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Toxigenic Clostridium difficile 배양에서 종동정과 독소확인 다중 PCR 검사의 적용

Implementation of Multiplex PCR for Species Identification and Toxin Typing in Toxigenic Clostridium difficile Culture

김미나|정재우|박숙자|백승미|성흥섭|장윤하
대한임상미생물학회 (Korean Society of Clinical Microbiology)|2009.03|Annals of Clinical Microbiology|제12권 제1호|pp. 11-16

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키워드

Polymerase chain reactionToxinClostridium difficile

초록

배경: Toxigenic C. difficile 배양검사(TCDC)의 민감도를 높이고 검사소요시간을 단축하기 위해 종동정과 독소유전자형 검사를 하는 다중 PCR 검사를 평가하였다. 방법: C. difficile 특이 tpiF 유전자와 tcdA, tcdB 독소유전자에 특이적인 시발체를 사용하여 다중 PCR를 실시하였다. 2008 년 1월 14일부터 3월 8일까지 검사실에 C. difficile 독소와 배양이 함께 의뢰된 528개 대변 검체에 대해 Tox A/B II로 직접독소검사(DT)와 TCDC 검사를 함께 실시하였다. TCDC 검사에서 전반부 288개 검체에서 배양된 분리주는 VIDAS C. difficile Toxin A/B (CDAB; bioMérieux, Marcy-l’Etoile, France)를 이용한 효소면역검사법과 PCR법을 동시에 시행하여 비교하였고, 후반부 240개 검체는 PCR 검사로만 독소생성을 확인하였다. 결과: 전반기 검체에서 29주의 C. difficile이 분리되었고 독소 양성률은 PCR 검사에서 65.5%, CDAB 검사에서 44.8%였다 (P＜0.05). 전체 528검체 중 DT+/TCDC+, DT+/TCDC-, DT-/TCDC+ 결과를 보이는 검체가 PCR을 이용하여 독소확인을 했을 때 각 32 (6.1%), 33 (6.3%), 10 (1.9%)개였다. 총 75개의 양성 검체 중 13.3%를 TCDC에서만 검출하였다. 전체 42주 의 독소양성 분리주 모두 tpi 양성으로 종동정을 확인하였고, 30주(71.4%)는 tcdA+/tcdB+, 12주(28.6%)는 tcdA-/tcdB+였다. 결론: TCDC에서 종동정과 독소형 확인에 다중 PCR법을 적용함으로써 민감도, 신속성을 향상시켜서 진단검사로서 수행 능을 개선할 수 있을 것이다.

Abstract

Background: We evaluated multiplex PCR for species identification and toxin typing to improve the sensitivity and turnaround time of toxigenic Clostridium difficile culture (TCDC). Methods: We performed multiplex PCR using primers targeting the species-specific gene, tpi, and the toxin genes, tcdA and tcdB. From January to March 2008, 528 stool specimens were tested with direct toxin assay (DT) using C. difficile Tox A/B II (Techlab, Blacksburg, USA) and TCDC. For 288 specimens from early study period, toxin production by C. difficile isolates of TCDC was measured by enzyme immunoassay with culture supernatants using VIDAS C. difficile Toxin A&B (CDAB; bioMérieux, Marcy-l'Etoile, France) and multiplex PCR with isolated colonies. For 240 specimens from late period, only multiplex PCR was used to test toxin production by the isolates. Results: During the early period, 29 C. difficile were isolated and their toxin-positive rates were 65.5% by PCR and 44.8% by CDAB (P＜0.05). Among 528 stool specimens, the results of DT+/TCDC+, DT+/ TCDC-, and DT-/TCDC+ were 32 (6.1%), 33 (6.3%), and 10 (1.9%), respectively, when tested with PCR. 13.3% of total 75 positive specimens was detected only by TCDC. Of the 42 toxigenic C. difficile isolates, all were positive for tpi, 30 (71.4%) were tcdA+/tcdB+, and 12 (28.6%) were tcdA-/tcdB+. Conclusion: TCDC using multiplex PCR for species identification and toxin typing is sensitive and rapid to be used as a routine diagnostic test.

참고문헌 (25)

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