Study On The Detection Methods Of Pasteurella Multocida And Haemophilus Parasuis

Pasteurella multocida(Pm)and Haemophilus parasuis(Hps)are the main pathogens of porcine respir atory disease syndrome.It is also one of the common pathogens of swine pneumoconiosis,Haemophilus pa rasuis,and collective disease on farms.The pig industry has been greatly affected,which has seriously hind ered the healthy development of the pig industry.Therefore,establishing a rapid detection method for Pm and Hps is the key to successful prevention and control.In this paper,Pm and Hps were used as research objects to establish dual and multiplex PCR detection methods and LAMP detection methods.The main research content is as follows:(1)By designing primers for conserved sequences of porcine Pasteurella multocida and Haemophilus parasuis,the method of isothermal amplification of loop-mediated porcine Pasteurella multocida and Haemophilus parasuis was established,and perform corresponding specificity and sensitivity verification tests.The results showed that porcine Pasteurella multocida could amplify corresponding specific DNA fragments in the loop-mediated isothermal amplification method,and there was no specific DNA amplification for other related pathogens.The minimum detection limit was 85.2pg/u L,20 times more sensitive than normal PCR.H.parasuis can amplify the corresponding specific DNA fragments in the loop-mediated isothermal amplification method.There is no specific DNA amplification for other related pathogens.The minimum detection limit is 74.8 pg/u L,and the sensitivity is 20 times higher than ordinary PCR.Reaction can be completed in 45 min.(2)According to the GenBank Haemophilus parasuis 16 S rRNA,PILA,LIPO gene conserved sequences and Pasteurella multocida 16 SrRNA,KMT1 gene conserved sequences established double and multiplex PCR detection methods,through the optimization of the method and annealing temperature screening,the optimal annealing temperature was determined,and the method was finally verified.The results showed that the optimum annealing temperature for PILA gene and LIPO gene of Haemophilus parasuis was 59.0℃,and the optimum annealing temperature for the double-PCR of the 16 S rRNA gene of Haemophilus parasuis and Pasteurella multocida was 55.0℃.The optimum annealing temperature formultiplex PCR of KMT1,PILA and LIPO was 58.0℃.Specific amplification of relevant pathogenic bacteria did not amplify any target bands,but both Pm and Hps could amplify specific bands,indicating that this method has better specificity.In this study,the two rapid detection methods established for Pasteurella multocida and Haemophilus parasuis were highly specific and sensitive,and in a relatively short period of time,rapid and accurate identification of pathogenic bacteria can be achieved.It provides a new technology for the rapid detection of Pasteurella multocida and Haemophilus parasuis in clinical and primary laboratory.