title: Direct identification and discernment of Mycobacterium avium and Mycobacterium intracellulare using a real-time RNA isothermal amplification and detection method.
abstract: The purpose of this work was to establish a real-time simultaneous amplification and testing method for identification and discernment of Mycobacterium avium and Mycobacterium intracellulare (SAT-MAC assay) and to evaluate the efficiency with which this method can detect isolated strains and clinical sputum specimens. The specific 16S rRNA sequences of M. avium and M. intracellulare were used as targets to design RNA probes and a reverse transcription primer containing T7 promoter. RNA isothermal amplification and real-time fluorescence detection were performed at 42 °C. SAT-MAC assay, culture tests on Lowenstein-Jensen (L-J) culture medium and PCR-sequencing were used to test the clinical isolated strains and sputum specimens. The limit of detection (LOD) of M. avium and M. intracellulare by SAT-MAC was found to be 30 CFU/mL and 20 CFU/mL. SAT-MAC showed high specificity in 21 species of mycobacteria standard strains and 5 species of non-mycobacteria bacteria. Using PCR-sequencing as the reference method, both rates of SAT-MAC assay for identifying M. avium and M. intracellulare from clinical isolates were 100% (259/259). Consistent with the results of L-J culture combined PCR-sequencing, the coincidence rate of SAT-MAC assay in clinical sputum specimens was 100% (369/369) for M. avium and 99.19% (366/369) for Mycobacterium intracellular. The SAT-MAC assay can identify and distinguish M. avium and M. intracellulare rapidly and accurately. It may be suitable for use in clinical microbiology laboratories.
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: cuizhenling@163.com.
authors: Zhenling
authors: Z
authors: Cui
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: liyuanyuan19880804@163.com.
authors: Yuanyuan
authors: Y
authors: Li
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: songcheng0317@sina.cn.
authors: Song
authors: S
authors: Cheng
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: yanghua97065@163.com.
authors: Hua
authors: H
authors: Yang
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: lujunmeisakura@hotmail.com.
authors: Junmei
authors: J
authors: Lu
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: 1561819341@qq.com.
authors: Honglei
authors: H
authors: Zhu
authors: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, School of Medicine, Tongji University Shanghai, China. Electronic address: shtblab@163.com.
authors: Zhongyi
authors: Z
authors: Hu
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D003937:Diagnosis, Differential; D006801:Humans; D057230:Limit of Detection; D008163:Luminescent Measurements; D009162:Mycobacterium avium / Q000235:genetics* / Q000302:isolation & purification; D015269:Mycobacterium avium Complex / Q000235:genetics* / Q000302:isolation & purification; D015270:Mycobacterium avium-intracellulare Infection / Q000175:diagnosis* / Q000382:microbiology; D011237:Predictive Value of Tests; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021521:Ribotyping / Q000379:methods*; D013183:Sputum / Q000382:microbiology; D013696:Temperature; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Identification; Mycobacterium avium; Mycobacterium intracellulare; RNA isothermal amplification