Detection of mutations associated with resistance to rifampicin and isoniazid in <italic>Mycobacterium tuberculosis</italic> by polymerase chain reaction-ligase detection reaction

Abstract

Mycobacterium tuberculosis (MTB) infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB (MDR-TB), defined as being resistant to at least rifampicin (RMP) and isoniazid (INH), and extensively drug-resistant TB (XDR-TB), defined as being additionally resistant to any fluoroquinolone and one injectable secondline drug, are associated with higher treatment failure rates. China is the second largest nation in terms of the number of the MTB infection, and 9.3% of the cases are MDR-TB [2]. MDR-TB arises from spontaneous chromosomal mutations at low frequency, but clinical MDR-TB largely occurs as a result of man-made selection during disease treatment of these genetic alterations through erratic drug supply, suboptimal physician prescription, and poor patient adherence [3]. S315T/M mutation in katG and mutations in the promoter region of inhA operon are the main mechanisms of INH resistance [4,5]. Mutations at positions 516, 526, and 531 in rpoB are the most frequent mutations in RMP-resistant MTB strains [6]. Since the transmission of the selected MDR-TB strains causes a large number of MDR-TB cases [7,8], the need for rapid, reliable, and cost-effective drug susceptibility testing (DST) is urgent. Polymerase chain reaction (PCR) followed by ligase detection reaction (LDR) method has been widely used to detect various single nucleotide polymorphisms and drug-resistant mutations with high sensitivity and low cost [9–12]. In the present study, we established a PCR-LDR assay for the simultaneous detection of the mutations related to MDR-TB, and evaluated its performance using isolates and clinical samples. Here, a total of 120 MTB isolates obtained from pulmonary TB patients at The Third Hospital of Changzhou, during July, 2011 and March, 2012 were collected and subjected to DST for RMP and INH in the BACTEC MGIT 960 instrument with medium, and DST supplement (Becton Dickinson and Company, Sparks, USA) as recommended by the manufacturer. All the 120 MTB isolates were detected by phenotypic DST, PCR-LDR, and DNA sequencing. The specific primers and probes (Supplementary Tables S1 and S2) for both wild type and mutant type targeting codon 315 of the katG gene, codon 15 of the inhA promoter region, codons 516, 526, and 531 of rpoB. All the primers and probes were synthesized by Invitrogen (Shanghai, China). Genome DNA of MTB was extracted by a commercial kit (Fosun Diagnostics, Shanghai, China) according to manufacturer’s instruction. All isolates were detected by both the PCR-LDR assay and sequencing. The multiplex PCR-LDR method was the same as described in previous studies [11,12]. The amplifications and LDR were performed in a PE 9600 thermal cycler (Life Technology, Foster City, USA). The LDR products were electrophoresed on an ABI 377 DNA Sequencer (Life Technology) for 30 min. Results were analyzed using the GeneMapper software (Life Technology). Meanwhile, the PCR products of rpoB and inhA promoter region were purified and sequenced by Life Technology. The results of phenotypic DST, PCR-LDR, and DNA sequencing were summarized in Table 1. Phenotypic DST of MTB isolates indicated that 26 (21.7%) at low level (1 mg/ ml) and 11 (9.2%) at high level (10 mg/ml) resistance to INH, while 17 (14.2%) at low-level and 9 (7.5%) at highlevel resistance to RMP. Among them, 13 (10.8%) were resistant to both INH and RMP at low level, or MDR. PCR-LDR assays for the detection of MDR-TB were carried out on 120 isolates. The S315M in katG was detected in 16 (13.3%) of 120 isolates. No S315T mutation in katG and G15T mutation in the promoter region of inhAwas detected. The D516V/G, H526Y/D, S531/L/W mutations in rpoB were detected in 1 (0.8%), 3 (2.5%), and 8 (6.7%) of 120 isolates, respectively. The typical results of PCR-LDR were shown in Fig. 1 and Supplementary Fig. S1. Acta Biochim Biophys Sin 2014, 46: 78–81 |a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt119. Advance Access Publication 11 November 2013