Establishment of a Multiplex RT-PCR Method for the Detection of Five Known Genotypes of Porcine Astroviruses

Abstract

Porcine astroviruses (PAstVs) are prevalent in pigs worldwide, and five genotypes have been reported to circulate in China. However, little is known about the coinfection status of PAstVs. For differential and simultaneous diagnoses of these five genotypes of PAstVs, a multiplex RT-PCR method was established on the basis of the ORF2 gene of type 1 PAstV, and the ORF1ab genes of type two to five PAstVs. This quintuple PCR system was developed through optimization of multiplex PCR and detection sensitivity and specificity. The results showed that this multiplex RT-PCR method could specifically detect all the five PAstV genotypes without cross-reaction to any other major viruses circulating in Chinese pig farms. The detection limit of this method was as low as 10 pg of standard plasmids of each PAstV genotype. In addition, a total of 275 fecal samples collected from different districts of Guangxi, China, between April 2019 and November 2020, were tested by this newly established multiplex RT-PCR. Moreover, the sensitivity and specificity of monoplex and multiplex RT-PCR methods were compared by detecting the same set of clinical positive samples. The results revealed that PAstV1 (31/275), PAstV2 (49/275), PAstV3 (36/275), PAstV4 (41/275), and PAstV5 (22/275) were all detected, and dual (PAstV1+PAstV2, PAstV1+PAstV3, PAstV2+PAstV3, PAstV2+PAstV4, PAstV3+PAstV4, and PAstV4+PAstV5) or triple genotypes (PAstV1+PAstV2+PAstV3 and PAstV2+PAstV3+PAstV4) of coinfections were also unveiled in this study. The detection result of multiplex PCR was consistent with that of monoplex PCR. Compared with monoplex PCR, this multiplex PCR method showed obvious advantages such as time and cost efficiency and high sensitivity and specificity. This multiplex RT-PCR method offered a valuable tool for the rapid and accurate detection of PAstV genotypes circulating in pig herds and will facilitate the surveillance of PAstV coinfection status.

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

“…Although the positive rate of PAstV detection is high in many pig populations, infection by PAstVs is usually latent and can be easily missed under production conditions. 15,21 Our results also indicate that producers and diagnostic units should be vigilant about the prevalence of PAstVs. We detected ≥2 PAstV genotypes in 20 of 76 (26.3%) field samples.…”

“…This improvement may be attributed to factors that influence the efficiency of multiplex PCR assay, such as competition between reactions and appropriate annealing temperature. 7 A multiplex RT-PCR assay has been reported to detect the 5 PAstV genotypes, 15 but the sensitivity of the assay was lower, with a LOD of 10 pg/μL (~5 × 10 5 copies/μL), compared with the enhanced sensitivity of our multiplex PCR method. Similarly, 2 sets of multiplex real-time fluorescence quantitative PCR panels were established for these 5 genotypes with a lower LOD of single copies 30 ; however, our multiplex PAstV PCR assay has a significant advantage of low cost.…”

Isolation and Pathogenicity of a Chinese Porcine Astrovirus Type 5 Strain HNPDS-01 and Its Influence on Cecum Microbiota in Piglets

“…Phylogenetic tree for the ORF 2 gene was constructed with above method, of which red triangles indicate the ORF 2 of PAstV 5-HNPDS-01. can cause vomiting, diarrhea dehydration in piglets [10,34]. In this study, we isolated the PAstV 5-HNPDS-01 in LLC-PK from a clinical diarrheic sample and analyzed for its biological characteristics.…”

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways

“…They are divided into at least five distinct lineages, PAstV1 to PAstV5, based on their ORF2 sequences ( 20 – 22 ), and all of these genotypes were found to be prevalent in China ( 23 ). PAstV1 to PAstV5 are widespread in the pig population, and in recent years, incidences of coinfections with both dual and triple genotypes have also been shown to occur ( 22 , 24 ). The results of genetic evolutionary analyses suggest that PAstV may have crossed the species barrier between humans and other animals.…”