Establishment Of A TaqMan-based Real-time Fluorescent Quantitative RT-PCR Assay For Identifying Porcine Epidemic Diarrhea Virulent And Attenuated Strains

Porcine epidemic diarrhea?PED?caused by the porcine epidemic diarrhea virus?PEDV?is an highly contagious,enteric disease characterized by diarrhea,vomiting,and dehydration and high mortality in suckling piglets.Since 2010,the PED caused by mutant strains has been outbreaks in China and caused huge economic losses.The live vaccines represented by the attenuated strains of CV777 are more common in clinical applications,which makes it impossible to rapidly distinguish between field strains and vaccine strains in laboratory diagnosis.ORF3 gene is an auxiliary gene of PEDV.The partial deletion of ORF3 gene can lead to attenuation of PEDV virulence,and it is also an important target for identifying virulent and attenuated strains of PEDV.In this study,ORF3 was used as a target to establish a TaqMan probe real-time fluorescent quantitative PCR?TaqMan FQ-PCR?method for identification of PEDV virulent and attenuated strains,and to provide an important technical support for the diagnosis and control of PED.The nucleic acid sequences of PEDV CV777 attenuated strain and mutant virulent strain?Hebei Quyang strain,HBQY?were used as templates,the complete gene sequence of PEDV ORF3 was amplified by RT-PCR.The obtained target gene fragments from strain CV777 or HBQY were connected with the pET-28a vector respectively,and the recombinant plasmid pET-28a-QY-ORF3 and pET-28a-CV777-ORF3 were constructed and identified by PCR,EcoR I and Xho I endonuclease digestion and sequence analysis.It was proved that the constructed recombinant plasmids contained the ORF3 genes of the mutant strain and the attenuated strain,respectively.According to the difference of ORF3 gene sequence between CV777 attenuated strain and virulent strain,a TaqMan probe primer for identifying virulent and attenuated strains and a pair of specific primers for PCR were designed.Using recombinant plasmid pET-28a-QY-ORF3 and pET-28a-CV777-ORF3 as the templates,the optimum reaction system was established by optimizing the reaction conditions,and the corresponding standard curve was established.The Real-time PCR standard curve and linear regression equation for the specific detection of PEDV virulent strain were obtained,and the PEDV attenuated strain nucleic acid could not be detected.Sequence analysis of the amplified DNA fragments showed that they were identical to the reference sequences.The established TaqMan FQ-PCR method could detect at least 3.7 copies of viral nucleic acid and 7.96 TCID₅₀0 of PEDV,and appeared no cross reaction with porcine transmissible gastroenteritis virus,rotavirus,hog cholera virus,etc.Detection result was positive when the amplification threshold?Cq value?<36,and was negatinve if Cq?36.The variation coefficient of inter repetitive test was less than 2.46%.The TaqMan FQ-PCR was further applied to detect 38 samples of the intestine and its contents,and fecal swabs from diarrhea piglets,and meanwhile PEDV was detected by indirect immunofluoresence assay?IFA?following virus blind passage three generations with Vero cells.The PEDV detection rate of TaqMan FQ-PCR and IFA were 31.58%?12/38?and 26.32%?10/38?,respectively.The results displayed that the TaqMan FQ-PCR was more sensitive than IFA.The above results showed that the TaqMan FQ-PCR based on PEDV ORF3 gene could identify and detect PEDV virulent strains rapidly and sensitively,which could provide important technical means for the rapid detection and prevention of PED.