关键词: 分支杆菌, 结核, 聚合酶链反应, 增强化学发光

Abstract: Objective Establishing a rapid PCR-ECL method of high sensitivity and high specificity for detecting M.tuberculosis .Methods The 419bp fragment existed in gene coding for Pab in M.tuberculosis and M.bovis was used as target.The sequence of the primer was 5′-ACCACCGAGCGGTTCGCCTGA-3′,5′-GATCT-GCGGGTCGTCCCAGGT-3′.The PCR system was optimized to select the proper concentration of primer,dNTPs and MgCl₂ and the annealing temperature and the cycle number to get high sensitivity and specificity.The polymerization production of 419bp fragment was purified and was used as probe.The probe was labeled by HRP with direct enzyme labeling methed.After polymerization the product was denatured and dotted on the nitrocellulose membrane,the DNA was detected using enhanced HRP luminol-H₂O₂-chemiluminescence system in the molecular hybridization test.Imitative specimen was made by suspending different concentration of M.tuberculosis with sputum of healthy person. M.tuberculosis was incubated with Lowenstein Jensen culture media at 37℃ for 2 weeks.The incubation was killed by being suspended in 70% alcohol for 15 minutes.Guanidinium thiocyanate silicar method was used to purify chromosomal DNA of M.tuberculosis of imitative specimen.Results The concentration of primer was selected as 0.3μmol/L,that of dNTP was 150μmol/L,that of Mgcl₂ was 3.5mmol/L the annealing temperature was 69-70℃,the cycle number was 35.In the standard strains of M.tuberculosis, M.bovis, BCG, M.avium, M.intracellulare, M.godonae, M.ulcerans, M.smegmatis, M.phlei, M.vaccae, M.gilvum and 13 clinical separated strains,the PCR Results of M.tuberculosis ,M.bovis,BCG were positive.The sensitivity of PCR system was 5fg purified chromosomal DNA,which was about the DNA of 1 M.tuberculosis cell.The sensitivity of ECL was 0.5pg DNA.Less than 10 M.tuberculosis cells could be detected with the imitative specimen.Conclusion A rapid PCR-ECL method for detecting M.tuberculosis was established.The detection could be finished in one and half days.

Key words: Mycobacterium,tuberculosis, Polymerase chain reaction (PCR), Enhanced chemiluminescence (ECL)