Simultaneous Determination of Mycobacterium leprae Drug Resistance and Single-Nucleotide Polymorphism Genotype by Use of Nested Multiplex PCR with Amplicon Sequencing
Simultaneous Determination of Mycobacterium leprae Drug Resistance and Single-Nucleotide Polymorphism Genotype by Use of Nested Multiplex PCR with Amplicon Sequencing
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DOI：
10.1128/jcm.00814-21
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发表时间：
2021-10-01
影响因子：
9.4
通讯作者：
Nakata, Noboru
中科院分区：
文献类型：
--
作者：
Iwao, Yasuhisa;Mori, Shuichi;Nakata, Noboru
麻风分枝杆菌是世界范围内麻风病的主要致病菌，其基因型可分为4种单核苷酸多态性（SNP）类型和16种亚型。确定M.麻风抗药性和基因分型通常通过PCR和桑格DNA测序完成，这需要大量工作。在这里，我们描述了一种快速的方法，包括多重PCR结合巢式扩增和下一代序列分析，允许同时测定M。麻风耐药性和SNP基因型。我们用这种方法分析了两个少杆菌，9个多杆菌，和6个类型未定的麻风病人的临床样本。folP1、rpoB、gyrA和gyrB中决定耐药性的区域以及M.从临床样品中扩增麻风基因组并测定其序列。结果表明，7份样品为1A亚型，3份为1D亚型，7份为3K亚型。3例3K亚型标本存在folp1突变。该方法可用于更快速的M.临床样本中的麻风。
Mycobacterium leprae is the predominant cause of leprosy worldwide, and its genotypes can be classified into four single-nucleotide polymorphism (SNP) types and 16 subtypes. Determining M. leprae drug resistance and genotype is typically done by PCR and Sanger DNA sequencing, which require substantial effort. Here, we describe a rapid method involving multiplex PCR in combination with nested amplification and next-generation sequence analysis that allows simultaneous determination of M. leprae drug resistance and SNP genotype directly from clinical specimens. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. Regions in folP1, rpoB, gyrA, and gyrB that determine drug resistance and those for 84 SNP-InDels in the M. leprae genome were amplified from clinical samples and their sequences determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. The method may allow more rapid genetic analyses of M. leprae in clinical samples.