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Virus Isolation in Tissue Culture
Probang Fluid
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Role in FMD diagnosis
The Reverse Transcription Polymerase Chain Reaction (RT-PCR) is an extremely sensitive method of detecting FMD viral genome. The detection of viral genome, rather than live virus or viral proteins, has the advantage that viable virus or intact viral antigen are not required. Together with high sensitivity comes the risk of false-positive reactivity. Very strict measures to prevent the possibility of contamination must be taken if RT-PCR is to be used as an additional technique to supplement antigen detection or virus isolation.
In the context of FMD diagnosis, RT-PCR is an additional and confirmatory test that can be performed, but is rarely used as the sole method on which a positive diagnosis is made, due mainly to the possibility of false-positive results. RT-PCR is no more effective than virus isolation in tissue culture at identifying FMDV in probang fluid samples from carrier animals. In fact some probang samples which are positive by virus isolation are negative by RT-PCR, presumably due to the presence of inhibitory substances.
Before a laboratory decides whether or not to introduce RT-PCR for FMD diagnosis, the following factors should be taken into account:
FOR AGAINST
• Rapid, sensitive and specific • Requires meticulous care,
strict working practices and
dedicated areas for different
stages of the procedure to
avoid the risk of false-
positive results.
• Can directly sequence the • Should directly sequence the
amplicon for amplicon if a diagnosis is
epidemiological studies to be based on RT-PCR to
eliminate false positive
result.
• Cheaper than maintaining • Estimates of cost must also
susceptible cell lines for take into account high costs
virus isolation of enzymes and dedicated
facilities required.
• Does not require presence • Virus isolation is always
of live virus in the sample required for strain
characterisation as the
nucleotide sequence does
not indicate the antigenic
properties of the virus.
• Presence of inhibitors (e.g., in
probang fluid) can lead to
false-negative results.
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