Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus.

Abstract

The authors evaluated the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in comparison with virus isolation and detection by an indirect immunoperoxidase assay (VI-IPA). To determine the specificity of the assay, samples from 60 spleens, 45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and four kidneys, collected from pigs of various ages which had been slaughtered in abattoirs in Canada (a population free from CSFV), were tested. All the samples tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples were passaged in porcine kidney (PK) 15 cells and retested by both assays. All were found to be negative, giving a specificity of 100%. To determine the analytical sensitivity of the assay, a similar comparative study was conducted, using CSFV grown in tissue culture and tonsil tissues from a CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per 100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue culture fluids, compared to one infectious unit per ml by VI-IPA. To determine diagnostic sensitivity, three coded panels (two internal and one external), comprising 45 samples from 14 pigs, were tested. The diagnostic sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal panels. The results of the external panel, apart from two samples that were missed by both RT-PCR and VI-IPA, were found to be in total agreement. These two samples remained negative after amplification in PK15 cells. All the RT-PCR results were based on a single test whereas, for the VI-IPA results, positive results were obtained for five samples only after an amplification round in PK15 cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable improved detection of the virus in a shorter time period.