Development and validation of reverse transciption polymerase chain reaction tests for classical swine fever virus detection
New methods for the detection of classical swine fever virus (CSFV) have been developed and validated. A variety of reverse transcription-polymerase chain reaction (RT-PCR) tests have been optimised and compared for ease of use and reliability, including comparisons with alternative methodologies. The methods offer advantages in terms of speed, specificity and sensitivity. Compared to previously described RT-PCR methods they are also simpler to use. For large scale usage, the tests would be best incorporated into systems for robotic sample processing. LRT-PCR has been applied to the detection of a very wide range of pathogenic micro-organisms including CSFV. CSFV causes a very serious and notable disease of pigs whose control is subject to legislation. Correct diagnosis is of the utmost importance and prescribed methods have to be very carefully validated. As well as diagnosis of the disease in the individual animal it would be very helpful to have sensitive methods for the early detection of disease in whole pig herds. This requires large numbers of samples to be analysed in a short time. Early RT-PCR methods were un-standardised and poorly validated. They involved multiple processing steps making them prone to sample cross-contamination. This rendered them unsuited to large-scale testing and prone to give false positive results. We have simplified RT-PCR testing for CSFV by incorporating several steps into a single process. The RT and PCR steps are now carried out in the same tube, as is a hybridization reaction involving fluorescent probes. This increases the specificity, reduces the risk of false positive results and enables automated reading of results. A series of ring tests were conducted to validate the procedures.
A standardised approach to phylogenetic analysis of classical swine fever viruses (CSFV) has been established and published. Using this methodology, a large number of CSFV isolates have been studied and conclusions drawn on the extent and geographic and temporal disposition of CSF viral diversity. A database has been established to hold background information on the viruses studied and the results of the phylogenetic analyses. This database will be maintained and updated with current information by the Community reference Laboratory for CSF (P5). The database will be accessible to the scientific community through the internet. The value of this is that when new outbreaks of CSF occur, diagnostic laboratories will be encouraged to use our methodology in typing their new virus isolates. By adopting this standardised approach they will be able to compare their results directly with those held in the database. This will give them insights into the likely origins of their outbreaks. When they deposit their new data in the database, this will expand the sum of information available for future users. A standardised phylogenetic methodology for the genetic typing of CSFV has been established. This involved selecting the most suitable regions of the genome for genetic comparison and the most reliable software for measuring viral similarity and producing reproducible phylogenetic trees describing the evolutionary relationships. The method was validated on a very large collection of viral isolates. These had been obtained in many different countries over the last 50 years. A comparison was made between the relationships predicted by this genetic approach and that suspected from conventional epidemiological studies. Additional data were generated on the genetic stability of the virus by experimental studies in pigs. An existing virus repository has been expanded to contain more than 600 isolates. Background information on these viruses has been stored in a specially designed computer programme, along with available genetic sequence data. This virus bank and the associated computerised database is held by the community reference laboratory for CSF in Hannover (P5) and will be maintained by them in the future. The database can be accessed by the scientific community via the internet.