Development of sensitive and specific real-time PCR assay for simultaneous diagnostics and genotyping of cytomegalovirus
Abstract number: p638
Vankova O., Morozova O., Ulanova T., Loparev V.
Human cytomegalovirus (HCMV) is an important pathogen capable of establishing lifelong persistent infections, which normally remain asymptomatic. Previous studies indicated that sequence variation among CMV strains frequently occurs even in highly conserved genes' regions. Genetic variation of functionally important genes may complicate CMV diagnostics.
The purpose of this work was designing primers and probes for simultaneously Real-Time PCR diagnostics and genotyping of CMV.
Materials and methods:
In this study 527 serum samples obtained from pregnant women and 3 samples from children with CMV congenital infection were used. Virus DNA was extracted by using the MagNA Pure DNA purification kit (Roche, Indianapolis, IN, USA). 4 sets of primers directed to IE2, gN, gO and gB CMV genes and 10 sets of probes for Real-Time detection with LidhtCycler instrument (Roche, Indianapolis, IN, USA) have been designed and evaluated. Genotyping was being carried out by sequencing analysis on ABI 3100 Avant Genetic Analyzer instrument (ABI, Foster City, CA, USA). Phylogenetic analysis of the nucleotide sequences was conducted with Clastal X, the Tamura-Nei substitution model, Grow tree based on Neighbour-Joining tree building method, and Maximum Parsimony method implemented in MEGA 3.0 package.
The Real-Time PCR analysis with IE2 gene detected CMV activity in 72 isolates among 527 analyzed samples The PCR tests with previously described primers to gN, gO and gB genes revealed 7, 1 and 2 positive samples accordingly. The PCR test sensitivity was defined with quantitative CMV control (ABI, Foster City, CA, USA). The sensitivity of PCR test on IE2 gene was 20 copies per 50 ml. The phylogenetic analysis of IE2 region sequences demonstrated that this region could be successfully used for virus genotyping. The Real-Time FRET test divided all analyzed samples into two groups, those that had a melting peak like laboratory strain Davies and those that had a melting peak like Towne and AD169 laboratory adapted strain. Two pairs of specific hybridisation probe covering two mutations inside IE2 gene's region were used to confirm it.
Nested PCR with primers to IE2 gene incorporated with Real-Time FRET analyse described here, provides a sensitive and specific assay for detecting CMV in clinical isolates. The IE2 gene can serve as a target for simultaneously detecting and genotyping of CMV using the Real-Time PCR opportunities.
|Session name:||XXIst ISTH Congress|
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