Establishment And Preliminary Application Of Nanoparticle-assisted Duplex PCR For Dog's Babesiosis And Dog's Ehrlichiosis

As two blood protozoa diseases of dog's Babesiosis and dog's Ehrlichiosis are cross-infected,which can cause zoonosis and pose a threat to human health.The purpose of this study was to establish a nanoparticle-assisted duplex PCR(Nano-d PCR)method for detecting Babesia and Eric in dogs,which would lay a technical foundation for the clinical diagnosis and mass screening of these two kinds of blood protozoa diseases.1.Establishment of duplex PCR method for detecting Babesia and Ehrlichia disease in dogs.Dog's blood samples of positive or suspected Babesiosis and Ehrlichiosis were collected,and DNA was extracted.Primers of 18 s r RNA for Babesia and 16 s r RNA for Eric were designed respectively.The corresponding DNA fragments were amplified by PCR,then purified by gel extraction,and TA cloned to p MD-18 T vector to construct p MD-BC and p MD-EC plasmids.The plasmids were used as templates for duplex PCR amplification and the optimum annealing temperature was selected.On this basis,a series of subdilution of the plasmids was done,and then duplex PCR was performed again to detect its sensitivity.The results showed that(1)the nucleotide sequences of both p MD-BC and pMD-EC share 100% homologous with the 18 s r RNA of Babesia Canis and 16 s r RNA of Ehrlichia Canis in Gen Bank;(2)the optimal annealing temperature of duplex PCR was 62?;(3)The minimum nucleic acid detection amounts of babesia and Eric were 2.07×10~5 and 1.92×10~5 copies/L,respectively.2.Establishment of Nano-dPCR method for detecting dog's Babesiosis and dog's Ehrlichiosis.On the basis of the previous experiment,the annealing temperature and primer dosage were optimized by using the nanoscale PCR kit.Positive DNA samples of canine rickettsia,toxoplasma,parvovirus and canine distemper virus were used as templates to verify the specificity of the nano-d PCR system.In addition,152 blood samples from dogs in Nanning city were collected,and the epidemiology of dog's Babesiosis and dog's Ehrlichiosis was investigated using the Nano-d PCR.The results showed that(1)a Nano-dPCR PCR method was successfully established for detecting dog's Babesiosis and dog's Ehrlichiosis.The optimum annealing temperature was 57?;(2)the Nano-d PCR system was specificic with the minimum detectable nucleic acid amounts of 2.07×10~3 copies/L for Babesiosis and 1.92×10~3 copies/L for Ehrlichiosis.The sensitivity of the Nano-d PCR was 100 times higher than that of the previous duplex PCR system;(3)the system was used for epidemiological screening of 152 clinical samples,and the results proved that the method was feasible.In summary,this study established a Nano-dPCR method for detecting dog's Babesiosis and dog's Ehrlichiosis,which was specific and sensitive,and feasible for the clinical diagnosis and mass epidemiological screen ing of Babesia Canis and Ehrlichia Canis.