역전사중합효소 연쇄반응-효소면역측정법을 활용한 장내바이러스 감염 진단
Application of a Diagnostic Method Using Reverse Transcription-PCR ELISA for the Diagnosis of Enteroviral Infections
1
조회
0
다운로드
1
인용
0
참고문헌
초록
배경 : 장바이러스(enterovirus)는 무균성 수막염의 주요 병 원체이다. 무균성 수막염이 의심되는 경우, 장바이러스에 대한 정확한 진단이 필수적이지만 기존의 세포배양을 통한 바이러스 의 분리법은 많은 시간과 노력이 필요하다. 그러므로 최근에는 장바이러스 진단을 위하여 신속하고 민감한 분자생물학적 방법 이 사용되고 있다. 방법 : 장바이러스의 유전자 중 잘 보존된 부분인 5’non-coding region에 대한 소식자를 이용하여 reverse transcription (RT)-PCR ELISA 키트를 제작하였다. 제작된 RT-PCR ELISA 키트는 RT-PCR법과 비교되었는데, 바이러스 분리주와 무균성 수막염이 의심되는 환자 검체에 대하여 민감도와 특이도를 측 정하였다. 결과 : RT-PCR ELISA법의 검출한계는 세포배양을 통한 바 이러스 분리법과 RT-PCR법보다 10-100배 높았으며, 또한 무 균성 수막염 환자의 임상 시료를 대상으로 한 RT-PCR ELISA 법의 검출률이 RT-PCR법 보다 7% 높게 나타났다(P=0.016). 결론 : 본 연구에서 활용된 RT-PCR ELISA법은 높은 민감 도와 특이도를 보여 장바이러스 진단법으로 이용할 것을 제안 한다.`
Abstract
Background : Enteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections. Methods : Reverse transcription (RT)-PCR ELISA kit for targeting 5’non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them. Results : The detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5’NCR (P=0.016). Conclusions : Our results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.
참고문헌 (20)
- Mayo M, "Virus taxonomy--1997", J Gen Virol, 79, pp.649~657 (1998)
- Pringle CR, "Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999", Arch Virol, 144, pp.2065~2070 (1999)
- Zell R, "Functional features of the bovine enterovirus 5′-non-translated region", J Gen Virol, 80, pp.2299~2309 (1999)
- Melnick JL, "Enteroviruses: polioviruses, coxsackieviruses, echoviruses and newer enteroviruses. in: Fields virology. 3rd ed", pp.655~712 (1996), Lippincott-Raven Publishers
- Kocwa-Haluch R, "Waterborne enteroviruses as a hazard for human health", Polish J Environ Studies, 10, pp.485~487 (2001)
- Roivainen M, "Enteroviruses: new findings on the role of enteroviruses in type 1 diabetes", Int J Biochem Cell Biol, 38, pp.721~725 (2006)
- Yagi S, "Spectrum of monoclonal antibodies to coxsackievirus B-3 includes type- and group-specific antibodies", J Clin Microbiol, 30, pp.2498~2501 (1992)
- De Clercq K, "Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA", J Virol Methods, 70, pp.7~18 (1998)
- Rotbart HA, "Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay", J Clin Microbiol, 32, pp.2590~2592 (1994)
- Egger D, "Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples", J Clin Microbiol, 33, pp.1442~1447 (1995)
- 김문보, "General -primer를 이용한 무균성뇌막염원인바이러스분석", 대한바이러스학회지, 26, pp.215~225 (1996)
- Taggart EW, "Enhancement of the AMPLICOR enterovirus PCR test with a coprecipitant", J Clin Microbiol, 36, pp.3408~3409 (1998)
- Ishiko H, "Molecular diagnosis of human enteroviruses by phylogenybased classification by use of the VP4 sequence", J Infect Dis, 185, pp.744~754 (2002)
- Zimmermann H, "Complete nucleotide sequence and biological properties of an infectious clone of prototype echovirus 9", Virus Res, 39, pp.311~319 (1995)
- Chomczynski P, "A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples", Biotechniques, 15, pp.532~537 (1993)
- Jones ME, "PCR-ELISA for the early diagnosis of invasive pulmonary aspergillus infection in neutropenic patients", J Clin Pathol, 51, pp.652~656 (1998)
- Chonmaitree T, "Comparison of cell cultures for rapid isolation of enteroviruses", J Clin Microbiol, 26, pp.2576~2580 (1998)
- Zoll GJ, "General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnosis routine andpersistent infection", J Clin Microbiol, 30, pp.160~165 (1992)
- Lomakina NF, "Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA", Arch Virol, 149, pp.1155~1170 (2004)
- Fong TT, "Enteric viruses humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools", Microbiol Mol Biol Rev, 69, pp.357~371 (2005)