Establishment Of A Porcine Rotavirus TaqMan Fluorescence RT-PCR Method And Virus Isolation、Identification

Infection with porcine rotavirus（Po RV）is one of the most common causes of acute dehydrating diarrhea in 1-week-old piglets.The global diversity of genetic variation among porcine rotaviruses,as well as the introduction of recombinant epidemic strains,has complicated porcine diarrhoea in recent years.Therefore,continual dynamic monitoring of epidemic strains and timely updates on epidemic status are critical for Po RV prevention and management.To effectively control the outbreak of Po RV in China,a Taq Man fluorescent quantitative detection method for porcine rotavirus was established.At the same time,a viral strain was isolated from Po RV-positive diarrhea samples,and the genetic diversity of its VP4,VP6,and VP7 genes was analyzed,and their pathogenicity was explored,with the goal of delivering data for the epidemiological and genetic evolution of porcine rotavirus.The main research contents are as follows:1.Establishment of a porcine rotavirus Taq Man fluorescence RT-PCR methodTwo pairs of primers and fluorescence quantitative probes were designed for the construction of standard plasmids and the establishment of a real-time RT-PCR system based on the existing VP6 sequence of porcine rotavirus type A in Gene Bank in order to establish a more sensitive detection method for the detection of porcine rotavirus.The target fragment VP6 was cloned to the p MD-19T vector by RT-PCR reaction,the positive plasmid standard was constructed,and the porcine rotavirus Taq Man fluorescence RT-PCR reaction system was established using the positive plasmid standard as the template.The reaction system were optimized,and the specificity,sensitivity,and stability of the technology were assessed.The results revealed that the optimal annealing temperature of the established real-time RT-PCR reaction was 52°C,and the optimal primer concentration and probe concentration were 0.3μM.The optimized real-time RT-PCR reaction system was used to establish a standard curve with the equation Y=-3.346x+40.034,with an amplification efficiency of 99.005%and a correlation coefficient of 0.996.The detection limit of this method can be as low as 3.25×10¹ copies/μL,and its sensitivity is about 100 times that of ordinary RT-PCR detection techniques.This method was used to specifically detect porcine epidemic diarrhea virus（PEDV）,porcine kobuvirus（PKV）,pseudorabies virus（PRV）,transmissible gastroenteritis virus（TGEV）and porcine reproductive and respiratory syndrome virus（PRRSV）and it was found that the response only had an amplification reaction against Po RV and had good specificity.2.Isolation and identification of porcine rotavirusTo isolate and obtain the circulating strain of porcine rotavirus in Guangxi,the diarrheal disease material collected from large-scale pig farms in Guangxi was routinely tested for diarrheal virus,and the positive Po RV diarrhea samples were treated with pancreatic enzymes with a final concentration of 30μg/m L and inoculated into Marc-145cells for virus isolation.The results showed that blind passage to the fifth generation of cells occurred cytopathic changes such as large detachment,grape clustering and reticular aggregation.The isolated virus was identified as Po RV by RT-PCR,western blotting and indirect immunofluorescence,and it was named GX9579 strain.Subsequently,the VP4,VP6 and VP7 sequence of GX9579 strain were sequenced and analyzed,and the nucleotide homology of the VP7 sequence,VP4 sequence and VP6 sequence of the isolated strain and the VP7 sequence of the TM-a strain in Hubei Province,the VP4 sequence of the SCYB-C2 strain in Sichuan Province and the VP6 sequence of the He NNY-01 strain in Henan Province were 96.49%,95.3%and 97.15%,and the genotype was G9P[23]I5.The growth curve and viral titer of the isolated strain were measured,and the viral titer was10^7.46 TCID₅₀/m L.3.Pathogenicity experiments of porcine rotavirusA piglet infection experiment was carried out in this study to explore the pathogenicity of the isolated strain GX9579.Three 1-day-old piglets in the infected group who did not eat colostrum were fed 2 m L(10^7.46 TCID₅₀/m L)of GX9579 virus fluid,and three piglets in the control group were fed the same amount of DMEM culture medium.Within 12 hours of infection,the piglets were depressed,their body temperature increased,they began to show diarrhea symptoms,they discharged yellow watery loose feces,and their weight continued to decrease;After 32 hours of infection,the piglets refused to take food,fell to the ground,and excreted fishy and flocculent watery feces;Death from dehydration within 48 hours of infection;The mortality rate within 120 hours of infection was 66.67%.swabs were collected every 24 hours after infection,and the porcine rotavirus Taq Man fluorescence RT-PCR method established in this study was used to detect viral shedding in feces,and it was found that the virus shedding in feces was the highest after 24 hours of infection,up to 10⁵ copies/μL,and then gradually declined.Histopathological observation of duodenum,jejunum and ileum was performed by HE staining,and Po RV was localized by immunohistochemistry.The results of HE staining showed that compared with the control piglets,the duodenal and jejunal villi of Po RV-infected piglets were partially broken and shed,the arrangement of ileal villi was disordered and shortened,the intestinal villi epithelial cells were shed into the intestinal lumen,the villi propria lamina had severe hyperemia and hemorrhage,and edema appeared in the submucosa.Immunohistochemistry showed that the positive signal was distributed in intestinal villus epithelial cells and small intestinal glandular cells.In conclusion,a quantitative fluorescence detection method based on Taq Man probe for the detection of porcine rotavirus VP6 gene was designed,which not only has highly sensitive,good specificity and reproducible,but can also be used for a large number of clinical tests.The isolated GX9579 strain has strong pathogenicity to newborn piglets,which can cause diarrhea,dehydration and even death of piglets.GX9579 strain infects piglets with great damage to the morphology of piglets’small intestinal villi.It may also colonize in duodenum,jejunum and ileal villi epithelial cells and small intestinal glandular cells,thereby causing diarrhea processes.