Patel R J, Fries J W, Piessens W F, Wirth D F
Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000.
J Clin Microbiol. 1990 Mar;28(3):513-8. doi: 10.1128/jcm.28.3.513-518.1990.
Analysis of the 1,016-base-pair sequence of a putative probe for identification of Mycobacterium tuberculosis revealed two almost identical fragments of 507 and 509 bases. From this sequence two pairs of primers were synthesized (MtbAB and MtbCD), ranging from 18 to 22 nucleotides, for use in polymerase chain reactions (PCRs) with DNA from six reference strains of M. tuberculosis, as well as type strains of M. bovis, M. bovis BCG, M. kansasii, M. avium, M. intracellulare, and M. scrofulaceum. Although there was amplification of DNA from all mycobacterial strains included in the study, when used as probes, a predominant band, fragment CD from M. tuberculosis H37Rv DNA, proved to be more specific for strains of M. tuberculosis than the original probe, pMTb4, was. Amplified fragments from as little as 1 fg of DNA (equivalent to one-fifth of an organism) could be resolved on ethidium bromide-stained gels loaded with a 1/10 volume of PCR. Furthermore, it was possible to amplify specific DNA sequences from frozen M. tuberculosis H37Rv organisms which were thawed prior to PCR.
对用于鉴定结核分枝杆菌的一个假定探针的1016个碱基对序列进行分析，发现了两个分别为507和509个碱基的几乎相同的片段。根据该序列合成了两对引物（MtbAB和MtbCD），长度为18至22个核苷酸，用于对6株结核分枝杆菌参考菌株以及牛分枝杆菌、卡介苗、堪萨斯分枝杆菌、鸟分枝杆菌、胞内分枝杆菌和瘰疬分枝杆菌的标准菌株的DNA进行聚合酶链反应（PCR）。尽管该研究中所包含的所有分枝杆菌菌株的DNA均得到了扩增，但当用作探针时，结果表明，来自结核分枝杆菌H37Rv DNA的片段CD这一主要条带，相较于原始探针pMTb4，对结核分枝杆菌菌株具有更高的特异性。在加入1/10体积PCR产物的溴化乙锭染色凝胶上，可以分辨出低至1 fg DNA（相当于五分之一个生物体）扩增得到的片段。此外，有可能从PCR前解冻的冷冻结核分枝杆菌H37Rv生物体中扩增出特定的DNA序列。
