Establishment and Clinical Application of a Droplet Digital PCR Detection Method for Streptococcus agalactiae in Tilapia
A droplet digital PCR(ddPCR)method for detection of Streptococcus agalactis in tilapia was established by designing and screening primers and probes,optimizing reaction concentration and annealing temperature,the sensitivity,specificity and repeatability of the method were analyzed,and finally the method was applied to clinical samples for detection.The results showed that the ddPCR method established here had obvious distribution boundary between negative and positive droplets,high average copy number and high amplification efficiency at primer concentration of 0.9 μmol·L-1,probe concentration of 0.3 μmol·L-1 and the annealing temperature of 56.9 ℃.The linearity was found to be good(R2=0.997 3),with the minimum detection limit of 2.56 copies·μL-1,and without cross reaction with Streptococcus suis type 2,S.dolphinus and other five common pathogens of aquatic animal diseases.There was repeated coefficient variation of 3.15％and 100％of the coincidence rate between the results of clinical samples and real-time fluorescence PCR method.The coincidence rate between the results of bacterial isolation and identification method was shown to be 94.12％,indicating that the established ddPCR method for detection of S.agalactis in tilapia has high sensitivity,strong specificity and good reproducibility,and can be used for quantitative detection of clinical samples infected by S.agalactis in tilapia,which provides a useful reference for the study of S.agalactis in tilapia.
Streptococcus agalactiaedroplet digital PCRquantitative detectiontilapiaclinical application
万方数据
维普
