Study On The Rapid Detection Of Mycobacterium Tuberculosis Using A Molecular Beacon Real-time PCR And Commercial Application

ObjectiveTo establish a molecular beacon real-time PCR, which was specific, sensitive, standardized, and automated, for rapid detection of mycobacterium tuberculosis directly from clinical samples. To evaluate the application of the method and appraise the feasibility in monitoring the therapy effect of tuberculosis, and to develop commercial kit based on the established method. MethodsThe study was divided into three sections:1. Design of primers and molecular beacon probes:We select target gene of MTBC according to reference, and figure out the high conservative sequence of target gene in Genbank. Primers and probes were designed and synthesized, the activity of primer and probe was confirmed as well. We also confirmed the optimal method of DNA extraction.2. Establishment and evaluation of real-time PCR for detection of mycobacterium tuberculosis: A molecular beacon real-time PCR was established, and the PCR system was optimized in anneal temperature, the concentration of Mg²⁺ and Taq polymerase, the circulate parameter of real-time PCR, and so on.Evaluation of the real-time PCR:Specificity: 10 reference strains and 150 clinical isolates were diagnosed by the real-time PCRSensitivity: Templates contained 10⁶ to 10¹ DNA copies/mL were used to evaluate the sensitivity of the real-time PCR.Anti-interference: Two samples were detected simultaneously by the real-time PCR, one was inoculated with reference strains and mycobacterium tuberculosis, another one was inoculated with equivalent mycobacterium tuberculosis only.Reproducibility: The same sample was detected for 3 times and the results were compared.3. Evaluate the application of the real-time PCR and the prospect of commercial kit:272 sputum samples were collected from suspicious patients with tuberculosis. Molecular beacon real-time PCR, compared with acid-fast staining and cultivation, was carried to detect mycobacterium tuberculosis directly from the clinical samples. We compared the detection rate among three methods, and analyzed the cost/benefit. Finally, we appraise the prospect of the commercial kit.Results1. The IS6110 gene was selected; two primers and probes were designed using Primer Express software. A 107bp fragment was amplified using the primers (5'-gtcgcccgtctacttggtg-3', 5'-gcggattcttcggtcgtg-3') and probe (5'-ccgacggtgcgtaagtgggtgcgtcgg-3').2. The thermal cracking was the optimal method for genome DNA extraction, which was convenient, quickly and efficient.3. The PCR mixtures contained 1×PCR buffer, 3.0mmol/L Mg2+, 200μmol/L dNTP, 0.2μmol/L of each primer, 0.2μmol/L Molecular Beacon and 2.5U Taq polymerase. The themocycling conditions were: an initial denaturation of 94℃for 3 min, followed by 40 cycles of denaturation at 94℃for 20s, annealing at 52℃for 20s and extension at 72℃for 20s.4. The molecular beacon real-time PCR was specific, reproducible and good anti-interference; the detection limit for mycobacterium tuberculosis was 10³ DNA copies/ mL.5. 66.91% (182/272) sputum samples were positive by molecular beacon real-time PCR. The results indicated that the real-time PCR is more sensitive than acid-fast staining (10.75%) and cultivation method (43.01%).ConclusionThe molecular beacon real-time PCR established in this study could detect mycobacterium tuberculosis from clinical samples in 2h. It was a beneficial supplement of traditional methods, which could increase the accuracy and sensitivity of tuberculosis diagnosis.