应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

Study on Detection of the Mycobacteria DNA in Formalin-fixed, Paraffin-embedded Tissue Samples by Triplex Polymerase Chain Reaction

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感染。1 6例病理诊断为可疑淋巴结结核病人的标本 ,1 5例检测结果符合结核分枝杆菌复合体感染 ,1例符合非结核分枝杆菌感染。此种多重PCR方法可?

To supply an additional differential diagnostic method for pathological diagnosis of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infections in formalin-fixed, paraffin-embedded tissue samples by triplex-PCR. Three pairs of oligonucleotide primer were used in triplex-PCR. A 383bp DNA fragment encoding part of the 65kD mycobacterial surface antigen, a 123bp fragment corresponding to a specific Mycobacterium tuberculosis complex sequence which was the insertion sequence 6110 (IS6110) and a 268bp fragment for human β-globin were amplified by triplex-PCR respectively. The sensitivity of the triplex-PCR-electrophoresis for the mycobacteria DNA was 0.6 picogram. The specific bands of 383bp and 123bp among the amplified DNA from Mycobacterium tuberculosis, M.bovis, M.bovis BCG and M.simiae were present in the agarose gel. By contrast, only a band of 383bp was found among the nontuberculosis mycobacteria which contained M.avium, M.chelonae, M.scrofulaceum, M. xenopi, M.kansasii, M.intracellulare and M.smegmatis.Compared with the standard strains, there was an additional 268bp band in simulated clinic samples infected by mycobacteria, 209 formalin-fixed, paraffin-embedded tissue samples of the patients diagnosed as scrofula by clinic doctor at first visit were examined by triplex polymerase chain reaction. Among them, 193 tissue samples of the patients pathologically diagnosed as scrofula, tuberculous granulomatous tissue or tuberculous granulomatous inflammation were positive: the specific hands of 383bp, 123bp and 268bp were present in the agarose gel and this tallied with Mycobacterium tuberculosis complex infection. Of 16 tissue samples of the patients pathologically diagnosed as suspicious scrofula, 15 samples were same positive results and this tallied with Mycobacterium tuberculosis complex infection, too; 1 sample could find the specific bands of 383bp and 268bp which were present in the agarose gel and this tallied with nontuberculous mycobacteria infection. The results showed that the triplex-PCR could detect and identify the DNA of Mycobacterium tuberculosis complex and nontuberculous mycobacteria except M.simiae. It is a valuable detecting method which has high sensitivity and specificity.