Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons

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Abstract

A rapid real-time polymerase chain reaction (PCR) assay using molecular beacons has been developed for the simultaneous detection of wild-type and mutant strains of cytomegaloviruses (CMV) with respect to codon 460 of the UL97 gene has been developed. The molecular beacons were designed to complement the wild-type codon 460 or the mutant sequence arising from a single base-pair difference (point mutation). Discrimination between wild-type and mutant templates was demonstrated as the beacons did not generate fluorescence with their respective mismatch targets but only with those that they were designed to perfectly anneal with. Samples that harbor mixed populations of CMV could also be readily recognized. Applied to a small number of clinical samples, the retrospective screening by this assay are in general concordance with that obtained by PCR-RFLP. Using molecular beacons strategy, codon 460 mutation was detected in ten out o the total number of 40 samples, whereas the latter method identified nine samples as containing the mutation. The discrepant result arose from the genotyping of one clinical sample as mixed (containing both wild-type and mutant CMV strains) by molecular beacons but as wild-type by PCR-RFLP, suggesting that this real-time strategy is possibly more sensitive for mutation analysis.

Introduction

Prolonged antiviral prophylaxis or therapy is often associated with the emergence of resistant viruses [9]. Ganciclovir-resistant strains of cytomegalovirus (CMV) arise from mutations, mainly point mutations and also deletion of entire codons, in the UL97 or DNA polymerase gene, or both viral genes concurrently [7], [8], [12], [16], [17]. The UL97 gene encodes a protein kinase and a consequence of single or multiple amino acid changes to the protein is impaired monophosphorylation of ganciclovir leading to inability form ganciclovir triphosphate, the active form of the drug [5]. To date, the majority (94%) of all ganciclovir-resistant clinical CMV isolates contain mutations are clustered at three sites within the codon 400–707 region of UL97, with the majority of mutations occurring at codons 460, 594 and 595 [11].

A variety of molecular laboratory assays for diagnostic screening of ganciclovir-resistant CMV isolates have been developed, replacing phenotypic assays such as plaque reduction assay (PRA), that are frequently time consuming and laborious, and not amenable to automation. For instance, owing to the slow growth of the virus, a typical PRA for CMV resistance to ganciclovir requires at least three weeks before results can be reported. This laboratory diagnostic method is therefore not useful for timely management of patients with CMV infection. To date, molecular assays that have been developed for identification of CMV mutations known to confer ganciclovir resistance frequently involve conventional polymerase chain reaction (PCR) followed by restriction enzyme digestion and gel electrophoresis [6] and/or DNA sequencing [3].

The introduction of real-time PCR amplification that utilizes fluorogenic reporter probes has allowed for rapid and convenient sequence variation detection. Because hybridization of these probes to their complementary target is highly specific, real-time PCR techniques have already been demonstrated for the genotyping of acquired genetic alterations in human diseases and infectious agents [4], [13]. In the current study, we investigated the use of a real-time PCR platform using molecular beacons for the specific detection of antiviral drug resistance. The focus of our study was a point mutation (ATG to GTG) in codon 460 of the UL97 CMV gene. This frequently occurring mutation results in an amino acid substitution (methionine to valine) and has previously been shown by marker transfer studies to confer ganciclovir resistance in CMV [6]. Here we propose a real-time PCR strategy for the simultaneous detection and genotyping of CMV DNA for the wild type and mutant strains of clinical isolates of the virus.

Section snippets

Specimens and DNA isolation

DNA extraction was performed directly from blood samples using a modified phenol-chloroform method, and spectrophotometrically quantified as previously described. The 40 samples were from a cohort of pediatric solid organ transplant patients admitted to the National University Hospital, Singapore. Prior to coding for unbiased reexamination, all samples were analyzed for the presence of CMV by quantitative conventional PCR, as previously described [2]. This was followed by codon 460 mutation

Results

Nucleic acids isolated from 40 blood samples were subject to real-time PCR molecular beacon analysis for the presence of the A to G mutation in codon 460 of the UL97 CMV gene. Each sample was simultaneously analyzed in two separate reaction wells, each well containing PCR reaction mixture with either the wild-type beacon or the mutant beacon duplexed with primers and molecular beacon for β-globin. Fluorescence was measured during every cycle of the PCR using the appropriate excitation and

Discussion

A rapid real-time PCR assay using molecular beacons for the simultaneous detection of wild-type and mutant CMV viruses with respect to codon 460 of the UL97 gene has been developed. The molecular beacons were designed to complement the wild-type codon 460 or the mutant sequence arising from ATG to GTG change (methionine to valine amino acid substitution). We elected to perform the detection of wild-type and mutant CMV strains in each sample simultaneously but in separate reaction tubes in order

References (18)

  • S.K. Poddar

    Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus

    Mol Cell Probes

    (2000)
  • K. Abravaya et al.

    Molecular beacons as diagnostic tools: technology and applications

    Clin Chem Lab Med

    (2003)
  • M.M. Aw et al.

    Quantitation of peripheral blood cytomegalovirus DNA for monitoring recurrent cytomegalovirus retinitis in pediatric solid organ transplant recipients

    Pediatr Transplant

    (2000)
  • F. Baldanti et al.

    Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS

    J Virol

    (1996)
  • P.S. Bernard et al.

    Real-time PCR technology for cancer diagnostics

    Clin Chem

    (2002)
  • K.K. Biron et al.

    Metabolic activation of the nucleoside analog 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus

    Proc Natl Acad Sci USA

    (1985)
  • S. Chou et al.

    Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance

    J Infect Dis

    (1995)
  • S. Chou et al.

    A nine-codon deletion mutation in the cytomegalovirus UL97 phosphotransferase gene confers resistance to ganciclovir

    Antimicrob Agents Chemother

    (2000)
  • S. Chou et al.

    A deletion mutation in region V of the cytomegalovirus DNA polymerase sequence confers multidrug resistance

    J Infect Dis

    (2000)
There are more references available in the full text version of this article.

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