Development and Validation of a Multiplex, Real-Time RT PCR Assay for the Simultaneous Detection of Classical and African Swine Fever Viruses

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.

Practical Guidance for Clinical Microbiology Laboratories: Viruses Causing Acute Respiratory Tract Infections

“…Multiplexing of molecular assays was traditionally restricted by the number of targets that could be efficiently amplified within a single reaction vessel (247)(248)(249). The earliest approaches were often batch-based assays that relied on a single nucleic acid extraction followed by one or more molecular assays.…”

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

“…Additionally, multiple tests are required to detect coinfections or to differentiate between species with clinically indistinguishable signs. Although multiplex real‐time RT‐PCRs have been described for many swine pathogens (Haines et al., ; Li et al., ; Rasmussen et al., ; Shi et al., ; Wernike et al., , ), the diagnosis of FMDV, SVDV, CSFV and ASFV still requires the performance of multiple single tests. Automated multiplex assays can potentially reduce labour cost and handling time.…”

“…User‐friendly reverse transcription insulated isothermal PCR (RT‐iiPCR) assays performed on compact, field‐deployable instruments with automatic display of “+” or “−” results have also been described for CSFV (Lung et al., ) and FMDV (Ambagala et al., ). Real‐time multiplex PCR assays have been reported for the detection of FMDV and CSFV (Shi et al., ; Wernike et al., ) and other targets (Haines, Hofmann, King, Drew, & Crooke, ; Li et al., ; Rasmussen et al., ; Wernike et al., ). Although RT‐iiPCR and real‐time RT‐PCR are rapid and highly sensitive, separate single‐target reactions are normally performed by the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease when testing for the presence of foreign animal disease pathogens.…”

Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

“…For this field test, the primers and probes have already been extensively evaluated (Hoffmann et al., ; Haines et al., ); therefore, this study started with an adaptation of the assay to the portable T‐COR4. The AgPath‐ID One‐Step RT‐PCR kit was used in this assay as it has previously been shown to give high sensitivity (Stephens et al., ).…”

“…An AgPath‐ID One‐Step RT‐PCR kit and primers and probes (Haines et al., ) were used in the real‐time PCR assay. The reaction contained 0.8 μ m ASFV primers, 0.6 μ m CSFV primers, 0.2 μ m FAM‐labelled CSFV probe and Cy5‐labelled ASFV probe, 1 μ l enzyme mix, 12.5 μ l 2 × buffer, 4 μ l water and 3 μ l viral nucleic acids.…”