Development And Preliminary Application Of TaqMan Fluorescence Quantitative Real Time RT-PCR Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is an acute intestinal infectious disease of pigs, which is characterized by diarrhea, vomiting and dehydration. The morbidity and mortality of newborn piglets are very high. The disease causes severe economic losses to swine-breeding industry around the world. Currently, PEDV can be diagnosed by immunefluorescence, ELISA, immunohistoche and RT-PCR method, which are time-consuming and low specificity and sensitivity, moreover early detection is also not available. Real time RT-PCR, a method with high accuracy and high sensitivity, can remedy almost the shortcomings of conventional method, and at the same time allows forhigh-throughput screening and quantification of viral loads using small volumes.PEDV is a kind of enveloped positive single-strand RNA virus, the M gene, a very conservative region, is generally used as a target gene for detection method. Taking PEDV CV777in the GenBank as reference, we designed a pair of primers clonning the full-length of M gene, and a pair of specific primers and TaqMan probe amplifying a196bp fragment. The681bp gene fragment (M) were amplified by RT-PCR from the feces of affected piglets. Sequence alignment analysis indicated that the homogeneities between M gene we gained and PEDV CV777’s can be as high as98%, and had more than97%homology with other PEDV strains published. We cloned the target gene into the pMD18-T vector, the recombinant plasmid identified correctly serves as standard plasmid, then we established a rapid Taqman fluorescent quantitation real-time RT-PCR methodology. After testing the specificity, sensitivity and repeatability of the method, the results showed that concentration of reaction templates and the fluorescent quantitative CT value were well linearly related. The established equation was y=-3.656gx+42.42with a correlation coefficient of0.999. The sensitivity of the real time RT-PCR was200copies/μL for plasmid DNA,100fold higher than conventional RT-PCR. Thirty diarrhea samples were collected, twenty-six samples and twenty-two samples were detected as positive case by real-time RT-PCR and conventional RT-PCR method respectively.Three PEDV variants which were FJ-9,YN-1and GD-01contained in intestines were respectively inoculated orally into two colostrum-deprived three-day-old PEDV negative piglets. Another two piglets inoculated with MEM as the control. All the infected piglets occured diarrhea, vomiting occasionally and severe dehydration, while the control group showed normal. Rectal swabs were collected every8hours daily (three cotton-tipped swabs per pig) after inoculation. Then the viral copies were measured by fluorescent quantitation RT-PCR developed by the author. Moreover, we had analyzed the rule of fecal shedding virus. The results showed piglets were detected fecal virus-positive first in the post-infection (PI)8-20h respectively, and since then had maintained a higher amount of virus in feces. We anatomyed the piglets when they were dead or dying, and pathological changes were mainly observed in the small intestine and the stomach. Intestines were transparent, thinning and lack of flexibility, distended with yellowish watery contents; stomachs were distented containing flecks of curdled undigested milk, and the walls were thinning. Tissues and organs were collected and detected by HE staining and immunohistochemical SABC method. The results of HE staining indicated that the small intestines of infected piglets were significantly shorter, thicker, lamina propria inflammatory cells infiltration, mainly lymphocytes; thinning of the intestinal serosa; villus epithelial cells were cuboidal, partially emptybubble, degeneration, necrosis and shedding; another change was the increase of goblet cells in the intestinal villi; almost no significant pathological changes were observed in the colon and cecum. In addition, gastric mucosa was shedding, submucosa was edematous and inflammatory cells infiltration. Immunohistochemical results showed that the positive signals were detected in duodenum, jejunum, ileum, the ileocecal valve, colon and stomach in the infected piglets. Positive signals presented in the cytoplasm of epithelial cells of the small intestinal villi and crypt epithelial cells, the presence of a small amount of virus in the colon villus epithelial cells and the lamina propria of intestinal villi and the gastric mucosal epithelium. Intestines and other tissues of the control group piglets had no obvious pathological changes, and no virus was detected as well.