Development of highly efficient protocols for extraction and amplification of cytomegalovirus DNA from dried blood spots for detection and genotyping of polymorphic immunomodulatory genes

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.

Fig 1. Overview of target genes and placement of all primers tested.

Yellow arrows symbolize CMV genes and green arrows primers tested (F = forward, R = reverse). Striped regions were not suitable for primer placement due to nucleotide repeats, GC content, or inter-strain variations. Dark grey regions are the target areas for amplification. The best performing primers are marked in light green color and bold text. All elements are in a 1:1 scale.

Fig 2. Extraction of CMV DNA from dried blood spots using four different methods.

(A-C) Extraction of CMV DNA from two 3.2 mm diameter filter paper discs punched from dried blood spots with different CMV concentrations. (D) Relative extraction yield of the different treatments compared to the reference treatment (treatment A). P values were determined by one-way ANOVA; ns = not significant. n = 3.

Fig 3. Amplicon yield of specific PCRs on diluted amniotic fluid samples with varying CMV concentrations.

Successful amplifications are marked with black dots (●) and unsuccessful amplifications with white dots (○). Amplification of UL146 at 100 cp/ml was positive in two of three assays and has been marked with a grey dot ($●$). Grey zones mark areas above and below the limit of quantification of the R-gene CMV kit. n = 3.

Fig 4. Specific PCRs for UL40, UL111A, UL146, and US28 on DNA extracted from DBSs.

PCR amplification from positive sample (amniotic fluid, 100,000,000 cp/ml, Pos.), treatment D extractions from filter paper discs (1:10, 1:100, and 1:1,000 dilution; 8,500–850,000 cp/ml), and no template control (NTC). Gel representation of capillary electrophoresis reads (S1 File). Green lines represent the alignment marker used for calibration of band sizes.