Establishment Of RAPD Combined With QPCR For Detecting Mycobacterium Tuberculosis

Background:Tuberculosis is a chronic respiratory infectious diseases caused by mycobacterium tuberculosis.It seriously damages human health,has claimed the lives of hundreds of millions of people.But in recent years,with the increase of the population flow,the popularity of HIV infection,the emergence of multiple drug resistance loci,the popularity of TB rebound.Therefore,early、rapid and accurate diagnosis of mycobacterium tuberculosis infection in the treatment of diseases and dissemination has the vital significance.Traditional laboratory diagnosis of TB have low sensitivity and take a long time,operation is cumbersome.The immunological detection is affected by the subjects’ immune status,prone to false positives and false negatives.Molecular biological detection method such as fluorescence quantitative PCR,nested PCR,loop-mediated isothermal amplification and Xpert MTB/RIF test has greatly increased the efficiency of TB diagnosis.But the sensitivity of diagnosis of smear negative TB,extrapulmonary tuberculosis,pediatric tuberculosis and tuberculosis with HIV infection,is not ideal.Objective:With combined advantage of Random Amplified Polymorphic DNA(RAPD)and fluorescence quantitative PCR(qPCR),to develop a rapid and exact method to detect mycobacterium tuberculosis.Methods:According to the domestic and foreign literature,we designed a random primer for RAPD.We optimized the reaction condition of RAPD using H37Rv as a template and then 3 distinct and bright bands appear on the agarose gel electrophoretic plate.The DNA of 3 bands was extracted,purified and processed for DNA sequencing.The results correlated with the ones published on NCBI and proved that the matching rate with mycobacterium tuberculosis reached 99%.Now we choose one of them and design its specific primer and probe to process qPCR.The reaction condition of qPCR were optimized.Take the H37Rv and other negative control bacteria as templates,to analyze the sensitivity and specificity of qPCR and RAPD-qPCR.Take 152 clinical tuberculosis specimens and 26 non tuberculous clinical specimens(including sputum and bronchoalveolar lavage fluid,pleural effusion),respectively,with mycobacterium tuberculosis nucleic acid detection kit(PCR-fluorescence)and RAPD-qPCR methods.The reaction condition of RAPD-qPCR were optimized,the random primer IS986F-T2 was optimized for 288-F,288-R.Evaluate the difference of two methods detection rate.Results:qPCR can detect 30 pg/μl of mycobacterium tuberculosis.Whereas RAPD-qPCR increases the sensitivity to 3 fg/μl.And the Ct values are lower(6.58±0.19 to 14.95±0.36)between 30 pg/μl to 30 fg/μl of TB DNA in the samples by RAPD-qPCR,So that the results are stable and easy to judge.The detection of all other clinically common bacteria was negative by both qPCR and RAPD-qPCR,and this illustrates the specificity of around 100%.RAPD-qPCR of 150 specimens of tuberculous detection rate reached 27.33%,mycobacterium tuberculosis nucleic acid detection kit(PCR-fluorescence)detection rate was 22.67%,the difference is statistically significant.Two methods of detecting 26 non tuberculous clinical specimens results are negative,The specificity of two methods reached 100%.Conclusion:This method has higher detection rate for clinical mycobacterium tuberculosis infection,superior to the traditional mycobacterium tuberculosis nucleic acid detection kit(PCR-fluorescence).Its specificity of around 100%.It can be used in molecular biological detection of clinical tuberculosis...