Development and evaluation of novel molecular assays for the detection of economically important viruses for farm animals

• Main Author: Mckillen, J.
• Published: Queen's University Belfast 2012
• Subjects: 636.08944
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557963

In this project a number of novel molecular assays were developed and evaluated as potential tools for detection, genotyping and quantifying economically important viruses of farm animals. Molecular Beacon assays were developed for an African swine fever Virus, Aujeszky's disease virus, porcine circovirus type 2 and porcine parvovirus. The technology was found to be capable of the rapid, sensitive and specific detection of viral nucleic acids to an equivalent or better standard than the conventional peR assays used in our laboratories. 5'-conjugated minor groove binder assays were applied to the detection of African swine fever virus, porcine circovirus type 2, and porcine parvovirus, swine vesicular disease virus and foot and mouth disease. The performance of these assays was equivalent to those of published assays and, in the case of foot and mouth disease virus and swine vesicular disease virus, to the assays currently used in the UK reference laboratories. A 5'-conjugated minor groove binder assay and a Primer Probe Energy Transfer (PriProET) assay were developed for the differential detection, and potential genotyping of avian infectious laryngotracheitis virus. These assays were compared to TaqMan and conventional peR assays. The 5'-conjugated minor groove binder assay did not perform as well as the TaqMan assay in terms of diagnostic sensitivy. The PriProET assay proved to be equivalent to the TaqMan assay. Both assays were capable of detecting single base changes in the viral target and could therefore potentially function as genotyping assays. The porcine circovirus type 2 5'-conjugated minor groove binder assay was applied to the quantification of viral load in a vaccine study. The assay was capable of quantifying viral load in vaccinated and non-vaccinated groups of animals, in tissues, sera and faeces. The assay performed better in terms of efficiency than a SYBR Green I assay but would require further validation in order to establish the accuracy of quantification, particular if it was to be used for diagnosis of "histopatological" post-weaning multisystemic wasting syndrome. A loop-mediated isothermal amplification assay was applied for the detection of avian infectious laryngotracheitis virus. The assay had 1 Q-fold reduced analytical sensitivity when compared-to real-time peR assays but showed good diagnostic sensitivity. Initial evaluation of the use of Eva Green for possible pen-side detection was promising.