중합효소연쇄반응을 이용한 결핵균과 비정형 결핵균의 감별동정
Mycobacterium tuberculosis and NontuberculousMycobacteria by PCR Assay
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배경: 본 실험에서는 신속하고 정확하며 결핵 및 비결핵균을 검출하고 동정할 수 있는 PCR 결과와 염색결과를 비교하여 그 검출률을 비교하고자 하였다. 방법: 255개의 항산성염색 양성 호흡기 검체를 대상으로 하였다. rpoB gene을 이용한 one-tube nested PCR로써 결핵균 및 비정형 결핵균에 모두 존재하는 360 bp와 결핵균에만 존재하는 190 bp의 부위를 증폭했으며 이 실험을 통하여 음성으로 판독되었던 34검체에 대하여 16S-23S rRNA spacer gene을 target으로 한 two-tube nested PCR을 시행하였다. One-tube nested PCR 결과 비정형 결핵균으로 판독된 검체를 대상으로 PCR-RFLP법을 이용한 마이코박테륨 동정을 시행하였다. 결과: Two-tube nested PCR을 시행하여 one-tube nested PCR에 의한 221 검체 약 87%의 결핵균 검출률을 237 검체 93%까지 향상시킬 수 있었다. PCR-RFLP법을 이용한 마이코박테륨 동정을 시행한 결과 임상적으로 의의가 있는 M. avium 3 검체와 M. intracellulare 1 검체가 동정되었다. 결론: 항산성 염색 결과 양성인 검체를 대상으로 PCR 검사법을 이용한 결핵 및 비정형 결핵균의 감별동정을 시행하는 것이 정확한 진단에 유용함을 확인하였다.
Abstract
Background: The purpose of this study was to evaluate the usefulness of direct PCR for a rapid detection of Mycobacterium tuberculosis (MTB), the differentiation of MTB from nontuberculous mycobacteria (NTM), and the identification of NTM species in acid-fast bacilli (AFB) smear-positive specimens. Methods: A total of 255 AFB smear-positive respiratory specimens were studied. For the differentiation of MTB from NTM, the bands of the 360 bp, which exists both in MTB and NTM, and the 190 bp, which exists only in MTB, were amplified using the onetube nested PCR targeting regions of the rpoB gene. The two-tube nested PCR targeting 16S-23S rRNA spacer gene was done with 34 specimens that were negative by one-tube nested PCR. The specimens that were tested positive for NTMs were subsequently subjected to PCR-restriction fragment length polymorphism (PCR-RFLP) analysis based on the rpoB gene for mycobacterial species identification. Results: Detection rate of MTB and NTM was 87% after the one-tube nested PCR. The detection range of MTB and NTM increased up to 93% after the two-tube nested PCR. The results of PCR-RFLP analysis identified those NTMs as M. avium and M. intracellulare. Conclusion: This result seems to suggest strongly that the PCR testing especially aiming for differentiation of MTB from NTM, and identificatin of mycobacterial species using AFB smear-positive specimens would be highly importnat in clinical settings for effective treatment of patients.
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