모세관 중합효소연쇄반응에 의한 결핵균검출

Abstract

Background :Recently, polymerase chain reaction(PCR) has become widely used in clinical laboratory for diagnosis of tuberculosis. However, the PCR has some ingenious problems, it is necessary to investigate the DNA purification, PCR processing and analysis of PCR product for using PCR as routine diagnostic tests. Method :PCR for detection of Mycobacterium tuberculosis DNA was performed on M.tuberculosis H37Rv strain and 77 acid-fast stainnegative specimens from clinically suspetted tuberculous patients, using hot-air capillarymthermocycler (FTC-2000, Daehan Medical Inc., Korea). Comparison study was made among three different methods of DNA extraction, such as proteinase K-chloroform, chaotropic agent-silica and bead-beating method. In addition various methods of PCR and of PCR product analysis, such as one-step amplification of a 123mbp sequence of IS6110 with primer P1-P2 and detection by agarose gel electrophoresis (IS611 0 method), two-step nested-amplification of 188-bp sequence of IS986 with primer sets INSHNS2 and Pt3mPt6 and detection by agarose gel electrophoresis(IS986 method), and amplification of 396-bp of mtp40 with biotin labeled primer PTl-PTBB and calorimetric detection using RNA probe(mtp?lO method) were evaluated for their value of clinical applicability. To minimize the loss of mycobacterial DNA during DNA extraction or interference with PCR which may cause false-neg&ive result, each sample spiked with M. tuberculosis suspension (lb bacilli per mL) . Result :Bovine serum albumin(500μg/mL), MgCl2 concentration(2-4 mM) and annealing temperature(40-50°C) eggected critically on capillary-PCR. By chaotropic agent-silica method, chromosomal DNA from a sample containing one organism of M. tuberculosis could be extracted in 1.5-2hrs and recovered in the initial reaction vessel with low risk of cross-contamination. Onthe other hand by IS986 method, 0.5fg of M. tuberculosis chromosomal DNA, in the other words one mucobacterial DNA per capillary-PCR vessel could be detected. Utilizing the above PCR methods, 26(33.8%) of 77 acid-fast stain-negative clinical specimens were positive for M. tuberculosis. Conclusion :In summary, we concluded that two-step nested-PCR of IS986 with primer sets INSI-INS2 and Pt3-Pt6 using hot-air capillary-thermocycler after DNA extraction by chaotropic agent-silica method appeared to be most useful for early and sensitive diagnostic method detedting M. tuberculosis in acid-fast stain-negative specimens.