Establishment And Evaluation Of Taqman PCR Assay For Detecting Mycobacterium Tuberculosis

Tuberculosis (TB) is a kind of old chronic infectious disease of animals andhumans, which is mainly caused by Mycobacterium tuberculosis. According to thelatest report published by the WHO, in2009，there were9.4million incident caseseach year,14million prevalent cases,1.3million deaths among HIV-negative peopleand0.38million deaths among HIV-positive people. In our country, the number ofthe patients of active tuberculosis is about4.5million, and to the number of theinfected patients, the ranking is the second in the world only to that of India. ALL ofthis seriously impact the national economy and people`s live. New effective drugsagainst either replicating or latent bacilli, better vaccines and new diagnosticmethods are desperately needed to change and overcome this situation.In recent years,the sensitivity and specificity of existing diagnostic toolsthemselves are limited in determining the pathogens of TB, so we need to build arapid diagnosis method to guide the accurate medical treatment. Traditionallaboratory diagnostic methods have different aspects of disadvantages, sodiagnosis early is important to accurate treatment. Polymerase chain reaction (PCR)was progressively applied to detect a variety of respiratory trect infection pathogens.Currently, real-time PCR based on development of the traditional PCRtechnology.,such as Taqman PCR. It is integrated high sensitivity of PCR, highspecificity of DNA hybridization probe and exact quantitation of spectrum technical.Detection of target gene can appear in real time and the complete process can beaccomplished in1-2h. Therefore, the purpose of this study is to establishment andevaluation of Taqman PCR assay for detecting Mycobacterium tuberculosis.Targets and Methods: Clinical research objects sequentially selected43 non-tuberculosis patients of the first hospital of Jilin university and132tuberculosispatients of Changchun infectious disease hospital from September2010to Juanary2012. Collect sputum and bronchoalveolar lavage fluid (BALF) for testing. Thediagnostic standards of tuberculosis are based on sputum culture. According toforeign documents, synthetize primers and probes on the basis of genetic sequencesof MTC and M. tuberculosis. Constructing real-time fluorescence amplificationcurve. The optimization of the reaction system, specificity and sentitivity ofexperiments are done. Established and evaluation of Taqman PCR Assay fordetecting mycobacterium tuberculosis.The research results show that positive rates of sputum smear, bronchoscopylavage fluid smear and histological examination from tuberculosis, respectively are11.4%,17.4%,33.3%. The results of sputum and BALF detected by Real-time PCRare50%and66.7%, and false positive rate is0. After statistics analysis, real-timePCR is better than the other two methods, P=0.000(P<0.05), and detecting to BLAFis better than sputum,P=0.006(P<0.05). The results show this method can be usedto detect mycobacterium tuberculosis.