거대세포바이러스와 엡스타인바바이러스 DNA 정량검사를 위한 핵산 추출 = Nucleic Acid Extraction for the Quantification of Cytomegalovirus and Epstein-Barr Virus

다국어 초록 (Multilingual 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.

국문 초록 (Abstract)

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.

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