Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR

• Main Author: Mohamed, Nahla
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Klinisk virologi 2006
• Subjects: Molecular biology; Microbiology; Virology; rTth DNA polymerase; UNG; QPCR; RNA virus; broadly targeted PCR; Molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7118; http://nbn-resolving.de/urn:isbn:91-554-6639-7

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.