Abstract
The human cytomegalovirus (HCMV) is an important human pathogen primarily affecting immunocompromised patients, like transplant recipients or HIV- infected individuals. Early diagnosis of cytomegalovirus (CMV) infection in high-risk patients is essential in order to start preemptive treatments. pol (UL54) gene encoding for HCMV viral DNA polymerase is a well-defined target for HCMV detection in clinical samples and identifying most highly conserved regions for primer design remains crucial. Though real-time polymerase chain reaction (qPCR) is a rapid and sensitive method for HCMV detection, failure to detect some HCMV strains due to primer and target mismatches have led the researchers to explore more sensitive and reliable methods. Hence, to understand the broader diversity of the pol mutations in HCMV and to specify the most suitable region for primer-probe design to be used in qPCR assay, we studied both nucleotide and amino acid heterogeneities in 60 HCMV positive samples that were collected to represent national mutational prevalence of pol gene of HCMV in Turkey. The test was designed with a new set of primers- probe for HCMV detection and quantification based on the sequencing data which revealed the most conserved region on the pol gene. Statistical probit analysis was applied on qPCR studies which revealed a 95% detection limit of 100 copies/mL. In addition, linearity, reproducibility, and precision of the new test were assessed for diagnostic purposes.
Highlights
The human cytomegalovirus (HCMV) is a double-stranded DNA virus that belongs to the Herpesviridae family
Submitted: 20 April 2015 / Accepted: 11 June 2015 immunocompromised patients and in the pediatric population, it is further shown that a prolonged periods of antiviral therapy with those drugs leads to the gain of resistance mutations on genes like DNA polymerase gene of HCMV and these mutations are the major cause of treatment failure [8,9]
Analyzes on different isolates resulted in the finding that specimens carry many variations that represent the mutational prevalence of pol gene of HCMV in Turkey compared to the AD169 strain (Figure 1)
Summary
The human cytomegalovirus (HCMV) is a double-stranded DNA virus that belongs to the Herpesviridae family. Antiviral drugs like ganciclovir (GCV), cidofovir (CDV), foscarnet (PFA) and more recent oral prodrug valganciclovir are anti-HCMV drugs approved for the treatment of HCMV-infected individuals [5,6,7]. These drugs are proven to reduce the severity of the disease especially in Submitted: 20 April 2015 / Accepted: 11 June 2015 immunocompromised patients and in the pediatric population, it is further shown that a prolonged periods of antiviral therapy with those drugs leads to the gain of resistance mutations on genes like DNA polymerase (pol) gene of HCMV and these mutations are the major cause of treatment failure [8,9]. Molecular diagnosis techniques like polymerase chain reaction (PCR) were commonly used for the early diagnosis, but accuracy of detecting HCMV was reported to be prone to failures due to primer and target mismatches since sequence variation among HCMV strains is very common even on highly conserved regions [11]
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