| In resent years, tuberculosis has a rising tendency on global. In our country, tuberculosis is a common disease. But the prevalence rate of nontuberculous mycobacteria (NTM) in developed countries is higher than developing countries. With the prevalence of AIDS in the world and the increase of immunity impairment which is caused by medicine treatment, the incidence of NTM is rising rapidly.Since the clinical symptoms and X-ray image changes of NTM is similar as tuberculosis, the differentiation of NTM and tuberculosis is very difficult. Especially many nontuberculous mycobacteria are primary drug resistance to anti- tuberculosis drugs, so is it an urgent demand for a method or technique that can detect and differentiate the DNA of Mycobacterium (M.) tuberculosis and nontuberculosis mycobacteria.Now Ziehl-Neelsen staining still is a common used method in clinical detection for mycobacteria. This method has a lower sensitivity and can't differentiate dead bacteria and live bacteria. Culture method also has a lower sensitivity especially after a few days when the patient has administered anti-tuberculosis drugs.In our country in 1990s, it has a development of the study of rapid diagnosis for Mycobacterium infection by classical polymerase chain reaction (PCR), nest PCR, and so on. But they all can't detect and differentiate the DNA of Mycobacterium tuberculosis and nontuberculosis mycobacteria at the same time.Most of Homo sapiens specimens are formalin-fixed, paraffin-embedded historic specimens. This kind of archive specimens could be preserved for a long term. The PCR technique could amplify short DNA fragments and concept degraded DNA in a certain degree. Its high sensitivity could provide a good condition for the molecular biology study of paraffin-embedded historic specimens.On the common sections of paraffin-embedded historic specimens, it is difficult to differentiate Mycobacterium tuberculosis infection and nontuberculosis mycobacteria infection under a light microscope. There in no record about specific pathology character in pathology diagnosis as yet. We can only see simple description in the section of granulomatous inflammation: when observe the historic section, we can find that nontuberculosis mycobacteria is longer and thicker than Mycobacterium tuberculosis . Final diagnosis still need to based on the results of culture and biochemistry tests.In the first section of our study, we plant to detect clinical samples by the triplex polymerase chain reaction ( triplex-PCR ). We believe that it will provide pathological grounds to physicians for Mycobacterium tuberculosis infection and nontuberculosismycobacteria infection. In the secend section of our study, we plant to detect and analyse formalin-fixed, paraffin-embedded historic specimens by this method. In this way, we are going to have an ability of the retrospective study about mycobacteria infection. At the same time, we will provide a complement differentiation method for the pathological diagnosis of Mycobacterium tuberculosis complex infection and nontuberculosis mycobacteria infection.Part 1 Detection and identification of the DNA between Mycobacterium tuberculosis andnontuberculosis mycobacteria by triplex polymerase chain reaction techniqueObject To improve the specificity and sensitivity of polymerase chain reaction (PCR) technique for detecting and identification of DNA of Mycobacterium (M.) tuberculosis and nontuberculosis mycobacteria. Method three pairs of oligonucleotide primer were used in triplex-PCR. A 383bp DNA fragment encoding part of the 65KDa mycobacterial surface antigen, a 123bp fragment corresponding to a specific M. tuberculosis sequence which was the insertion sequence 6110 (IS6110) and a 268bp fragment for human -globin gene were amplified by triplex-PCR, respectively. Results The sensitivity of the triplex-PCR-electrophoresis for the DNA of mycobacteria was 0.6pg. The specific bands of 383bp and 123bp among the amplified DNAfrom M. tuberculosis, M. |
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