Sharma R K, Katoch K, Shivannavar C T, Sharma V D, Natrajan M, Bhatia A S, Saxena N, Katoch V M
Central JALMA Institute for Leprosy (ICMR), Taj Ganj, Agra, India.
Int J Lepr Other Mycobact Dis. 1996 Dec;64(4):409-16.
Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.
对130例不同麻风病谱的病例（56例结核样型/界线类偏结核样型/未定类，73例瘤型/界线类偏瘤型/麻风瘤型，以及1例神经炎型病例）和50名健康接触者的活检及皮肤刮片标本进行研究，以评估基因扩增的应用。通过综合冻融——优化的溶菌酶/蛋白酶K处理——纯化及分级分离程序，从这些临床标本中提取核酸。来自培养微生物的核酸通过本实验室先前标准化的逐步程序进行分离。使用引物并按照其开发者描述的程序对18-kDa蛋白基因的360-bp片段进行基因扩增，Southern印迹上的360-bp片段被作为阳性标准。聚合酶链反应产物通过电泳、溴化乙锭（EB）染色和印迹（B）杂交进行分析。总体敏感性范围从抗酸菌检测不到的标本中的71%到可证实有抗酸杆菌的标本中的100%。在结核样型/界线类偏结核样型/未定类标本中观察到73%的阳性率，在瘤型/界线类偏瘤型/麻风瘤型标本中为93%。辨别出四种组合：EB阳性，B阳性（71%）；EB可疑，B阳性（14%）；EB阴性，B阳性（3%）和EB阴性，B阴性（12%）。与单独的EB染色相比，通过将印迹杂交与EB染色相结合，敏感性可显著提高。通过对照标本以及从这些标本中培养出的分枝杆菌和非分枝杆菌生物体的纯化DNA均未出现任何假阳性，发现该检测具有绝对特异性。建议为获得最佳敏感性和特异性，应同时进行EB染色和印迹杂交。
