Description:
The aim of this project is to investigate the possibility of using isothermal amplification techniques in veterinary (on-site) diagnostics. We will try to develop a prototype test for one viral animal disease. Test specifications like sensitivity and specificity will be investigated. Fitness for purpose will be judged and if found in order the test will form a example for the development of similar test for other viral diseases.
Research objectives:
In the first phase we have tried to proof the principles of the test system. The combined action of a nicking endonuclease and a DNA-polymerase was tested with an artificial template based on the ISO-accredited sheep-and-goatpox real-time PCR test developed in our lab. In the same phase the detection system using a molecular beacon was tried out and evaluated. In the next phase the three-way-junction (3WJ) the formation of a T-DNA using two long oligonucleotides- was tested. In this phase we could demonstrate target independent amplification of a small DNA amplicon that could be detected using a molecular beacon.
Converting the system to a field situation however proved to be very difficult. The system was much too insensitive, and upon trying to improve sensitivity by introducing an exponential amplification step we also introduced aspecific reactions.
We decided to try to optimalize the system, and in the meantime to broaden our horizon by looking for other potential isothermal amplification reactions. In this pilot study the so called helicase-dependent-amplification (HDA) looked very promising. Because of the continuing problems with the 3WJ we decided to focus our attention on the HDA system in the final phase of the project.
Results and products:
In the final phase of the project we will try to develop a diagnostic test for sheep-and-goatpox virus (DNA virus) based on the HDA method. If succesfull other viral diseases, preferrably an RNA virus such as Avian Influenza, will be tested with the method. |
