sybr-green-i and Cytomegalovirus-Infections

Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use.. Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl2 and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products.. Optimal values for most studied primers were found to be 3 mM for MgCl2 concentration, 0.5 microM and 0.6 microM for primers concentration, and 55 degrees C for annealing temperature.. Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.

Topics: Benzothiazoles; Cytomegalovirus; Cytomegalovirus Infections; Diamines; DNA Primers; DNA, Viral; Humans; Magnesium Chloride; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Temperature

Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 10(7)copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.

Topics: Antiviral Agents; Benzothiazoles; Cytomegalovirus; Cytomegalovirus Infections; Diamines; Drug Resistance, Viral; Ganciclovir; Humans; Mutation; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity

Topics: Benzothiazoles; Computer Systems; Cytomegalovirus; Cytomegalovirus Infections; Diamines; DNA, Viral; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Infections; Laboratories, Hospital; Nucleic Acids; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reagent Kits, Diagnostic

The aim of this study is to assess a real-time PCR technique on the LightCycler 2.0 with SYBR-Green I detection as compared to another real-time PCR method based on detection with FRET (fluorescence resonance energy transfer) probes for the quantification of CMV DNA.. The two real-time PCR methods were used to test plasma samples from immunocompromised patients with clinically suspected CMV disease, patients under follow-up without symptoms, and healthy adults. A standard curve for quantitative analysis by the SYBR-Green I method was performed with 10-fold diluted solutions of DNA from the CMV Towne strain (ATCC VR-977) cultured in MRC-5 monolayer. In addition, frozen samples from patients positive for CMV pp65 antigenemia were also analyzed and results compared using the two real time PCR methods.. The real-time PCR technique using SYBR-Green I on the LightCycler 2.0 was a highly specific, fast, simple and reliable test to quantify CMV; moreover, it was cost-effective.. Quantification of CMV DNA in plasma using this sensitive, fast, low-cost method was advantageous for the diagnosis and follow up of patients with opportunistic CMV infection, which are increasingly more frequent in our daily hospital clinical practice.

Topics: Adult; AIDS-Related Opportunistic Infections; Antigens, Viral; Benzothiazoles; Bone Marrow Transplantation; Cell Line; Child; Computer Systems; Cytomegalovirus; Cytomegalovirus Infections; Diamines; DNA, Viral; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Immunocompromised Host; Infant, Newborn; Neoplasms; Neutrophils; Opportunistic Infections; Organic Chemicals; Phosphoproteins; Polymerase Chain Reaction; Postoperative Complications; Quinolines; Reagent Kits, Diagnostic; Reference Standards; Sensitivity and Specificity; Viral Matrix Proteins; Viremia