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
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.
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.
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.
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.
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