Abstract
A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.
Highlights
Classical swine fever (CSF) and African swine fever (ASF) are two contagious viral diseases of swine affecting both domestic and wild Suidae populations of all breeds and ages
Probes and optimised parameters for simultaneous detection of African swine fever virus (ASFV), Classical swine fever virus (CSFV) and an internal control Different combinations of a variety of primers and probes that detect ASFV or CSFV, including those described by Hoffmann et al (2005), King et al (2003) and McGoldrick et al (1999) and novel primers that target the VP72 gene of ASFV, were evaluated using a selection of real-time polymerase chain reaction (RT-PCR) kits
The spread of ASFV close to, and into, regions where CSFV is endemic in wild boar populations [33] and the emergence of CSFV in countries such as South Africa where ASFV is endemic [34], further substantiates the need for a simple differential diagnostic test that can detect both diseases without additional effort
Summary
Classical swine fever (CSF) and African swine fever (ASF) are two contagious viral diseases of swine affecting both domestic and wild Suidae populations of all breeds and ages. Both diseases are notifiable to the World Organisation for Animal Health (OIE) due to the high mortality rates associated with the acute forms of both diseases, and the potential for rapid spread and huge economic loss incurred by affected countries, along with impact for international trade. CSF is caused by Classical swine fever virus (CSFV), an enveloped single-stranded, positive-sense RNA virus belonging to the Pestivirus genus of the Flaviviridae family [1]. The approximately 12.3 kb CSFV genome encodes a single large open reading frame (ORF) flanked by highly conserved 59- and 39-untranslated regions (UTRs). CSFV has a worldwide distribution and, the virus has been eradicated in many countries, the disease was present in 46 regions or countries between January 2005 and August 2012 [3]
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