Development of a liquidchip assay for simultaneous detection of Mycobacterium tuberculosis complex and M. avium and M. paratuberculosis

Abstract:

A microsphere-based LiquidChip assay was developed for rapid and simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium avium subsp. paratuberculosis (MAP). Four sets of oligonucleotide primers and probes which respectively targeted at IS6110, IS1081, IS1245 and F57 specific genes of mycobacterium were selected to establish the quadruplex assay. The quadruplex assay specifically identified target strains from a total of 54 strains of 13 species of mycobacterium, and 23 species of non-mycobacterium microorganisms were all detected as negative. The sensitivity on detecting cloned plasmid DNA by the quadruplex assay was 2.1×101?2.5×102 genomic copies or 0.06?0.74 fg DNA per reaction. The intra-assay and inter-assay variations of the quadruplex assays were both lower than 10%. The assay detected 75.6% (99/131) and 94.9% (37/39) positive from TB suspected human sputum samples and bovine tissue samples respectively, compared to culture methods that detected 38.9% and 53.8% positive from human and bovine samples respectively. The quadruplex assays also detected 24 MAP positive from 29 bovine blood specimens detected as positive by real time PCR specific for MAP. The tests on quadruplex mixed templates showed that the assay could identify mix infections. Clinical detection by the assay could be finished within one day, and the detection on purified DNA template could be completed in 2?3 h.

Key words:LiquidChip technique, M. tuberculosis complex, M. avium, M. a. subsp. paratuberculosis