title: Rolling circle amplification for direct detection of rpoB gene mutations in Mycobacterium tuberculosis isolates from clinical specimens.
abstract: Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.
authors.affiliation: Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing, China.
authors.forename: Xiaoyou
authors.initials: X
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Wen
authors.initials: W
authors.lastname: Yang
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.forename: Chuanyou
authors.initials: C
authors.lastname: Li
authors.forename: Zhaogang
authors.initials: Z
authors.lastname: Sun
authors.forename: Peter
authors.initials: P
authors.lastname: Jelfs
authors.forename: Gwendolyn L
authors.initials: GL
authors.lastname: Gilbert
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D007538:Isoniazid; D012293:Rifampin