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

Heungsup Sung; Sang-Hyun Hwang; Young-Jin Koh; Mi-Na Kim

doi:10.3343/lmo.2016.6.3.165

Journal List > Lab Med Online > v.6(3) > 1057312

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Sung, Hwang, Koh, and Kim: Nucleic Acid Extraction for the Quantification of Cytomegalovirus and Epstein-Barr Virus

Original Article

Laboratory Medicine Online 2016; 6(3): 165-170.

Published online: 1 July 2016

DOI: https://doi.org/10.3343/lmo.2016.6.3.165

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

Heungsup Sung, M.D.¹, Sang-Hyun Hwang, M.D.², Young-Jin Koh, M.D.¹, Mi-Na Kim, M.D.¹

¹Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.

²Department of Laboratory Medicine, Center for Diagnostic Oncology and Hematologic Malignancy Branch, Division of Translational & Clinical Research II, National Cancer Center, Goyang, Korea.

Corresponding author: Heungsup Sung. Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, 88 Olympic-ro 43-gil, Seoul 05505, Korea. Tel: +82-2-3010-4499, Fax: +82-2-478-0884, sung@amc.seoul.kr

Received 28 April 2015 Revised 14 December 2015 Accepted 25 January 2016

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Nucleic acid extraction, Cytomegalovirus, Epstein-Barr virus, MIQE guideline

Figures and Tables

Fig. 1

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

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).

Table 1

Effects of DNA concentration on determination of EBV DNA load and DNA purity

	EBV DNA
	Negative (N = 164)	<3 log₁₀ copies/mL (N=80)	≥3 log₁₀ copies/mL (N=56)
DNA concentration (µg/mL)^*	39.1±31.7	48.4±26.0	47.7±22.4
A260/A280	1.93±0.28	1.92±0.13	1.98±0.21
Internal control Cq (ΔCq)	-0.14±0.82	-0.14±0.66	0.08±0.87

^*P=0.198 by One-way analysis of variance (ANOVA) with Bonferroni correction.

Abbreviation: ΔCq, internal control Cq of sample - internal control Cq of negative control sample.

Table 2

Comparison of ΔCq and DNA purity of EBV determined by qPCR

	A260/A280 ratio			P value^*
	Ratio < 1.7 (N = 25)	1.7 ≤ ratio ≤ 1.9 (N = 114)	Ratio > 1.9 (N = 161)	P value^*
ΔCq	0.01±1.13	-0.07±0.79	-0.15±0.74	0.52

^*By One-way analysis of variance (ANOVA) with Bonferroni correction.

Abbreviation: ΔCq, internal control Cq of sample - internal control Cq of negative control.

Notes

This article is available from http://www.labmedonline.org

References

1. Razonable RR, Emery VC. Management of CMV infection and disease in transplant patients. Herpes. 2004; 11:77–86.

2. Cherikh WS, Kauffman HM, McBride MA, Maghirang J, Swinnen LJ, Hanto DW. Association of the type of induction immunosuppression with posttransplant lymphoproliferative disorder, graft survival, and patient survival after primary kidney transplantation. Transplantation. 2003; 76:1289–1293.

3. Baldanti F, Lilleri D, Gerna G. Monitoring human cytomegalovirus infection in transplant recipients. J Clin Virol. 2008; 41:237–241.

4. Gärtner B, Preiksaitis JK. EBV viral load detection in clinical virology. J Clin Virol. 2010; 48:82–90.

5. Hodinka RL. Human cytomegalovirus. In : Versalovic J, editor. Manual of clinical microbiology. 10th ed. Washington, DC: ASM Press;2011. p. 1558–1574.

6. Gärtner BC. Epstein-Barr virus. In : Versalovic J, editor. Manual of clinical microbiology. 10th ed. Washington, DC: ASM Press;2011. p. 1575–1584.

7. U.S. Food and Drug Administration. Nucleic acid based tests. Updated on Jan 2015. http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm330711.htm.

8. Fryer JF, Heath AB, Anderson R, Minor PD. the collaborative study group. Collaborative study to evaluate the proposed 1st WHO International Standard for human cytomegalovirus (HCMV) for nucleic acid amplification (NAT)-based assays. WHO ECBS Report 2010. WHO/BS/10.2138.

9. Fryer JF, Heath AB, Wilkinson DE, Minor PD. the collaborative study group. Collaborative study to evaluate the proposed 1st WHO International Standard for Epstein-Barr virus (EBV) for nucleic acid amplification (NAT)-based assays. WHO ECBS Report 2011. WHO/BS/11.2172.

10. Demeke T, Jenkins GR. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem. 2010; 396:1977–1990.

11. Clinical and Laboratory Standard Institute. Molecular diagnostic methods for infectious diseases. Approved guideline. MM03-A2. 2nd ed. Wayne, PA: Clinical and Laboratory Standard Institute;2006.

12. Clinical and Laboratory Standard Institute. Quantitative molecular methods for infectious diseases. Approved guideline. MM6-A. Wayne, PA: Clinical and Laboratory Standard Institute;2003.

13. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55:611–622.

14. Kraft CS, Armstrong WS, Caliendo AM. Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin Infect Dis. 2012; 54:1793–1797.

15. Kim S, Park SJ, Namgoong S, Sung H, Kim MN. Comparative evaluation of two automated systems for nucleic acid extraction of BK virus: NucliSens easyMAG versus BioRobot MDx. J Virol Methods. 2009; 162:208–212.

16. Lee KS, Park DS, Yoon KH, Lee YJ, Cho JH. Evaluation of ExiPrep16 automated system for the extraction of nucleic acids from nasopharyngeal swabs for the detection of respiratory viruses. Lab Med Online. 2013; 3:227–233.

17. Huggett JF, Novak T, Garson JA, Green C, Morris-Jones SD, Miller RF, et al. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes. 2008; 1:70.

18. Riemann K, Adamzik M, Frauenrath S, Egensperger R, Schmid KW, Brockmeyer NH, et al. Comparison of manual and automated nucleic acid extraction from whole-blood samples. J Clin Lab Anal. 2007; 21:244–248.

19. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev. 2006; 19:165–256.

20. Costa C, Mantovani S, Balloco C, Sidoti F, Fop F, Cavallo R. 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. 2013; 193:579–582.

21. Pillet S, Bourlet T, Pozzetto B. Comparative evaluation of the QIAsymphony RGQ system with the easyMAG/R-gene combination for the quantitation of cytomegalovirus DNA load in whole blood. Virol J. 2012; 9:231.

22. Bravo D, Clari MÀ, Costa E, Muñoz-Cobo B, Solano C, José Remigia M, et al. 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. 2011; 49:2899–2904.

23. Gouarin S, Vabret A, Gault E, Petitjean J, Regeasse A, Hurault de Ligny B, et al. Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay. J Clin Virol. 2004; 29:194–201.

24. Vincent E, Gu Z, Morgenstern M, Gibson C, Pan J, Hayden RT. Detection of cytomegalovirus in whole blood using three different real-time PCR chemistries. J Mol Diagn. 2009; 11:54–59.

25. Forman M, Wilson A, Valsamakis A. Cytomegalovirus DNA quantification using an automated platform for nucleic acid extraction and real-time PCR assay setup. J Clin Microbiol. 2011; 49:2703–2705.

TOOLS

Similar articles