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



Majid Talkhabifard1, Naeme Javid2, Abdolvahab Moradi2, Amir Ghaemi2, Alijan Tabarraei3, *
1 Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran
2 Department of Microbiology, Golestan University of Medical Sciences, Gorgan, Iran
3 Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran


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© 2017 Talkhabifard et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran, Tel: +98 17 32422652, +98-17-32421289, Fax: +98 17 32440225, Email: alijant@yahoo.com


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.

Keywords: PCR-ELISA, Human cytomegalovirus, Glycoprotein B, Semi-quantitative detection, Genotyping.