Rapid and Accurate Identification of Mycobacterium tuberculosis Complex and Common Non-Tuberculous Mycobacteria by Multiplex Real-Time PCR Targeting Different Housekeeping Genes

• Published in: Current microbiology 2012-11, Vol.65 (5), p.493-499
• Main Authors: Nasr Esfahani, Bahram, Rezaei Yazdi, Hadi, Moghim, Sharareh, Ghasemian Safaei, Hajieh, Zarkesh Esfahani, Hamid
• Format: Article
• Language: English
• Subjects: Antibiotics; Bacterial Proteins - genetics; Bacterial Typing Techniques - methods; Bacteriology; Biochemical tests; Biomedical and Life Sciences; Biotechnology; Clinical isolates; DNA Primers - genetics; DNA topoisomerase; DnaJ gene; genes; Genes, Essential; Genetics; Humans; internal transcribed spacers; Life Sciences; Melting; Melting curve; melting point; Microbiology; Mycobacterium - classification; Mycobacterium - genetics; Mycobacterium - isolation & purification; Mycobacterium avium; Mycobacterium tuberculosis; Mycobacterium tuberculosis - classification; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Mycobacterium tuberculosis complex; Polymerase chain reaction; Primers; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; ribosomal DNA; rRNA 16S; Sensitivity and Specificity; Spacer; Temperature effects; therapeutics; Tuberculosis; Tuberculosis - microbiology
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-012-0188-2

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T m) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.