Development of a standardized quantitative real time PCR panel for respiratory viral diagnosis

Traditional viral diagnostics such as viral culture and various serological techniques tend to be slow, insensitive and labour-intensive. A large proportion of viral pathogens still go undetected using these techniques. This thesis concerns the development of a rapid and sensitive technique - standardized real-time quantitative PCR. Individual qPCR assays and synthetic plasmid controls were developed for 12 common respiratory viruses including influenza types A and B, parainfluenza (PIV)-l, -2 and -3, respiratory syncytial virus (RSV) A and B, metapneumovirus (MPV), human coronavirus (HCoV) 229E and OC43, human rhinovirus (HRV) and adenovirus. A reference gene assay using hypoxanthine phosphoribosyl transferase (HPRT) was also developed. A retrospective analysis on nasopharyngeal aspirates from patients previously diagnosed was conducted. The results demonstrated that the respiratory viral qPCR panel was sensitive, efficient, and had a large dynamic range of detection. Some cross-reactivity was noted for HRV with an enterovirus (coxsackievirus B3). HPRT proved to be a stable reference gene with the additional benefit that qPCR viral loads could be interpreted based on copy number per unit volume of specimen. One hundred culture negative specimens were examined and viral nucleic acid was amplified in 43 of them. There was a statistically significant relationship between viral load and whether or not the same specimen was positive by culture for influenza A , PIV-3, RSV A and B, HRV and adenovirus. Mean viral load was highest in patients with lower respiratory tract infections (LRTI) compared to those with fever or upper respiratory tract infections (URTI) and 95% confidence interval (CI) between these patients did not overlap. These results suggest that patients with more severe clinical disease had higher viral loads. This study highlights the developmental phase of a technique that has the potential to increase the detection rate of viral pathogens involved in respiratory illnesses.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat