RNA isothermal transcription-mediated amplification and real-time detection combined melt curve analysis for the identification of Mycobacterium intracellulare
LI Yuan-yuan1,2,CHENG Song2,LU Jun-mei2,HU Zhong-yi2,CUI Zhen-ling2
1.School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, China; 2.Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital,School of Medicine, Tongji University, Shanghai 200433, China
Abstract：The aim of the study is to establish and evaluate a RNA isothermal transcription-mediated amplification and real-time detection combined melt curve analysis method (RIARD-MCA) for the identification of Mycobacterium intracellulare in clinical isolates. Specific primers incorporating the T7 promoter sequence, and RNA probes was designed based on M. intracellular 16S rRNA gene and undergone successive cycles of amplification using T7 RNA polymerase at 42 ℃. Then we used 21 reference strains, 5 non-mycobacterium strains and 229 clinical strains to evaluate the specificity and sensitivity of the new assay. At the same time, we compared the results with the 16S rRNA and hsp65 gene sequencing. In this test, only M. intracellular was positive, with the sensitivity of 100% (63/63) and specificity of 100% (166/166), comparing to PCR gene sequencing. It’s suggested that RIARD-MCA is a sensitive, specific and rapid method in identification of M. intracellular, which may be used for rapid identification of M. intracellular in clinical isolates.
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