Establishment And Evaluation Of Quantitative Nested Real-time Polymerase Chain Reaction Assay For Detection Of Mycobacterium Tuberculosis DNA

Objective: Molecular diagnosis based on genomic amplification methods hasbeen reported as an alternative to conventional culture for early detection oftuberculosis in sputum. Recent studies suggest that quantitative nestedreal-time polymerase chain reaction(NQRT-PCR) assay is a more sensitive andspecific way to detect Mycobacterium tuberculosis（MTB）DNA. We attempt toestablish NQRT-PCR assay, evaluate its sensitivity and specificity and identifyMTB DNA in clinical specimen from tuberculosis pleuritis patients.Methods:1、 The preparation of standards for MTB DNA detectionWe applied MPT64gene of H37vRV standard strains as a template,designed primer（sIF-1and IR-2）.After amplification、purification，we connectPCR product to pGM-T vector,through cloning and identifing,and finally got aplasmid containing a target fragment, formula of plasmid copy numberconcentration of plasmid (copies/ml)=quality∕molecular weight*6.02*102.The multiple proportion dilution of the above mentioned plasmid were selectedas the quantitative standards of MTB DAN positive references.2、Establish a nested PCR assay to detect MTB DNA We designed the internal and external set of primers, applicated nestedPCR in a two-step way, then used2%agarose gel lectrophoresis for qualitativeanalysis. The target fragments were241bp and201bp, respectively.3、Establish a real-time PCR to detect MTB DNAEstablished real-time PCR is a way which is using SYBR Green Ⅰfluorescent to mark and testing in PCR amplification, target fragment is201bp.The above inter primer was used as primer and made a standards followed theabove method.4、Establish quantitative nested real-time PCR to detect MTB DNAPleural effusion is a sample being a trace specimen of MTB,we applied anested PCR step before real-time PCR,in order to improve the positive rate andspecificity. It could be been accurate quantification at the same time.5、Quantitative detection of MTB DNA in sputum coming from pulmonarytuberculosis patients and pleural effusion coming from tuberculosis pleurisypatientsWe used three PCR methods to detect MTB DNA of those samples,andcompared the result. We enrolled24cases sputum from pulmonarytuberculosis, cases pleural effusion from tuberculosis pleurisy,75casessamples from the control group containing sputum and pleural effusion.6、We complete the correlation analysis between quantitative nestedreal-time PCR and inflammatory index、immune index、 the yield of effusion.Meanwhile, we made the correlation analysis among five etiology methods which include Culture、smear、the nested PCR、the real-time PCR andquantitative nested real-time PCR.Results:1、Successful construction of plasmid standards, we contrast two sequenceshomology in GeneBank （the recombinant plasmid and mycobacteriumtuberculosis strain H37Rv）, than is100%. We measure the concentration of theplasmid is7.09×1012copies/ml, by the multiple proportion dilution, accepteight gradient,7.09×102to7.09×109copy/ul. No non-specific amplifiedproduct was observed and melting curve was single–peak, providing100%specificity from1×103to1×107,with r value between0.99to1.0,providingstable amplified efficiency.All RT-qPCR were run on the Cycler III System(Roche, Switzerland).2、Positive sputum culture is the Gold Standard as diagnosis tuberculosis,wetested24cases of MTB DNA of sputum from pulmonary tuberculosis patients.The Sensitivity、specificity、 positive predictive value、negative predictivevalue of quantitative nested real-time PCR respectively were95.83%,61.54%,69.70%, and95.83%; However,taking medical thoracoscopy as the goldstandard, the sensitivity and specificity were75%and100%, respectively.Thepositive rate of three PCR methods are significant difference (P <0.05), thesensitivity of the quantitative nested real-time PCR is the highest. Between theCt value of two kinds of quantitative PCR, the quantitative nested real-timePCR is smaller. 3、Further more,we applied quantitative nested real-time PCR to detect MTBDNA of pleural effusion which came from27cases of the clinical diagnosis oftuberculosis pleurisy,with positive rate70.37%, and differently, the positiverate of the real-time PCR and the nested PCR were66.67%and11.11%,respectively.4、We did the correlation analysis between the quantitative nested real-timePCR and other related indexes.The results only showed a positive correlationbetween the quantity of MTB DNA of pleural effusion and ESR、AFB、cultule、the nested PCR、T–SPOT and PPD test with a significant difference.Concludes:1、We established the quantitative nested real-time PCR,which was confirmedwith a high sensitivity and specificity;2、Compared to nested PCR、real-time quantitative PCR and quantitativenested real-time PCR, we found the sensitivity of the quantitative nestedreal-time PCR is highest and could be considered as an alternative method for detecting MTB infection in clinical specimens.