거대세포바이러스와 엡스타인바바이러스 DNA 정량검사를 위한 핵산 추출
Nucleic Acid Extraction for the Quantification of Cytomegalovirus and Epstein-Barr Virus
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배경: 거대세포바이러스(cytomegalovirus, CMV)와 엡스타인바바이러스(Epstein-Barr virus, EBV) 국제 표준물질이 이용 가능해짐으로써, CMV와 EBV 정량 PCR (qPCR)의 표준화는 향상되었지만, 핵산 추출 방법 또한 qPCR 결과에 영향을 줄 수 있다. 이 연구에서는 바이러스 qPCR 전에 DNA 농도와 순도 측정이 일상적으로 필요한지를 살펴보았으며, 수기법인 QIAamp DNA Blood Mini 키트(키트(Qiagen, USA)와 자동화법인 QIASymphony DSP DNA Mini 키트(Qiagen)를 비교하여 핵산 추출법을 도입할 때 고려해야 할 사항을 살펴보고자 하였다. 방법: DNA 정량과 순도는 QIAamp DNA Blood Mini 키트로 수기 추출한 후 EBV qPCR을 시행한 300개 전혈 DNA를 대상으로 하였으며, NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, USA)에서 측정하였다. 수기 핵산추출과 자동화된 핵산추출의 비교는 CMV qPCR이 의뢰된 72개의 전혈과 EBV qPCR이 의뢰된 54개의 전혈을 대상으로 하였다. CMV와 EBV DNA는 artus CMV 및 Artus EBV PCR로 정량하였다. 결과: DNA 농도와 EBV DNA 정량값 사이에는 일관된 상관성이 없었으며, DNA 순도와 Cq (내부대조물질의 Cq값에서 동일한 qPCR 배치에서 사용한 음성 대조검체에 첨가한 내부대조물질의 Cq값을 뺀 값) 사이에는 상관성이 없었다. 수기 핵산추출과 자동화된 핵산추출 후 CMV와 EBV qPCR값은 두 방법 간에 높은 상관 관계가 있었다(CMV, rho=0.946; EBV, rho=0.887). 두 방법 모두에서 CMV DNA가 양성이었던 29검체 중 8검체(27.6%)는 QIASymphony에서 추출한 핵산 정량값이 유의하게 높았다. 결론: 검사실에서 CMV와 EBV qPCR 전에 일상적으로 DNA 농도와 순도를 측정할 필요는 없을 것으로 판단되었다. 전혈에서 CMV와 EBV DNA를 추출할 때 자동화 기기인 QIASymphony법의 정량값이 수기법인 QIAamp DNA Blood Mini 키트보다 유의하게 높았다.
Abstract
Background: Availability of an international standard will improve the standardization of quantitative PCR (qPCR) for cytomegalovirus (CMV) and Epstein-Barr virus (EBV); however, nucleic acid extraction methods may affect qPCR results. This study was designed to determine whether routine measurement of DNA concentration and purity is required in qPCR for CMV and EBV. In addition, the performance of the automated QIASymphony DSP DNA Mini kit (Qiagen, USA) and the manual QIAamp DNA Blood Mini kit (Qiagen) in extracting DNA from whole blood samples was compared. Methods: The concentration and purity of 300 extracted DNA samples were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA). A total of 72 and 54 whole blood samples were tested by artus CMV and EBV qPCR (Qiagen), respectively. Results: No correlation was found between DNA concentration and EBV DNA load or between DNA purity and the PCR inhibition measured by ΔCq (the difference between internal control Cq of the sample and that of the negative control). Quantification of CMV and EBV DNA using the two extraction methods showed highly similar results (rho=0.946 and 0.887, respectively). Of the 29 specimens that yielded CMV DNA by both methods, however, 8 specimens (27.6%) yielded higher CMV DNA loads with QIASymphony. Conclusions: Routine measurement of DNA concentration and purity is not necessary for qPCR of CMV and EBV. The automated QIASymphony outperformed the manual QIAamp Blood Mini kit in extracting CMV and EBV DNA from whole blood samples.
참고문헌 (25)
- Razonable RR, "Management of CMV infection and disease in transplant patients", Herpes, 11, pp.77~86 (2004)
- Cherikh WS, "Association of the type of induction immunosuppression with posttransplant lymphoproliferative disorder, graft survival, and patient survival after primary kidney transplantation", Transplantation, 76, pp.1289~1293 (2003)
- Baldanti F, "Monitoring human cytomegalovirus in fection in transplant recipients", J Clin Virol, 41, pp.237~241 (2008)
- Gärtner B, "EBV viral load detection in clinical virology", J Clin Virol, 48, pp.82~90 (2010)
- Hodinka RL, "Manual of clinical microbiology", pp.1558~1574 (2011), ASM Press
- Gärtner BC, "Manual of clinical microbiology", pp.1575~1584 (2011), ASM Press
- "Nucleic acid based tests" (2015)[URL]
- Collaborative study to evaluate the proposed 1st WHO International Standard for human cytomegalovirus (HCMV) for nucleic acid amplification (NAT)-based assays, "Fryer JF", WHO ECBS, ~, pp.WHO (2010), BS[DOI]
- Collaborative study to evaluate the proposed 1st WHO International Standard for Epstein-Barr virus (EBV) for nucleic acid amplification (NAT)-based assays, "Fryer JF", WHO ECBS, ~, pp.WHO (2011), BS[DOI]
- Demeke T, "Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits", Anal Bioanal Chem, 396, pp.1977~1990 (2010)
- Clinical and Laboratory Standard Institute, "Molecular diagnostic methods for infectious diseases. Approved guideline. 2nd ed. MM03-A2", Clinical and Laboratory Standard Institute, ~ (2006)
- Clinical and Laboratory Standard Institute, "Quantitative molecular methods for infectious diseases. Approved guideline. MM6-A", Clinical and Laboratory Standard Institute, ~ (2003)
- Bustin SA, "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments", Clin Chem, 55, pp.611~622 (2009)
- Kraft CS, "Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective", Clin Infect Dis, 54, pp.1793~1797 (2012)
- Kim S, "evaluation of two automated systems for nucleic acid extraction of BK virus: NucliSens easyMAG versus BioRobot MDx", J Virol Methods, 162, pp.208~212 (2009)
- 이경선, "비인두면봉 검체에서 호흡기바이러스 검출을 위한 ExiPrep16 자동핵산추출기 평가", Laboratory Medicine Online, 3(4), pp.227~233 (2013), 대한진단검사의학회
- Huggett JF, "Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon", BMC Res Notes, 1, pp.70~ (2008)
- Riemann K, "Comparison of manual and automated nucleic acid extraction from whole-blood samples", J Clin Lab Anal, 21, pp.244~248 (2007)
- Espy MJ, "Real-time PCR in clinical microbiology: applications for routine laboratory testing", Clin Microbiol Rev, 19, pp.165~256 (2006)
- Costa C, "Comparison of two nucleic acid extraction and testing systems for HCMV-DNA detection and quantitation on whole blood specimens from transplant patients", J Virol Methods, 193, pp.579~582 (2013)
- Pillet S, "Comparative evaluation of the QIAsymphony RGQ system with the easyMAG", R-gene combination for the quantitation of cytomegalovirus DNA load in whole blood, 2012, pp.9, 231~
- Bravo D, "Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients", J Clin Microbiol, 49, pp.2899~2904 (2011)
- Gouarin S, "Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay", J Clin Virol, 29, pp.194~201 (2004)
- Vincent E, "Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries", J Mol Diagn, 11, pp.54~59 (2009)
- Forman M, "Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup", J Clin Microbiol, 49, pp.2703~2705 (2011)