Rapid and accurate identification of mycobacterium tuberculosis complex by simultaneous detection of 16S rRNA and IS6110 sequence in FFPE samples

Backgrounds: Conventional biochemical tests for Tuberculosis M. (MTB) identification are not only timeconsuming but also none applicable for Formalin Fixed Paraffin Embedded (FFPE) samples. In this study, we developed a rapid multiplex qPCR (M-qPCR) assay for identification of MTB, which was confirmed sensitive and specific for FFPE sample in conventional surgical pathology works. Materials and methods: A single-tube M-qPCR was designed to simultaneously detect tuberculosis specific 16S rRNA hypervariable region and IS6110 sequence in the tuberculosis genome. Short length of PCR products of 110 bp and 134 bp were designed for 16S rRNA and IS6110 respectively. The specificity of this assay for MTB was also confirmed by a discriminative panel for MTB from Institutes for Food and Drug Control (NIFDC) combined with several other respiratory tract bacteria. Further we tested on 532 clinical FFPE samples which were suspected for infection with MTB. Results: The analytical sensitivity of our assay was 10 fg of purified mycobacterial DNA (estimated 4.6 fg each M. tuberculosis) and the specificity was found to be 100% in being able to distinguish from all the reference bacterials. For the clinical FFPE samples, our assay can increase about 10% sensitivity compared with Ziehl-Neeslen staining. Conclusion: This M-qPCR assay might be a quick, specific and cost-effective test for detecting of M. tuberculosis in conventional surgical pathology works.