，以检测这两个物种的该基因内部是否存在异质性。最后进行逆转录聚合酶链反应（RT-PCR）以检测这两个物种中该基因的转录情况。我们的数据表明，这两个物种中均检测到了tonB基因。已知该基因在流感嗜血杆菌中编码一种毒力蛋白TonB；然而，在副流感嗜血杆菌中发现该基因则是新的发现。巢式聚合酶链反应和限制性内切酶分析表明，这两个物种的tonB基因在结构上显然相同，但某些副流感嗜血杆菌分离株可能存在差异。对选定数量的流感嗜血杆菌和副流感嗜血杆菌进行的RT-PCR表明，这两个物种中均转录了tonB基因。这表明，如果TonB蛋白表达，它在副流感嗜血杆菌的毒力中可能发挥不同的作用，因为它不像流感嗜血杆菌那样需要摄取血红素或血红素复合物。
