Evaluation Of The Isothermal RNA Amplification Assay For Common Pathogenesis Nontuberculous Mycobacterium

Objective To establish and evaluate the isothermal RNA amplification assay foridentification of common pathogenesis nontuberculous mycobacterium.Methods Based on16S rRNA gene of M.avium, M.intracellulare, M.fortuitum,M.xenopi, M.scrofulaceum and23S rRNAgene of M.kansassi, M.marinum, RNAprobesand specific primers which incorporated the T7promoter sequences were designed. Thedetection undergoed successive cycles of amplification using T7RNA polymerase at42℃. Then we used22reference strains,5non-mycobacterium strains and259clinicalstrains to evaluate the SAT assay, at the same time, we compared the results with PCRgene sequencing. The369sputum samples were detected by the SAT assay, at the sametime, we compared the results with L-J culture+PCR sequencing. The samples withdifferent results between SAT and L-J culture+PCR sequencing were detectedrepeatedly.Results The SAT assay of M.avium, M.intracellulare, M.kansassi, M.fortuitum,M.xenopi and M.scrofulaceum probes had no cross-reactivity with21othermycobacterium and5common non-mycobacterium strains, and the specificity allreached100%. In order to exclude false positive, the positive samples of M.marinumhad to be detected repeatedly due to M.marinum probe had cross-reactivity withM.scrofulaceum. The sensitivity of SAT for identification of the seven commonpathogenesis nontuberculous mycobacterium were30CFU/ml,20CFU/ml,30CFU/ml,60CFU/ml,20CFU/ml,240CFU/ml,110CFU/ml respectively. The SAT results ofM.avium, M.intracellulare, M.kansassi, M.fortuitum, M.xenopi, M.scrofulaceum,M.marinum correlated with100%(259/259),100%(259/259),99.61%(258/259),99.61%(258/259),100%(259/259),100%(259/259),98.07%(254/259) of PCR sequencing inclinical isolates respectively. The SAT results of M.avium, M.intracellulare, M.kansassi,M.fortuitum, M.xenopi, M.scrofulaceum, M.marinum correlated with100%(369/369), 99.19%(366/369),99.73%(368/369),100%(369/369),100%(369/369),100%(369/369),100%(369/369) of the L-J culture+PCR sequencing in sputum samples respectively.Conclusions The SAT assay is a sensitive, specific and rapid method foridentification of M.avium, M.intracellulare, M.kansassi, M.fortuitum, M.xenopi,M.scrofulaceum, M.marinum. It may be used as a new molecular identification methodof this seven common pathogenesis nontuberculous mycobacterium.