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


Autoři: Christian Berg aff001;  Martin B. Friis aff003;  Mette M. Rosenkilde aff002;  Thomas Benfield aff004;  Lene Nielsen aff003;  Hans R. Lüttichau aff001;  Thomas Sundelin aff003
Působiště autorů: Unit for Infectious Diseases, Department of Medicine, Herlev-Gentofte Hospital, University of Copenhagen, Herlev, Denmark aff001;  Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark aff002;  Department of Clinical Microbiology, Herlev-Gentofte Hospital, University of Copenhagen, Herlev, Denmark aff003;  Department of Infectious Diseases, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark aff004
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222053

Souhrn

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.

Klíčová slova:

Biology and life sciences – Molecular biology – Molecular biology techniques – Artificial gene amplification and extension – Polymerase chain reaction – Organisms – Viruses – DNA viruses – Herpesviruses – Human cytomegalovirus – Microbiology – Medical microbiology – Microbial pathogens – Viral pathogens – Anatomy – Body fluids – Blood – Physiology – Genetics – Gene amplification – DNA – Biochemistry – Nucleic acids – Research and analysis methods – Extraction techniques – DNA extraction – Medicine and health sciences – Infectious diseases – Viral diseases – Cytomegalovirus infection – Pathology and laboratory medicine – Pathogens – Engineering and technology – Equipment – Laboratory equipment – Filter paper


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