A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis.
Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9 %, 95.5 %, and 92.4 % with ropB in rifampin resistance, 67.3 %, 97.1 %, and 84.3 % with rpsL and rrs in streptomycin resistance, 60.0 %, 96.0 %, and 91.4 % with embB in ethambutol resistance, 68.4 %, 94.9 %, and 86.3 % with inhA and katG in isoniazid resistance, and 74.1 %, 98.9 %, and 93.2 % in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.
Division of Life Science and Applied Genomics Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Yisuo
Y
Sun
Shufen
S
Li
Lin
L
Zhou
Lin
L
Zhou
Qiu
Q
Zhong
Shisong
S
Fang
Tao
T
Chen
Lijun
L
Bi
Wai-Kin
WK
Mat
Cunyou
C
Zhao
Hong
H
Xue
D000995:Antitubercular Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D005454:Fluorescence Polarization / Q000379:methods*; D005798:Genes, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000382:microbiology
D000995:Antitubercular Agents