Lab Med Online.  2016 Jul;6(3):165-170. 10.3343/lmo.2016.6.3.165.

Nucleic Acid Extraction for the Quantification of Cytomegalovirus and Epstein-Barr Virus

• Sung H
• Hwang SH
• Koh YJ
• Kim MN

• KMID: 2308732
• DOI: http://doi.org/10.3343/lmo.2016.6.3.165

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.

Keyword

Nucleic acid extraction; Cytomegalovirus; Epstein-Barr virus; MIQE guideline

MeSH Terms

Cytomegalovirus*
DNA
Herpesvirus 4, Human*
Polymerase Chain Reaction
DNA

Figure

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  Fig. 1 Distribution of ΔCq (the difference between internal control Cq of sample and that of negative control) in terms of DNA purity.

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  Fig. 2 Correlation between copy number of CMV DNA in whole blood after manual extraction by QIAamp DNA Blood Mini kit and automated extraction by QIASymphony DNA Mini kit (Qiagen). Spearman's coefficient of rank correlation (rho)=0.946 (95% confidence interval [CI], 0.916-0.966, P<0.0001).

• 

  Fig. 3 Correlation between copy number of EBV DNA in whole blood after manual extraction by QIAamp DNA Blood Mini kit and automated extraction by QIASymphony DNA Mini kit (Qiagen). Spearman's coefficient of rank correlation (rho)=0.887 (95% CI, 0.812-0.933, P<0.0001).

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