| Porcine coronavirus infection has always been highly concerned about the harm of porcine coronavirus infection to the pig breeding industry,and its causes are complex and diverse,which makes the clinical differential diagnosis of this kind of disease become a difficult point in grass-roots farms.The results of serological and epidemiological investigations show that porcine coronavirus infection occurs all over the world,sometimes even in an explosive manner.Porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV)and porcine hemagglutinating encephalomyelitis virus(PHEV)are three common coronaviruses in piglets,which have high mortality to young piglets.Because PEDV,TGEV and PHEV infection all show obvious symptoms of vomiting and diarrhea,they are often confused and misdiagnosed in the differential diagnosis of traditional diseases.Visual chip technology is a detection method produced by applying the basic mode of traditional biochip and adapting to visual chromogenic method,which includes visual protein chip and gene chip.It has the advantages of high speed,visibility,easy operation,low equipment requirements,low cost and so on.In recent years,this technique has been widely used in animal and plant pathogens,gene mutation or typing detection,and plays an important role in biology,medicine,pharmacology,genetics and other disciplines.In this study,the nuclear protein(N)of the above three viruses was prokaryotic expressed and purified,and the purified N protein was added to the microarray vector,and the reaction systems such as drying,fixation,blocking and washing were optimized to prepare a visual protein chip which can detect the specific antibodies of the above three pathogens at the same time.During the detection,the serum to be tested was added to the standardized protein chip,washed,dried,incubated with secondary antibody,washed,dried,coloured with chromogenic solution,and the results could be observed with naked eyes after washing and drying.Its preliminary application in clinical detection shows that the standardized protein chip has strong specificity and no cross-reaction in a variety of complex serum environments;the sensitivity is good,and the lowest detection limit is 12 800 times dilution;in addition,the coincidence rate between this method and ELISA is more than 90%.The visual antibody chip prepared in this study can quickly,efficiently and massively differentiate and diagnose porcine epidemic diarrhea,porcine transmissible gastroenteritis and porcine hemagglutinating encephalomyelitis,and is expected to be used to monitor the level of specific antibodies against PEDV,TGEV and PHEV 3 pathogens in clinical outpatients and grass-roots farms.The establishment of this detection method can play a positive role in the early diagnosis and prevention of this kind of epidemic disease under the condition of insufficient equipment conditions such as grass-roots clinic and breeding farm,and provide an important reference for the innovation of detection methods of other animal viral diseases.It is helpful to meet the current market demand for the detection of animal diseases.It will play a positive role in promoting the intensification,scale and automation of the pig breeding industry,and is expected to bring an important improvement to the human food safety environment. |
