This paper should be cited as: Talkhabifard, M. Javid, N. Moradi, A. Ghaemi, A. Tabarraei, A.

Optimization of PCR-ELISA in Detection of Human Cytomegalovirus Infection

 

Talkhabifard, M. (BSc)1 ,Javid, N. (MSc)2 ,Moradi, A. (PhD)3 ,Ghaemi, A. (PhD)4 ,Tabarraei, A. (PhD)5

 

1. MSc Student of Virology, Department of Microbiology, Golestan University of Medical Sciences, Gorgan, Iran

2. MSc of Microbiology, Department of Microbiology, Golestan University of Medical Sciences, Gorgan, Iran

3. Professor of Virology, Department of Microbiology, Golestan University of Medical Science, Gorgan, Iran

4. Assistant Professor of Virology, Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran

5. Associate Professor of Virology, Infectious Diseases Research Center, Department of Microbiology, Golestan University of Medical Sciences, Gorgan, Iran

 

 

Abstract

Background and Objective: Human Cytomegalovirus (CMV) is an important cause of congenital viral infection that can lead to serious diseases and complications in infants. Application of rapid, sensitive, and specific HCMV detection methods is necessary for congenital infection detection. We aimed to optimize the use of PCR and ELISA for detection of HCMV in infants.

Material and Methods: PCR–ELISA was performed by using specific primers and probe for detection of the HCMV glycoprotein B gene. First, the extracted DNA from urine samples and controls were labeled by digoxigenin during DIG-labeling PCR. After that, Biotin-labeled probe captured the DIG-labeled PCR products. The probe-PCR product hybrid is immobilized on a streptavidin-coated Microtiter plate, and detection was confirmed by proxidase-conjugated anti-digoxigenin antibody, and calorimetric substrate.

Results: The clinical Human CMV strains isolated from16 patients were detected by this method. The optimized PCR-ELISA method was able to detect less than100 copies of HCMV genome. There was no non-specific reaction.

Conclusion: PCR-ELISA can be applied as a sensitive, specific and reliable method for Semi-quantitative CMV detection in clinical samples.

Keywords: Cytomegalovirus, Glycoprotein B, PCR-ELISA, Semi-Quantitative

 

 

 

 

 

Corresponding Author: Tabarraei, A.

 

Email: alijant@yahoo.com

 

Received: 12 May 2013
Revised: 19 July 2013
Accepted: 20 Jul 2013