Abstract
Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.
Highlights
Cytomegalovirus (CMV) is a widespread human herpesvirus that establishes lifelong latent infection and is a major cause of birth defects as well as morbidity and mortality in immunocompromised individuals [1]
We show that this method can be used together with sensitive polymerase chain reaction (PCR) procedures designed to identify functional knockouts and polymorphisms of the four CMV genes UL40, UL111A, UL146, and US28
Extracting viral DNA from dried blood spots (DBS) for subsequent PCR analysis can be troublesome as the sample volume is often limited and the paper will retain some of the sample
Summary
Cytomegalovirus (CMV) is a widespread human herpesvirus that establishes lifelong latent infection and is a major cause of birth defects as well as morbidity and mortality in immunocompromised individuals [1]. Extraction and amplification of CMV DNA from DBSs. CMV infection may cause a multitude of debilitating and life-threatening organ diseases, most notably retinitis, colitis, and pneumonitis. Congenital CMV syndrome is a potentially underdiagnosed disease with an estimated 87% of newborns with CMV infection being born asymptomatic [2]. These children are at risk of developing permanent sequelae at later stages of their childhood which occurs in as many as 13.5% of the cases [2].
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