Development of Real-Time PCR Based Methods for Detection of Viruses and Virus Antibodies

Abstract: Quantitative real-time PCR (QPCR) technology has been very useful for diagnosis of viral diseases. QPCR has recently reached a level of sensitivity, simplicity, and reproducibility which allows a large number of samples to be screened rapidly, make it a suitable tool for the clinical virology diagnostics.In this thesis, broadly targeted and degenerated quantitative QPCR assays were used. A somewhat novel single-tube real-time reverse transcription-polymerase chain reaction (QRT-PCR), with takes advantage of ability of rTth DNA polymerase to reverse transcribe RNA in the presence of Mn2+ at elevated temperatures and includes protection against amplimer contamination by using thermolabile UNG, was developed. A new technique for diagnostic of recent viral infection by detection of viral immunoglobulin M (IgM) was also developed.In the first paper, a sensitive single-tube QRT-PCR for detection of enteroviral RNA in patients with aseptic meningitis was presented. In the second paper, a single-serum-dilution real-time PCR-based PIA (PCR-enhanced immunoassay), called quantitative PIA (QPIA), to detect enterovirus IgM for diagnosis of EV infection in patients with aseptic meningitis, was also developed. In the third paper, a broadly targeted, simple, single tube degenerated quantitative QPCR technique for detection of JCV, BKV and SV40 DNA was developed. A conserved region of the VP2 gene of JCV, BKV and SV40 was targeted. A false positive result due to contamination with commonly used SV40 T-antigen plasmids was therefore avoided. In manuscript four, the QPIA assay provide a rational strategy for detection of EV IgM, allows the use of viral antigens isolate from newly diagnosed Type 1 diabetes patients (T1D-EV-QPIA) to measured IgM against diabetogenic viruses in serum from newly diagnosed T1D children, siblings, and healthy children.To conclude, novel broadly targeted real-time PCR methods for diagnosis of entero- and polyoma viral infections were developed.