Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus

Abstract

A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). The universal primers and specific TaqMan probes for each of the three genotypes, genotypes 1, 2, and 3, were designed within the 3'-UTR of the CSFV. Non-CSFV swine virus and clinical samples from specific pathogen-free (SPF) pigs were both demonstrated to be CSFV-negative by RT-MRT-PCR. The diagnostic sensitivity of RT-MRT-PCR was determined to be 1 viral copy/microl for each genotype of standard plasmid. For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. The intra-assay and inter-assay variations of RT-MRT-PCR were below 3% in all experiments. The sensitivity of RT-MRT-PCR was the same as the reverse transcription nested PCR (RT-nPCR) and higher than reverse transcription PCR (RT-PCR) and viral isolation from clinical samples. This assay was used further to evaluate the duration of viremia of wild-type CSFV in vaccinated exposed pigs. The results indicated that pigs vaccinated with the E2 subunit vaccine had longer viremia than pigs given the C-strain vaccine, which is compatible with the findings of previous studies. Thus, the new RT-MRT-PCR is a rapid, reproducible, sensitive, and specific genotyping tool for CSFV detection.

Fig. 1

Analytical and diagnostic specificity of reverse transcription multiplex real-time PCR. The HEX, Cy5, and FAM fluorescence were genotype 1 (black real line), 2 (green broken line), and 3 (red broken line), respectively. The x-axis represents cycle number. The y-axis is the amount of fluorescent signal detected. Sample 1: standard plasmid of genotypes 1, 2, and 3; samples 2–17: non-CSFV swine virus; samples 18–43: buffy coats and tissue emulsions from an SPF farm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

Diagnostic sensitivity and standard curve of reverse transcription multiplex real-time PCR based on a 10-fold serial dilution of standard plasmid, including plasmids of genotypes 1(a), 2(b), and 3(c).

Fig. 3

The linear correlations (R²) of C-strain (a), Q90-278 (b), and 83-19 (c) strains was calculated between TCID₅₀ and viral copy number of reverse transcription multiplex real-time PCR.