ABSTRACT
The aim of the study is to establish and evaluate a RNA isothermal transcription-mediated amplification and realtime detection assay (RIARD-MF) for the identification of Mycobacterium fortuitum in clinical isolates.RNA probes and specific primers of reverse transcription and amplification for T7 promoter were designed based on the sequence of M.fortuitum 16S rRNA.The isothermal successive cycles of amplification were performed for real-time detection by using T7 RNA polymerase at 42 ℃.Five non-mycobacterium strains,20 Mycobacterium strains and 259 clinical strains were detected by the established assay to evaluate the specificity and sensitivity,and the results were compared with those of PCR sequencing.In the test of 5 non-mycobacterium strains and 20 Mycobacterium strains,only M.fortuitum was positive,and the remaining 24 strains of bacteria were negative,which was consistent with PCR gene sequencing.The sensitivity and specificity of RIARD-MF reached 60 CFU/mL and 100％.In the test of 259 strains of clinical isolates,5 strains were identified to be M.fortuitum,the remaining 254 strains were not identified to be M.Fortuitum,which was also consistent with PCR gene sequencing.Both the specificity and sensitivity reached up to 100％ in the detection of clinical isolates.It suggested that the RIAR-DMF established in this study is a specific,sensitive,accurate and rapid method for the identification of M.Fortuitum and it may be hopeful for rapid identification of M.fortuitum in clinical isolates.
ABSTRACT
The aim of the study is to establish and evaluate a RNA isothermal transcription-mediated amplification and realtime detection assay (RIARD-MF) for the identification of Mycobacterium fortuitum in clinical isolates.RNA probes and specific primers of reverse transcription and amplification for T7 promoter were designed based on the sequence of M.fortuitum 16S rRNA.The isothermal successive cycles of amplification were performed for real-time detection by using T7 RNA polymerase at 42 ℃.Five non-mycobacterium strains,20 Mycobacterium strains and 259 clinical strains were detected by the established assay to evaluate the specificity and sensitivity,and the results were compared with those of PCR sequencing.In the test of 5 non-mycobacterium strains and 20 Mycobacterium strains,only M.fortuitum was positive,and the remaining 24 strains of bacteria were negative,which was consistent with PCR gene sequencing.The sensitivity and specificity of RIARD-MF reached 60 CFU/mL and 100％.In the test of 259 strains of clinical isolates,5 strains were identified to be M.fortuitum,the remaining 254 strains were not identified to be M.Fortuitum,which was also consistent with PCR gene sequencing.Both the specificity and sensitivity reached up to 100％ in the detection of clinical isolates.It suggested that the RIAR-DMF established in this study is a specific,sensitive,accurate and rapid method for the identification of M.Fortuitum and it may be hopeful for rapid identification of M.fortuitum in clinical isolates.