Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

Majid Talkhabifard,Abdolvahab Moradi,Naeme Javid,Alijan Tabarraei,Amir Ghaemi

doi:10.2174/1874285801711010083

Majid Talkhabifard, Abdolvahab Moradi + Show 3 more

Open Access

https://doi.org/10.2174/1874285801711010083

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Abstract

Background:Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance.Method:During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system.Results:PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system.In comparison with sequencing method, genotyping system was confirmed with kappa index of 1.Conclusion:PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.

Highlights

Human cytomegalovirus is an opportunistic infection with global prevalence that can act as a dangerous pathogen in immunocompromised persons
To assess the sensitivity of the CMV detection PCR-ELISA system, according to the Cut-off value, obtained results of different concentrations of the glycoprotein B (gB) control were shown a limit of less than 0.35 femtogram (Optical density = 0.22) of the plasmid for the detection of the virus genome, that was approximately equal to the results of the electrophoresis of the amplified products on 2% agarose gel and stained with ethidium bromide
To calculate the intra-assay coefficients of variation for absorbance value in CMV detection PCR-ELISA system, 2 negative and 2 positive urine samples were examined in four tests individually in a run

Summary

Introduction

Human cytomegalovirus is an opportunistic infection with global prevalence that can act as a dangerous pathogen in immunocompromised persons. Transplant recipients, patients with acquired immune deficiency syndrome and congenital infected newborns are at risk of developing CMV disease [1]. This virus is most important cause of congenital viral infection that commonly leads to serious complications including encephalitis, chorioretinitis, 1874-2858/17 2017 Bentham Open. Accurate early detection and quantification of CMV infection in clinical specimens is necessary for prognosis and control of the CMV disease in high-risk patients This is especially important in newborns with congenital CMV infection, because of serious irreparable clinical complications of CMV disease in this group [2 - 4]. PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance

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Results

Conclusion