Establishment Of BAS-ELISA And Luminex Methods For Viral Pathogens Detection Of Pig Diarrhea Disease

Porcine epidemic diarrhea virus(PEDV), Transmissible gastroenteritis virus(TGEV) and Porcine rotavirus(PRV)) were the main viral pathogens that caused pig diarrhea in clinic. PEDV and TGEV are commonly seen and both are belonging to the coronavirus family. The symptoms of the sick pigs were characterized with severe vomiting, diarrhea and dehydration. All ages of pigs can be infected and the mortality can be as high as 100%.Most area and regions in our country have been reported the outbreaks of the diseases. Furthermore, mixed infection or secondary infection in clinic caused great economic losses to the pig industry. The diagnosis in laboratory include virus isolation and RT-PCR detection.However,the virus isolation was time consuming and difficult in manipulation. RT-PCR detection was easy to manipulate, while with a number of false positive.Here we want to establish a more sensitive and specific method for detecting these two pathogens. A double-antibody sandwich ELISA method combined with biotin-streptavidin system(BAS-ELISA) for detecting PEDV、TGEV was developed using Monoclonal antibody as capture antibody, biotin-labelled Ig G as secondary antibody. Furthermore, Luminex detection method was established using the optimized condition of BAS-ELISA. The study provides the basis for further multiplex detection of diarrhea pathogens.1. The establishment of BAS-ELISA for PEDV detectionHyperimmune serum was prepared by inoculating rabbits with PEDV, then rabbit-anti-PEDV Ig G was purified and labeled with biotin. Monoclonal antibody against PEDV S protein was used as solid phase capture antibody and recombinant PEDV S protein was used as coating antigen to establish the BAS-ELISA. The detection limitation of PEDV S protein using BAS-ELISA was 20 ng/μl. Three of twenty-five clinical samples with diarrhea symptom were positive using BAS-ELISA detection, Coincidence rate of detection of BAS-ELISA with RT-PCR was 100%.2. The establishment of BAS-ELISA for TGEV detectionHyperimmune serum was prepared by inoculating rabbits with TGEV, then rabbit-anti-TGEV Ig G was purified and labeled with biotin. Monoclonal antibody(m Ab) against the TGEV S protein was used as solid phase capture antibody and purified transmissible gastroenteritis virus was used as coating antigen to establish the BAS-ELISA. The sensitivity assay showed that the minimum virus amount for detection was 17.5 ng/μl. Two of twenty-five clinical samples with diarrhea symptom were positive using BAS-ELISA detection, Coincidence rate of detection of BAS-ELISA with RT-PCR was 100%.3. Development of Luminex assay for PEDV, TGEV detectionBased on the principle of x MAP liquid chip and the optimized conditions of BASELISA, PEDV S protein m Ab coupled to fluorescent microspheres and the biotin labeled anti-PEDV- Ig G were used to establish a luminex liquid chip detection method for PEDV. By using TGEV S protein m Ab coupled to x Map fluorescent microspheres and the biotin labeled anti-TGEV-Ig G were used to establish a luminex liquid chip detection method for TGEV. Further studies on Luminex detection method were carried on.BSA- ELISA for PEDV, TGEV detection were a potentially valuable method with high sensitivity and specificity, which may provide a good choice for PEDV and TGEV surveillance and for further multiplex detection of diarrhea pathogen.