Victor T C, Jordaan A M, van Rie A, van der Spuy G D, Richardson M, van Helden P D, Warren R
Department of Medical Biochemistry, University of Stellenbosch, Medical School, South Africa.
Tuber Lung Dis. 1999;79(6):343-8. doi: 10.1054/tuld.1999.0222.
Mycobacterium tuberculosis isolates from patients in communities endemic for tuberculosis in South Africa.
To develop a reliable PCR-based dot-blot hybridization strategy to detect mutations conferring drug resistance.
Different loci in six genes associated with drug resistance to isoniazid, rifampacin, streptomycin and ethambutol were selected to develop the PCR-based dot-blot hybridization strategy.
Primers and probes to detect mutations at codons 315, 463 (katG) 269 (kasA), 531, 526 (rpoB) 43 (rpsL), 513 (rrs) and 306 (embB) were designed and used to develop a PCR-based dot-blot hybridization strategy. The dot-blot hybridization strategy with wild-type probes can efficiently be used to detect drug resistant mutations since these do not hybridize to mutant loci. Stripped blots and mutant probes can be used to identify the precise mutation. The embB gene (ethambutol resistance) was used to show how the dot-blot strategy can assist with the prediction of drug resistance more accurately. The method is rapid, reproducible, not technically demanding and samples can be done in batches. Additional loci can easily be incorporated.
A PCR-based dot-blot hybridization strategy is described which can accurately identify drug resistant strains and the method is useful for patients at risk and in areas endemic for tuberculosis.
从南非结核病流行社区的患者中分离出结核分枝杆菌。
开发一种可靠的基于聚合酶链反应（PCR）的斑点杂交策略，以检测赋予耐药性的突变。
选择与对异烟肼、利福平、链霉素和乙胺丁醇耐药相关的六个基因中的不同位点，以开发基于PCR的斑点杂交策略。
设计了用于检测密码子315、463（katG）、269（kasA）、531、526（rpoB）、43（rpsL）、513（rrs）和306（embB）处突变的引物和探针，并用于开发基于PCR的斑点杂交策略。带有野生型探针的斑点杂交策略可有效用于检测耐药突变，因为这些探针不会与突变位点杂交。去除探针的印迹和突变型探针可用于识别精确的突变。以embB基因（乙胺丁醇耐药）为例，展示了斑点杂交策略如何更准确地辅助预测耐药性。该方法快速、可重复，技术要求不高，样本可分批进行检测。还可轻松纳入其他位点。
描述了一种基于PCR的斑点杂交策略，该策略可准确鉴定耐药菌株，该方法对有风险的患者以及结核病流行地区有用。
