A Single-step Duplex qRT-PCR for the Identification of Porcine epidemic diarrhea virus Genotypes
HE Hai-Jian1*, WU Yuan1*, LIU Zheng-Kui2, WANG Zhi-Peng2, CHEN Lin2, WANG Lei2, SONG Hou-Hui2, WANG Xiao-Du2**
1 School of Agricultural and Biological Engineering, Jinhua Polytechnic, Jinhua 321007, China; 2 College of Animal Science and Technology, Zhejiang A&F University, Hangzhou 311300, China; 3 Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Hangzhou 311300, China; 4 Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Hangzhou 311300, China
Abstract：Porcine epidemic diarrhea virus (PEDV) is one of the important pathogens that affect severe diarrhea in 3~10 day-old piglets (Sus scrofa domestica). Now the variant strain (GⅡtype) causes serious loss in farms. In order to differentiate the genotypes of PEDV, a rapid and accurate diagnostic method is established and used to detect PEDV from clinical diarrhea samples, those are advantageous to the prevention and control of these diseases. In this study, a pair of primers based the N-terminal domain of the PEDV S gene were designed, and 2 probes were designed in the light of the different regions of PEDV spike gene (S) from the variant strains (GⅡ) and the classical strains (GⅠ). The 5' end of those probes were individually marked by 5-carboxyfluorescein (FAM) (GⅡ) and 5-hexachloro-fluorescein hosphoramidite (HEX) (GⅠ) fluorescent signals. One single-step duplex qRT-PCR based on specific probes was established to distinguish different genotypes of PEDV by experiments on amplification conditions, specificity, sensitivity, repeatability, and so on. Firstly, the standard curves were established. Within the range of 10-1~108 copies/μL, this method showed good amplification efficiency for GⅡstrain (R2=0.9892) and GⅠstrain (R2=0.9914), and there was a good linear relation-ship between Ct value and concentration as well. Secondly, the sensitivity was 7.34 copies/μL or 2.3×102TCID50 (tissue culture infective dose)/0.1 mL for GⅡstrain, 4.32 copies/μL or 4.3×103 TCID50/0.1 mL for GⅠstrain, respectively. Thirdly, there was no cross-reaction between GⅡand GⅠstrains, in addition between PEDV and other pathogens about Porcine deltacoronavirus (PDCoV), Classic swine fever virus (CSFV), Transmissible gastroenteritis virus(TGEV), Porcine rotavirus (RV), Japanese encephalitis virus (JEV), Porcine parvovirus (PPV). Therefore, the specificity of this method was good. In the end, the coefficient of variation between and within groups was low, so the reproducibility was good. In detecting clinical samples by this method, the results were 100% consistent with virus isolation. This study are useful tools for quantifying viral load, detecting PEDV, and differentiating PEDV genotypes, and providing one technique for the prevention and control of PEDV.
何海健, 吴瑗, 刘正奎, 王志鹏, 陈琳, 王磊, 宋厚辉, 王晓杜. 鉴别猪流行性腹泻病毒基因型的一步法双重qRT-PCR[J]. 农业生物技术学报, 2019, 27(7): 1311-1321.
HE Hai-Jian, WU Yuan, LIU Zheng-Kui, WANG Zhi-Peng, CHEN Lin, WANG Lei, SONG Hou-Hui, WANG Xiao-Du. A Single-step Duplex qRT-PCR for the Identification of Porcine epidemic diarrhea virus Genotypes. 农业生物技术学报, 2019, 27(7): 1311-1321.
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