Development of a loop-mediated isothermal amplification (LAMP) test for diagnosis of bovine herpesvirus-1

Dr.med.vet Thesis by Deborah Peltzer

Bovine Herpesvirus typ 1 (BoHV-1) belongs to the subfamily of the Alphaherpesvirinae and is the causative agent of infectious bovine rhinotracheitis (IBR) an epizootic disease that Switzerland is free of since a major eradication program was performed in the 1980s and 90s. However, since most of the neighbouring countries are not free of IBR, re-introduction is a constant risk. 
The virus is able to establish a latent infection in the host upon a short acute phase. While serological tests such as ELISA and SNT currently used for surveilling the absence of BoHV-1, detect also latently infected animals, they are not useful during an acute outbreak as many animals may not have seroconverted yet.  Specific real-time PCRs are available but require expensive equipment, time and experience. In contrast to PCR, LAMP requires no DNA extraction and no thermal cycler, only a standard heat block, and results may be visualised e.g. by difference in colour of the reaction buffer. Hence, it even enables pen-side testing and has proven helpful in virus-diagnosis under field conditions such as in regions without access to laboratory equipment.
Therefore, a LAMP-based diagnostic tool for detection of BoHV-1 during the acute phase using easy to get sample material such as nasal swabs will be developed and evaluated. Experiments using primers designed in a genome region with particularly low GC-content provide most promising results. After optimisation of conditions and primer concentrations, optical read-outs and control by measuring final DNA concentrations revealed specific amplification of BoHV-1. In next steps, bovine nasal swabs spiked with BoHV-1 and real positive material from abroad will be used to test the suitability of the test under field conditions.
 

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