Detection Of Mycobacterium Tuberculosis With Random Primer Combined Specific DNA Fragment

Background:In recent years, the global TB incidence increased year by year. The bacteriological smear, culture are still the most commonly used methods to diagnose tuberculosis, but is low sensitivity and take long time. Detection of anti-tuberculosis antibody in sera is often used as an ideal diagnostic method.but the detection of antibodies in vivo of tuberculosis patients are not reliable and accuracy in larger research. In recent decades, the molecular biological technology such as nested PCR, real-time PCR, loop mediated isothermal amplification and Xpert MTB/RIF greatly improve the efficiency of the diagnosis of tuberculosis, but are still not entirely satisfactory because of their low sensitivity (about34.9%-85.7%), especially in pediatric tuberculosis, sputum smear negative tuberculosis, extrapulmonary tuberculosis and tuberculosis with HIV infection.Objective:To explore a more sensitive and specific molecular biology method for the diagnosis of tuberculosis disease, on account of the limitation of currently laboratory diagnosis of tuberculosis.Methods:Ten strains of Mycobacterium tuberculosis (2×105-2×106/ml) being popular in Jiangsu area were extracted. DNA was diluted according to1:10series dilution as templates to evaluate sensitivity. Specificity were evaluated using the related bacteria and Mycobacterium as control. Refer to the relevant domestic and foreign literature, twelve random primers were selected for RAPD (Random Amplified Polymorphic DNA).12tubes of RAPD products were run by electrophoresis in2%agarose gel stained with DuRed and visualized in UV light. The high brightness and clear bands were selected and excised from the2%agarose gel by means of Gel-out kit. The products were extracted and cloned into the TA cloning vector (pEASYTM-T5Zero) and sequenced. The sequences obtained were blasted against the NCBI database to verify whether a MTB DNA fragment or not. A pair of MTB specific primers were designed and synthesized according to the determined sequence. The specific primers based PCR were performed using RAPD products as temples. The specific MTB bands were showed by PAGE. The sensitivity and specificity was compared between this novel method and routine real-time PCR test in our hospital.Results:Three DNA sequences from bands amplified with the random primer IS986F, S535and IS986R and MTB sequence from BLAST-nr were highly homologous. MTB DNA diluted in1:105can be detected by random primer IS986F combined its internal specific primers with100%of specificity. MTB DNA diluted in1:105can be detected by random primer S535combined its internal specific primers with90.0%of specificity. While MTB DNA diluted in1:103can be detected by random primer IS986R combined its internal specific primers with80%of specificity. MTB DNA diluted in1:104can be detected by routine real-time PCR.Conclusion:A new method for the detection of MTB based random combined specific PCR has been developed and has more sensitivity than routine real-time PCR with100%of specificity.