| In recent years,bovine respiratory disease(BRD)has become a major disease endangering the beef cattle breeding industry in our country,especially the beef cattle fattening mode of"North Cattle South and West Cattle East Transport"in our country,And communication provides more convenient conditions.According to statistics,fattening shelves cattle transported by long distance,about 60%BRD occurs.BRD is mainly caused by Mycoplasma bovis(M.bovis),Pasteurella mutocida(Pm)and Mannheimia haemolytica(Mh),and some cases are mixed infection.At the same time,the pathogens are fastidious bacteria,and the drug spectrum is different,especially the M.bovis,not only the long period of isolation and culture,but also for veterinary clinically used?-lactam antibiotics with natural resistance,if not correctly make a diagnosis,It will cause greater economic losses.Therefore,it is imminent to develop a rapid and accurate method to identify the main pathogen of BRD.Among them,the PCR-ELISA method is a combination of PCR reaction and the sensitivity of the digoxin detection system and has been widely used in the rapid detection of food and pathogenic microorganisms and has been favored by relevant scholars.The method comprises the following steps of:labeling a PCR primer with digoxigenin,labeling the biotin with a probe,binding the streptavidin to the ELISA plate,utilizing the high affinity of biotin and avidin to immobilize the hybridization product on the ELISA plate,Add the corresponding enzyme substrate,according to the color reaction or microplate read data to determine the results.The method can realize qualitative detection of multiple pathogens at the same time,which has the advantages of fastness,simple operation and the like.To this end,this study established a multiplex PCR-ELISA method for the simultaneous detection of three major pathogens of BRD by using probe primer double labeling.The specific experimental results are as follows:(1)High-throughput screening of species-specific genesThe whole genome sequence of the target strain was compared with the NCBI animal nt library and the bacterial nt library(the target strain was removed),the sequences which were not compared to the database were screened,and the target genes were further subjected to cluster analysis using orthomcl,Finally,667 Cattle A capsid-type Pasteurella multocida-specific genes and 380 Hemolymphis species-specific genes were obtained.(2)Multiplex PCR detection method was establishedBased on the selected species-specific genes,a multiplex PCR assay was established to optimize the primer conditions,annealing temperature,number of cycles and so on.Finally,three pairs of specific primers were selected.The annealing temperature was 54.5?a nd the optimal number of cycles was 30.The sensitivity of multiplex PCR Pm was 6.8×10~3 CFU/mL,Mh was 3.7×10~4 CFU/mL,and M.bovis was 1.1×10~6 CFU/mL.(3)Multiplex PCR-ELISA method was establishedA multiplex PCR-ELISA method was established by chemical denaturing hybridization and the conditions were optimized.The results showed that the concentration of streptavidin was 5?g/mL,the concentration of BSA was 0.5%For20min,peroxidase-labeled anti-digoxin antibody diluted 1000 times,color time 20min,the probe concentration of 0.5mmol/10mL.The detection method has a sensitivity of Pm of 6.8×10~1 CFU/mL,Mh of 3.7×10~3 CFU/mL,and M bovis of 1.1×10~4 CFU/mL. |
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