Establishment and Optimization of Droplet Digital PCR for Duck Enteritis Virus
In order to quickly and accurately detect duck enteritis virus(DEV),primers and probes for droplet digital PCR(ddPCR)were designed based on DEV UL6 gene sequence registered in GenBank,and a ddPCR method for detection of DEV was established after optimization of reaction conditions,followed by the verification of its sensitivity,specificity and reproducibility.The results showed that the virus concentration reached to the maximum(332 copies/µL)when the concentrations of primers and probes were 1.50 µmol/L and 0.45 µmol/L,respectively,at an annealing temperature of 57.1℃;the lowest detection limit was 0.7 copies/µL,and it couldn't generate positive microtitres for non-DEV pathogens,without cross-reactivity;the variable coefficients of both the intra-and inter-group repeated tests were less than 10%.In conclusion,the DEV ddPCR method established in this study was characterized by the advantages of high sensitivity,strong specificity,good reproducibility and high stability,and could be used for the detection of samples with low DEV content and early diagnosis and epidemiological investigation of the disease.