Restriction endonuclease analysis as a solution for determining rifampin resistance mutations by automated DNA sequencing in heteroresistant Mycobacterium tuberculosis strains
Restriction endonuclease analysis as a solution for determining rifampin resistance mutations by automated DNA sequencing in heteroresistant Mycobacterium tuberculosis strains
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DOI：
10.1089/mdr.2005.11.137
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发表时间：
2005-06-01
期刊：
影响因子：
--
通讯作者：
Akar, N
中科院分区：
文献类型：
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作者：
Karahan, ZC;Akar, N
结核分枝杆菌中的利福平耐药性主要是由于编码RNA聚合酶β亚基的rpoB基因中的少量突变。异源耐药性定义为同一培养物中野生型和耐药亚群的混合物，可通过导致假阴性或不确定结果来影响用于确定耐药性的分子测试的灵敏度。在我们的实验室中，我们在rpoB基因突变的测序过程中鉴定了一种异源耐药菌株，该突变是导致M.结核该分离物的序列分析表明不同菌株的混合物，并且不能确定突变的确切性质。为了解决这一问题，我们用Tsp 509 Ⅰ限制性内切酶对可能的突变位点进行了酶切，使不同菌株得以分离。分离的突变产物的测序揭示了导致利福平耐药性的突变。从这一结果，我们提出，当建议的突变位点创建和/或删除一个限制性内切酶识别位点的异源耐药群体，限制性内切酶分析可以用来简化通过测序的耐药突变的测定。
Rifampin resistance in Mycobacterium tuberculosis is mainly due to a small number of mutations in the rpoB gene coding for the beta subunit of RNA polymerase. Heteroresistance, which is defined as a mixture of wildtype and resistant subpopulations in the same culture, can influence the sensitivity of molecular tests for determining drug resistance by leading to false negative or indeterminable results. In our laboratory, we identified one heteroresistant strain during sequencing of the rpoB gene mutations, which are responsible for Rifampin resistance in M. tuberculosis. The sequence analysis of this isolate demonstrated the mixture of different strains, and the exact nature of the mutation could not be determined. In order to solve this problem, the possible mutation site was digested with Tsp509I restriction endonuclease, which made us separate the different strains. Sequencing of the separated mutated product revealed the mutation responsible for Rifampin resistance. From this result, we propose that, when the suggested mutation site creates and/or deletes a restriction endonuclease recognition site in heteroresistant populations, restriction endonuclease analysis can be used to ease the determination of resistance mutations by sequencing.