Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of porcine astroviruses

Abstract

Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1–5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously. The assay utilized specific primers for each genotype, producing fragments of 307, 353, 205, 253, and 467 bp, representing PAstV1–5, respectively. Our multiplex PCR assay amplified all 5 DNA fragments from single or mixed viral genomes without cross-reactions with other PAstV genotypes or other viruses in pigs. The limit of detection of the multiplex PCR assay was 5 × 102 copies/μL for PAstV1 and PAstV4, and 5 × 103 copies/μL for PAstV2, PAstV3, and PAstV5. We examined 76 pig fecal specimens with our multiplex PCR assay. PAstV was detected in 36 of 76 (47.4%) samples; ≥2 PAstVs were found in 20 of 76 (26.3%) samples. The multiplex PCR assay results were essentially the same as the results using a monoplex PAstV PCR assay, with a coincidence rate of >96%. Our multiplex PCR method provides a simple, sensitive, and specific detection tool for PAstV detection and epidemiologic surveys.