Establishment Three Kinds Of Nucleic Acid Detection Method For Equine Piroplasmosis

Equine piroplasmosis is caused by two pathogens,Theileia equi and Babesia caballi,and is transmitted by ticks.It is a blood protozoan disease that mainly infects red blood cells of equine animals.Clinical symptom basically is hemolytic anemia,icterus,when the symptom is serious,can cause the death of suffer from animal.The disease was once classified as a class ii animal epidemic in China and is now one of the diseases that must be reported by the OIE.Horses infected with Equine piroplasmosis can cause significant losses to the horse industry if they are not diagnosed and treated in time.T.equi is relatively more contagious than B.caballi.At present,although a lot of researches and explorations have been made on new methods for the diagnosis of Equine piroplasmosis around the world and some achievements have been made,a highly sensitive and specific diagnostic method is still needed at present.In this study,after consulting a large number of literature on the diagnosis and identification of piroplasmosis,the research objective was determined on the establishment of nested PCR nucleic acid detection method.Based on the conserved sequences of T.equi 18 S r RNA encoding sequence and B.caballi 18 S r RNA encoding sequence in Gen Bank,the first-round primers were designed.Meanwhile,the specific fragments of T.equi and B.caballi were used as the second-round primers.Through constant adjustment and optimization of the test methods,and the specificity and sensitivity tests,the nested PCR detection method for piriformis marsiformis was finally established.The results showed that the nested PCR method established in this study could specifically detect T.equi and B.caballi positive samples and mixed samples with good specificity and sensitivity.There was no response to,but the infection could not be distinguished.In view of this situation,this study also designed a dual-fluorescence quantitative PCR detection method and a RPA detection method for Equine piroplasmosis.At present,the simulated plasmid test of the two methods showed that T.equi or B.caballi infection could be specifically distinguished.The nested PCR detection method established in this study can be used for rapid detection of Equine piroplasmosis.The dual fluorescence quantitative PCR detection method and RPA detection method can be used for species classification and identification of Equine piroplasmosis positive samples.The comprehensive application of multiple methods for identification of Equine piroplasmosis has strong application value.