Establishment And Application Of Luminex Technology For Detection Of Porcine Diarrhea Viruses

Porcine diarrhea disease caused by the viruses was serious in most area.The disease cause highly infection to piglets and high mortality,with upto 100%.Moreover,multiple pathogen infection and synergistic infection are commonly seen in clinical.It is difficult to determine the main pathogen of the disease.Thus it is difficult to diagnose and control the disease.The viruses causing pig diarrhea disease are including Porcine epidemic diarrhea virus(PEDV),Porcine Deltacorona virus(PDCoV),Porcine transmissible gastroenteritis virus(TGEV),Porcine teschvirus(PTV),Bovine viral diarrhea virus(BVDV),Porcine Rotavirus(PoRV),Porcine sapelovirus(PSV),Porcine kobuvirus(PKV),Porcine astrovirus(PAstV),Porcine torovirus(PToV),Porcine Sapovirus(PSa V),etc.These viruses cause diarrhoea to a lesser degree and can cause severe diarrhea and death of pigs in the case of co-infection of multiple pathogens.Luminex xTAG technology is a detection methods using fluorescent-encoded microsphere as a carrier and different microspheres are encoded different numbers according to different fluorescent dyes.Unique TAG short sequences are bound to the microspheres to generate different color carrier for different sequence detection.Each coded microsphere can correspond to unique targets and realize the simultaneous detection of multiple molecules.PToV M gene,PDCoV M gene,PAstV RDRP gene,PKoV 3D gene,PSV RDRP gene,PTV 3D gene,Psa V RDRP gene,BVDV 5’URT gene,PEDV M gene,TGEV N gene and PoRV VP6 gene sequence were as target genes.According to the existing virus sequences in Gen Bank,DNAStar and Oligo7 software were used to design the PCR primer pairs of 11 pathogens:PTV,PAstV,PKV,PSa V,PToV,PDCoV,PSV,PEDV,PoRV,TGEV,and BVDV.A single PCR assay was performed using 11diarrhea pathogen detection primers,the obtained target sequence was recovered,and sequenced.Each primer sequence was selected.The primers were designed and synthesized using related software and then the primers were modified.Spacer C12 was added between the5’end of all upstream primers and the 3’end of the anti-TAG sequence,and the 5’end of all downstream primers was biotinylated(Biotin-)tag;selecting TAG microspheres complementary to anti-TAG sequence;hybr-idizing biotinylated tagged amplification product with TAG microspheres,and obtaining the hybridization product for liquid-phase chip detection,and detecting the detection result according to the MFI value.Samples were quantitatively tested.Based on a single detection system,multiple detection systems were mixed to establish a Luminex xTAG multiplex detection method.We continued to optimize the conditions and finally established the Luminex xTAG method for the simultaneous detection of 11 diarrheal viruses.The optimal of hybridization system and reaction conditions were as follows:20μL microsphere working solution,5μL PCR amplification product,and75μL SAPE report buffer;the results of optimal hybridization temperature was 42°C,and the best hybridization time was 30 min.The Luminex xTAG assay method specificity was test,which showed that each primer pair had good specificity and there was no cross-reaction between the primer pairs.The sensitivity test of the Luminex xTAG detection method showed that the minimum detection lines of this method were:PTV 3.12×10~3copies/μL,PKOV 2.92×10~3copies/μL,PDCoV2.79×10~3copies/μL,PSV 3.37×10~3copies/μL,PSa V 2.7×10~3copies/μL,PAstV 3.02×10~3copies/μL,PToV 2.65×10~3copies/μL,PoRV 2.57×10~3copies/μL,PEDV 1.74×10~3copies/μL,BVDV 2.41×10~3copies/μL,TGEV 2.75×10~3copies/μL;Specific tests showed that each primer pair had good specificity and there was no cross-reaction between the primer pairs.Using the Luminex xTAG multiplex detection method in this study,173 outbreaks of diarrhea in swine from 2017 to 2018 in Shanghai were tested.As a result,the positive detection rate of PoRV was 14.5%,the positive detection rate of PSV was 6.9%,the positive detection rate of BVDV was 6.4%,the positive detection rate of PEDV was 32.4%,and the positive detection rate of PKoV was 26.0%,PSa V positive detection rate was 11.6%,PToV positive detection rate was 2.9%,PAstV positive detection rate was 26.0%,TGEV positive detection rate was 3.5%,PDCoV positive detection rate The positive detection rate was 0.6%and PTV was 1.73%.Statistical analysis of the results showed the major virus types in different mixed infections.Among the 173 clinical samples,there were 7 viruses infected at the same time,including PKoV,PEDV,PoRV,PAstV,PSa V,PToV,and PSV.Quantitative analysis of the results speculates that the five viruses PSa V,PoRV,PAstV,PToV,and PEDV are the predominant viruses in the mixed infection sample.