Novel real-time simultaneous amplification and testing method to accurately and rapidly detect Mycobacterium tuberculosis complex.
The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.
Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Medical School, Tongji University, Shanghai, China.
Zhenling
Z
Cui
Yongzhong
Y
Wang
Liang
L
Fang
Ruijuan
R
Zheng
Xiaochen
X
Huang
Xiaoqin
X
Liu
Gang
G
Zhang
Dongmei
D
Rui
Jinliang
J
Ju
Zhongyi
Z
Hu
D001431:Bacteriological Techniques / Q000379:methods*; D003429:Cross Reactions; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011237:Predictive Value of Tests; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S