Establishment and evaluation of a fluorescence quantitative PCR assay for detection of Cytomegalovirus DNA from newborn dried blood spots

Objective To establish the method of fluorescence quantitative PCR for detection of Cytomegalovirus （CMV） DNA from newborn dried blood spots (DBS) and evaluate the limit of detection (LOD), precision, specificity and clinical application. Methods Primers and probes were selected from CMV-gB segment and standard plasmids were constructed to detect concentration of CMV strains.The LOD, within-run and between-run precisions were obtained by detection of 10-fold serial dilutions of CMV strains in DBS with quantitative PCR. This assay was further evaluated by detecting CMV DNA from 10 DBS with confirmed congenital CMV infection newborns and 120 DBS from healthy infants who were born within three days from September 2011 to February 2013. Results The LOD of CMV DNA was 4.44×103 copies/ml and the sensitivity was 2.66 copies/25 μl reaction system.The within-run and between-run precisions were 0.74% and 1.39% at a CMV strain concentration of 4.44×106 copies/ml, respectively, and 1.04% and 1.84% at a CMV strain concentration of 4.44×104 copies/ml respectively.Eight of ten newborns with congenital CMV infection and two of 120 neonatal DBS from healthy population were CMV-positive.All the positive products were verified by nested PCR and sequencing. Conclusions A method of fluorescence quantitative PCR were established successfully for CMV test in dried blood spots.This assay displayed the advantages of rapidness, good sensitivity and precision and could be used for large-scale screening for CMV infections.（Chin J Lab Med,2014,37:261-265） Key words: Cytomegalovirus infections; Cytomegalovirus; DNA, viral