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

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

Fig. (1)

Analytical sensitivity of CMV detection PCR-ELISA system. A. The results of the PCR-ELISA for 10-fold serial dilution of control plasmid containing gB2 sequence. The detection limit was 0.35 fg of plasmid DNA (~ 100 copies). The Pearson’s correlation coefficient between PCR-ELISA OD and logarithmic scale of plasmid DNA was 0.979 that represents the linear regression. B. Electrophoresis of the PCR products by 2% agarose gel with ethidium bromide staining (226 bp).

Fig. (2)

Analytical specificity of CMV detection PCR-ELISA system. Comparison the results of the CMV detection PCR-ELISA for specimens of; HCMV- negative human serum, urine samples, and HSV1- positive sample, and also 0.5 ng of each of the gB control plasmids, based on cut off value.

Fig. (3)

Genotyping models; Resulting from 10-fold serial dilutions of four gB types of control plasmids performed by genotype-specific probes in the genotyping system at hybridization temperature of 55°C.