Qian Guo1, Yan Yu2, Yan Ling Zhu3, Xiu Qin Zhao4, Zhi Guang Liu4, Yuan Yuan Zhang4, Gui Lian Li4, Jian Hao Wei5, Yi Mou Wu6, Kang Lin Wan7. 1. Pathogenic Biology Institute, University of South China, Hengyang 421000, Hunan, China; State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China. 2. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Dalian Maternity and Child Health Care Hospital (the Obstetrics and Gynecology Hospital of Dalian), Dalian 116000, Liaoning, China. 3. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 4. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China. 5. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;Key Lab of Laboratory Medicine, Wenzhou Medical College, Wenzhou 325000, Zhejiang, China. 6. Pathogenic Biology Institute, University of South China, Hengyang 421000, Hunan, China. 7. Pathogenic Biology Institute, University of South China, Hengyang 421000, Hunan, China; State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China; Key Lab of Laboratory Medicine, Wenzhou Medical College, Wenzhou 325000, Zhejiang, China.
Abstract
OBJECTIVE: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). METHODS: 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. RESULTS: The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. CONCLUSION: Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.
OBJECTIVE: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). METHODS: 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. RESULTS: The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. CONCLUSION: Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.