Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

Abstract

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.T he rapid and accurate diagnosis of pulmonary tuberculosis (PTB) plays a critical role in its successful management, as it is essential to treat patients with the appropriate therapy as soon as possible to minimize the risk of transmission. A number of tests based on nucleic acid amplification (NAA) have been developed to identify Mycobacterium tuberculosis in clinical specimens rapidly and directly (8,34,35,44). Among the approved commercial NAA methods, the amplified M. tuberculosis direct test (MTD test; Gen-Probe) detects M. tuberculosis RNA by using a specific isothermal transcription-mediated amplification method (21). The MTD test has been shown to be a sensitive, specific, and rapid method for use with clinical samples (9, 17, 18); however, it requires the use of expensive specialized detection equipment, which prevents the assay from being applied widely in clinical laboratories. The aim of this study was to report and evaluate a simultaneous amplification and testing method for detection of the M. tuberculosis complex (SAT-TB assay) for use with clinical sputum specimens, based on real-time fluorescence dete...

Application of real-time simultaneous amplification and testing method to accurately and rapidly detect extra-pulmonary tuberculosis

“…Simultaneous Amplification and Testing (SAT) is one of the molecular biological detection methods used by the tuberculosis laboratory in China in recent years. It is mainly used to detect MTB in sputum to assist diagnosis [4][5][6][7]. However, SAT has rarely been used to test EPTB specimens and therefore, its sensitivity and specificity for EPTB diagnosis are still not clear.…”

Using simultaneous amplification and testing method for evaluating the treatment outcome of pulmonary tuberculosis

“…Moreover, the SAT-TB assay is intended for point-of-care testing for TB [ 10 ]. Recent researches showed that sensitivity of the SAT-TB assay for the diagnosis of PTB with sputum samples was from 39.2 to 93% [ 11 , 12 ]. In patients with sputum-scarce, the sensitivity of the SAT-TB test using bronchial alveolar lavage fluid (BALF) specimens was 50.75% [ 13 ].…”

Clinical diagnostic value of simultaneous amplification and testing for the diagnosis of sputum-scarce pulmonary tuberculosis

“…It is fast, simple and has good reproducibility [ 8 ]. Previous studies of the SAT-TB assay that focused on its accuracy with sputum samples demonstrated that its overall sensitivity for the diagnosis of PTB was 67.7% (range 39.2% to 93%) [ 9 , 10 ].…”