Establishment and Application of a Fluorescent PCR for Edwardsiella tarda
In order to construct a rapid,specific and accurate method for detection of Edwardsiella tarda,specific primers and probe were designed targeting at gyrB gene,a fluorescent PCR was established after optimizing the reaction system and annealing temperature,followed by evaluation on its sensitivity,specificity and reproducibility,and then detection of clinical samples.The results revealed that the established method had a standard fluorescence curve with a good linearity when the concentrations of primers and probe were 250 and 200 nmol/L,respectively,and the annealing temperature was at 55℃.The lowest detection limit was 12.4 CFU/mL,and bacteria with the initial concentration of 1 CFU/mL could be detected by the method after being cultivated for 1 hour under 35℃;no cross reaction with 14 common aquatic animal pathogens such as Streptococcus suis type 2 and Streptococcus agalactiae was observed;the coefficients of variation of repeatability tests were all less than 0.95%;and 22 out of 100 clinical samples were detected positive,which was consistent with bacterial isolation and identification results.In conclusion,clinical diagnosis and research of Edwardsiella tarda disease were technically supported by the established method that was with high sensitivity,strong specificity and good reproducibility.
Edwardsiella tardafluorescent PCRlinearityspecificitycoefficient of variationclinical application
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