A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to 2012

Abstract

Since October 2010, clinical outbreaks of diarrhea in suckling piglets have reemerged in pig-producing areas of China, causing an acute increase in the morbidity and mortality in young piglets. Four viruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (GAR), and porcine circovirus 2 (PCV2), are the major causative agents of enteric disease in piglets. A novel multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of the four viruses in field samples from piglets. A mixture of four previously published pairs of primers were used for amplification of viral gene, yielding four different amplicons with sizes of 481bp for PCV2, 651bp for PEDV, 859bp for TGEV, and 309bp for GAR, respectively. The sensitivity of the mRT-PCR using plasmids containing the specific viral target fragments was 2.17×103, 2.1×103, 1.74×104 and 1.26×104copies for the four viruses, respectively. A total of 378 field samples were collected from suckling piglets with diarrhea in East China from October 2010 to December 2012, and detected by mRT-PCR. The PEDV-positive rates of the three years were 69.2%, 62.8% and 54.9%, respectively, suggesting that PEDV was a major pathogen in these diarrheal outbreaks. Taken together, all data indicated that this mRT-PCR assay was a simple, rapid, sensitive, and cost-effective detection method for clinical diagnosis of mixed infections of porcine diarrhea associated viruses.