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Click here for detailed information about the Standard Operating Procedures (SPOs) used in ASF diagnosis
VIRUS DETECTION
PCR
 PCR technique allows the specific detection of ASF virus DNA by enzyme-based amplification of a short viral genome fragment defined by a specific primer set. The described ASFV PCR method uses a primer set designed in a highly conserved region of the viral genome, VP72, that ensures the detection of a wide range of ASFV isolates.
PCR is a rapid method, that can be performed in less than four hours, and highly sensitive, allowing the viral detection even before the appearance of clinical symptoms in the infected animals. Furthermore, the PCR techniques can be applied in any kind of clinical sample (cell culture supernatants, EDTA-blood, serum, tissue homogenates), and they are particularly useful for identifying virus DNA in pig tissues that are unsuitable for virus isolation or antigen detection because they have undergone putrefaction, or when there is good reason to believe that virus may have been inactivated before samples are received in the laboratory.
Download the conventional PCR procedure for ASFV detection (PDF format)
Download the Real-Time PCR procedure for ASFV detection (PDF format)
DIRECT IMMUNOFLUORESCENCE
Direct Immunofluorescence is a common technique used for detection of infectious agents in organs or viscera from suspected animals.
Specific antibodies against a virus are required. Those antibodies are conjugated with fluorescein isothiocyanate (FITC). A fluorescence microscope visualises positive reaction of antibodies-FITC with the antigen on tissue section.
This technique is used since 1968 for ASFV detection. It is able to detect viral antigens on samples from organs of infected animals.
Virus identification of ASFV by this technique is easy, sensitive and quick to perform.
 Malmquist and Hay made one of the most important advances in the study of African swine fever virus (ASFV) in 1960. They showed that ASFV was capable of infecting and replicating in primary leukocyte cultures from pig peripheral blood.
When the virus replicates in such cultures, there is generally a haemadsorption reaction due to adsorption of pig red blood cells on ASFV infected leukocytes. Cell lysis follows after 48-49 hours of haemadsorption. The importance of this discovery relies on its specificity because none of the other pig viruses are capable of haemoadsorbing in leukocyte cultures.
OF THE AFRICAN SWINE FEVER (ASF) SOLUBLE CYTOPLASMIC ANTIGEN
A highly specific cytoplasmic soluble fraction of African swine fever (ASF) infected cells, is employed as the test antigen in the diagnosis of the ASF virus by ELISA, IEOP and Immunoblotting. This antigen is composed of all the ASF infection proteins, showing high sensitivity and it does not produce false positive reactions ( Pastor et al., 1988; Escribano et al., 1989; Pastor et al., 1990).
Download procedure in PDF [95 KB]
ANTIBODY DETECTION:
IMMUNOBLOTTING (IB)
The Immunoblotting technique is based on the possibility to achieve essentially the complete and quantitative transfer of most proteins, from SDS-gels to filters, while preserving the antigenic properties of the proteins under denatured conditions.
The easy handling of the technique, its sensitivity and the easy conservation of the reagents, make this technique suitable for antigenic and immune response studies.
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INDIRECT ELISA
 The enzyme immunoassay ELISA test is a very useful technique; it is widely used for the serological diagnosis of different animal diseases. Some of the most outstanding characteristics of this method are high sensitivity, specificity indexes, high speed and low cost. Large populations can be studied in short periods of time, thanks to the automatic equipment that today is available. This technique also provides easy interpretation of the results.
 The IFI for ASF, is a common serological diagnostic method, where the sensitivity of the histological techniques and the specificity of immunological techniques are combined.
This method uses fluorescein isothiocyanate conjugated against the immunoglobulins present in a sample serum, showing either immunological or fluorescent activity.
Download procedure in PDF [95 KB]
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Diagnostic procedures