Development of multiplex real-time PCR screens for the diagnosis of feline and canine infectious disease

SF600 Veterinary Medicine

Bennett, Susan ; Graham, Elizabeth

University:University of Glasgow

关键词: multiplex real time PCR, infectious diseases of cats and dogs;   

Others  :  http://theses.gla.ac.uk/5971/1/2014BennettMSc%28R%29i.pdf

来源: University of Glasgow

【 摘 要 】

Infectious disease is a significant cause of morbidity and mortality in cats and dogs.The diagnosis of the causative agent is essential to allow for the appropriate clinical intervention, to reduce infection spread, and also to support epidemiological studies which in turn will better the understanding of infectious disease transmission and control.The polymerase chain reaction (PCR) has replaced traditional detection methods as the gold standard diagnostic technique for many pathogens, however replacing traditional detection methods with panels of internally controlled multiplex real-time PCR screens designed along syndromic lines is not widespread.The aim of this thesis is to undertake the development phase of a wider project that intends to develop a broad panel of syndrome based real-time PCR multiplex assays for infectious diseases in cats and dogs.In order to achieve this, appropriate extraction and real-time PCR platforms and reagents were chosen.The aim of this project was to begin the development of five syndrome based panels of multiplex screens:- feline respiratory disease, feline conjunctivitis, feline anaemia, feline gingivitis, and feline and canine gastroenteritis.The multiplex assays were optimised and then evaluated through a series of experiments to determine the endpoint sensitivity, specificity and robustness of each multiplex in comparison to the single assays.The full optimisation and pre-clinical validation assessment of five multiplex assays was completed in the timescale of this project; the feline respiratory screen, the feline conjunctivitis screen and the feline/canine gastroenteritis 1, 2 and 3 screens.This study highlighted some of the difficulties that can be encountered when developing in-house multiplex real-time PCR assays.The main limitations were the lack of readily available positive control material, published assays and sequence data.The results of this study highlight that the development of an in-house multiplex real-time PCR diagnostic service can be at times difficult and occasionally time-consuming.A significant finding of this study was that PCR may not be as sensitive as virus isolation in the detection of feline calicivirus (FCV).It is hoped that further work including additional sequencing will aid in the development of a more sensitive FCV PCR assay.In-house multiplex real-time PCR should bring many advantages over current veterinary diagnostic assays.This will in turn aid in the treatment and clinical management of the animal, and increase our understanding of infectious disease in cats and dogs.