title: Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.
abstract: Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Guilian
authors.initials: G
authors.lastname: Li
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.|Department of Clinical Laboratory, Chinese People's Liberation Army General Hospital, Beijing 100853, P. R. China.
authors.forename: Chongyun
authors.initials: C
authors.lastname: Sun
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.|College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, P. R. China.
authors.forename: Xiaochen
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Haican
authors.initials: H
authors.lastname: Liu
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.|Institute for Plague Prevention and Control of Hebei Province, Zhangjiakou 075000, P. R. China.
authors.forename: Xinrui
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.
authors.forename: Ruifu
authors.initials: R
authors.lastname: Yang
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P. R. China.
authors.forename: Kanglin
authors.initials: K
authors.lastname: Wan
authors.affiliation: Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China.|Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071, P. R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D009841:Oligonucleotides; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D015342:DNA Probes; D009841:Oligonucleotides; C477371:locked nucleic acid; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin