Development of a fluorescent microsphere-based multiplex assay for simultaneous rapid detection of Mycobacterium tuberculosis complex and differentiation of M. tuberculosis and M. bovis in clinical samples.
A microsphere-based multiplex assay was developed, by using the xMAP technology, for the simultaneous rapid detection of the Mycobacterium tuberculosis complex (MTC) and the differentiation of M. tuberculosis and M. bovis. The assay simultaneously detected 4 target sequences, including specific insertion elements IS6110 and IS1081 of MTC, a 12.7- Kb fragment specific for M. tuberculosis, and an uninterrupted 229 bp sequence specific for M. bovis. The specificity of the assay was validated by testing on 13 species of mycobacteria reference strains, 22 isolated strains of M. tuberculosis and M. bovis, and 25 species of non-mycobacteria microorganisms. The limit of detection of mycobacteria by the assay was up to 6 to 10 bacteria per reaction, and the sensitivity of detecting cloned DNA templates was 0.37 to 0.74 fg DNA per reaction. The intra-assay and interassay variation of the assay on the 4 gene targets were low (1.5% to 8.5% and 4.1% to 9.2%, respectively). Clinical performance of the assay was validated on human sputum and bovine tissue samples and compared with the culture. The assay detected 69.8% (90/129) of MTC positive, as compared with 36.4% (47/129) positive by culture on sputum samples, and 75.5% (37/49) of MTC positive as compared with 42.8% (21/49) positive by culture on bovine tissue samples. The assay detected 100% of the culture positive in both human and bovine samples. The tests identified 98.9% (89/90) as M. tuberculosis from the MTC-positive sputum samples, and identified 91.9% (34/37) as M. bovis from the MTC-positive bovine tissue samples. The detection of mycobacterial DNA by the microsphere-based multiplex assay could be completed within 2 hours.
Chen
Ru
R
Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, China. ychenru@hotmail.com
Bi
Yingzuo
Y
Yang
Guohai
G
Liu
Zhiling
Z
Liu
Zhihui
Z
Zeng
Bijian
B
Tong
Tiezhu
T
D000704:Analysis of Variance; D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002417:Cattle; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D008168:Lung / Q000382:microbiology; D008863:Microspheres; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009163:Mycobacterium bovis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000382:microbiology
D004251:DNA Transposable Elements; D004269:DNA, Bacterial