The Variation Analysis And Establishment Of The Fluorescence Quantitative RT-PCR Method Of ORF3 Gene Of PEDV

Porcine epidemic diarrhea virus （PEDV） can cause porcine epidemic diarrhea （PED）, resulting in acute intestinal infectious diseases in sucking piglets. Because the enormous economic losses have been caused to pig industry, PEDV has become a common concern of global swine industry. It is significant to grasp the molecular characteristics and rapid diagnosis for controlling PED.In this study, ORF3 genes were cloned and genetic variation were evaluated. Meanwhile an Eva Green real time quantitative PCR（EG-qRT-PCR） was subsequently developed, which may provide a new technique for detecting PED.1 Variation analysis of PEDV ORF3 geneBased on the ORF3 gene sequence of PEDV CV777 in GenBank, the specific primer for amplifying ORF3 gene was designed. The 21 ORF3 genes of Sichuan, Chongqing, Fujian and Heilongjiang were cloned by RT-PCR, then compounded recombinant plasmid and sequenced, variation analysis.The 21 ORF3 genes were all 675 bp, which encode 224 aa. There were not nucleotides deletion and only mutations, which mostly was C→T、T→>C、G→>A、A→T. The amino acid mostly was Val→Ala、Phe→>Val、Ala→Thr、Asn→Ser. The nucleotide and deduced amino acid identity of 21 PEDV strains was 98.1%～100%,96.4%～100%, respectively. Compared with CV777, the identity of nucleic acid sequence and amino acid similarity was 95.9%～ 99.6% and 94.2%～99.6%. respectively. PEDVs of Sichuan, Chongqing, Fujian, Heilongjiang and reference strains were analyzed by phylogenetic analysis. They were separated into three groups:G1, G2, and G3. The 21 PEDV strains belonged to G1 group and showed a close relationship with Korea strains （2008-2012）, USA strains （2014）, Germany Strains （2014）, and partial other Chinese strains （2012-2014）, but differed genetically from British strains （Br1/87） and CV777 of G1 et al, attenuated DR13 of G2.2 The study of Eva Green real time quantitative PCR based on PEDVBased on the ORF3 gene sequence of PEDV of CV777 and attenuated DR13 in GenBank, a specific primer for EG-qRT-PCR was designed. The recombinant plasmids of virulent and attenuated PEDV were constructed and the concentration of virulent and attenuated, which was calculated by nucleic acid protein instrument measuring OD280 and OD260 value, was 4.79×10¹¹ copies/μL and 5.17×10¹¹ copies/μL, respectively.After optimizing conditions （primer concentration and annealing temperature）, the standard curve and melt curve of EG-qRT-PCR was established using the copy number over a range from 4.79×10³ copies/μL to 4.79×10⁸ copies/μL and 5.17×10³ copies/μL to 5.17×10⁸ copies/μL, respectively. Specificity was determined using PEDV and seven other viral pathogens of swine （TGEV, JEV, PoRV, PRRSV, PCV, PRV, PPV）, and only PEDV had amplification peak. The lowest limit of EG-qRT-PCR and RT-PCR of both virulent and attenuated PEDV was 10⁰ copies/μL,10³ copies/μL, respectively. The variability of the intra-assay and inter-assay were evaluated using standard solutions of each transcript for virulent （attenuated） PEDV, with coefficients of variation （CV） less than 0.56%（0.82%） and 1.05%（1.02%）, respectively.After validation, a total of 130 field samples in 2014-2016 were tested by the EG-qRT-PCR and RT-PCR method, with positive rate in 72.31% （94/130） and 58.46% （76/130）, respectively. Meanwhile, the positive samples were quantified between 5.13 ×10⁵ copies/μL and 6.31×10¹⁰copies/μL. By primitively detecting the viral loads of vaccines in the market, the bivalent inactivated vaccine was lower which the highest was 5.80×10² copies/μL, while the concentration of attenuated vaccine was up to 2.49×10⁸copies/μL. Besides, the viral loads of heart, liver, spleen, lung, kidney, intestinal lymph nodes and intestinal tissue were determined to 3.09×10⁵～4.47×10⁸copies/μL,8.91×l0⁴～1.38×10⁸copies/μL,0～ 6.61×10⁵ copies/uL,0～3.16×10⁶ copies/μL,4.79×10⁵～6.31× 10⁸ copies/μL,1.12×10⁷～ 9.12×10⁹copies/μL and 3.55×10⁹～3.16x10¹²copies/μL by EG-qRT-PCR, respectively. The results showed that the content of intestinal tissue was most, followed by the intestinal lymph nodes. The detection rates of spleen and lung was the lowest, which occasionally can be detected.