Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

J Clin Virol. 2010 Aug;48(4):278-81. doi: 10.1016/j.jcv.2010.04.011. Epub 2010 May 31.


Background: Infection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5-2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).(7)

Objectives: Evaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards.

Study design: We compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol-chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR.

Results: The highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol-chloroform method in combination with the nested-PCR and 6mm, 3x3mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR.

Conclusion: These results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Blood / virology*
  • Cytomegalovirus / genetics
  • Cytomegalovirus / isolation & purification*
  • Cytomegalovirus Infections / diagnosis*
  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification*
  • Desiccation
  • Humans
  • Polymerase Chain Reaction / methods*
  • Reagent Kits, Diagnostic
  • Sensitivity and Specificity
  • Specimen Handling / methods*
  • Virology / methods*


  • DNA, Viral
  • Reagent Kits, Diagnostic