Multiplex RT-PCR for inclusive detection of major diarrheal viruses in pigs with multiple infections

Viral diarrhea severely damage pig industry, causing tremendous economic loss worldwide, especially during the porcine epidemic diarrhea virus (PEDV) outbreaks in recent years. The most common viruses causing diarrhea are PEDV, transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRV-A). In the past years, some new viruses like porcine kobuvirus (PKV) and porcine sapovirus (PoSaV) were discovered from pig intestinal contents. It is very difficult to make differential diagnosis on porcine viral diarrhea due to complicated pathogens in the gastrointestinal track and high similarity in clinical signs and pathological changes presented when diarrhea occurs. In this report, we developed a multiplex RT-PCR for rapid detection and differential diagnosis on viral diarrhea caused by the above mentioned viruses.

Specific primer sets targeted to TGEV and PEDV N genes, PoRV-A gene, and PKV and PoSaV polyproteins were designed based on highly conserved regions after extensive sequence analysis on target genes. Viral RNAs were extracted and subjected to reverse transcription using hexamer random primers. PCR amplification was performed with individual primer sets and different combination of all primers using total cDNA as a template. Amplified segments were in different sizes for each virus and vary from 200 bp to 1000 bp. Primer concentrations and annealing temperature were optimized to obtain a better amplification. Lately, limit of detection and specificity of this multiplex RT-PCR were carried out for validation purpose. Finally, around 400 clinical fecal samples were tested with this multiplex RT-PCR.

The results showed that the primer sets we designed functionally worked for all these five diarrheal viruses under the same amplification conditions. This multiplex RT-PCR could detect as less as 10 target cDNA copies and have no cross reactivity with other porcine viruses like porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, classical swine fever virus, pseudorabies virus, and reovirus. Field samples analysis using this multiplex RT-PCR revealed that viral diarrhea in pig herd in China was mainly caused by PEDV, with some extent of combination with other diarrhea viruses.

These results suggest that the multiplex RT-PCR is useful for rapid and accurate identification of five major pathogenic viruses in pigs with multiple infections.

Disclosure of Interest: None Declared.