The Establishment Of Diagnostic Method Based On Loop-Mediated Isothermal Amplification For PHEV Detection And Its’ Clinical Application

Porcine hemagglutinating encephalomyelitis is an acute infectious diseasecommon in piglets,which has the characters of highly contagious.The disease iscaused by the virus of Coronavirus genus,which is called hemagglutinatingencephalomyelitis virus,HEV. Clinical data suggest that it mainly against1to3weekspiglets,the main features are vomiting,exhaustion and obvious neurologicalsymptoms,death rate as high as20to100%.Therefore, in the control of the disease,timely and accurate diagnosis is particularly important.At present,there are severalcommonly used diagnostic methods for the disease currently:isolation andidentification of the virus,nest PCR,Realtime-PCR,HA/HI,VN,enzyme-linkedimmunosorbent test,polymerase chain reaction and so on. These methods have playeda huge role in the actual production,but there are all kinds of insufficient, theoperation is complex,takes time and costs high and so on.it is unsuitable for pig farmersin rural areas. Therefore, the establishment of a rapid and accurate new diagnostic technology isurgently needed.In this study,we use M consensus sequence of the HEV gene synthesizes twocouples of LAMP primers,to create a HEV LAMP method for detecting.At the sametime,we conduct the optimization of reaction conditions,specificity test,sensitivitytest.The results show that this method can complete the reaction at63°C water bathin1h and the reaction is excellent. Compared to porcine transmissible gastroenteritisvirus (TGEV), porcine epidemic diarrhea virus (PEDV), classical swine fever virus(CSFV), pseudorabies virus (PRV) and porcine circovirus (PCV),there has nocross-reactivity,and it can detect a minimum concentration of RNA about ten copies of PCR.In additon,the study applies the established LAMP rapid diagnostic methodsuccessfully to the porcine hemagglutinating encephalomyelitis for15preliminarydiagnosis of the disease clinically suspected samples of a farm in jilin province, and toevaluate its clinical testing results, the positive corresponding rate is100%. Theresearch results will provide important clinical for the diagnosis of HEV technology.