Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan® systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases. The use of QPCR as a tool for diagnostic virological and viral research laboratories has greatly increased in recent years. It often replaces conventional PCR and amplicon detection systems which are more complex and laborious, with a higher risk of amplicon carry-over contamination. The new QPCR methods presented here utilize broadly targeted primers and probes for rational and sensitive detection and quantification of variable RNA viruses. They take advantage of the dual properties, both RNA and DNA dependent DNA polymerase activities, of the rTth thermostable polymerase, and thermolabile UNG with dUTP to protect against inadvertent contamination of samples with amplimers. In paper one, a novel QPCR approach to detect and quantify human enteroviral (EV) RNA in patients with neurological disorders such as aseptic meningitis is presented. In the second paper, the development of a novel serological technique, quantitative PCR enhanced immunoassay (QPIA), for serodiagnosis of EV infection, is described. In paper three the subject is the development of a touch-down QPCR (TD-QPCR) for detection and preliminary genogrouping of norovirus (NV), a group of Caliciviruses. In paper four a rational, broadly targeted, system for detection of diverse influenza viruses, yet being able to discriminate between influenza A, B and C, is designed and evaluated. In the last paper, another rational broadly targeted system, for detection of corona viruses in humans and animals, is described. The technologies described in this collection of papers have common features. They are a platform for further development of diagnostic tools for screening and detection of viruses in known viral diseases, maybe also for discovering new viruses.

As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR). In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer. In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential. Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

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.