Establishment of a Recombinase Aided Isothermal Fluorescencerapid Detection Method for Acute Hepatopan-creatic Necrosis and Its Application in Environmental Samples
To establish a real-time fluorescence Recombinase Aid Amplification(RAA)rapid detection method for Acute Hepato Pancrentic Necrosis Disease(AHPND)in shrimp,three pairs of primers and one probe were designed based on the conserved sequence of the PVA1 plasmid in Vibrio parahaemolyticus(VpAHPND)causing acute hepatopancreatic necrosis disease in NCBI.The primers were screened,reaction temperature was optimized and the sensitivity and specificity of the method were evaluated using fluorescence value and peak time.This method was applied to detect VpAHPND in clinical simulated environment DNA(eDNA)samples.The results showed that the F2/R2 primer set of VpAHPND exhibited strong specificity and could only amplify AHPND positive nucleic acids.There was no cross reaction with common shrimp pathogens such as Enterocytozoon Hepato Penaei(EHP),White Spot Syndrome Virus(WSSV),infectious hypodermal and haematopoietic necrosis virus(IHHNV),Infection with Hepatobacter Penaei(NHPB),Decapod Iridescent Virus 1(DIV1)or Vibrio Parahaemolyticus(VP).The detection method demonstrated high sensitivity and the lowest detection limits is 1.4×102 copies/μL.The detection of VpAHPND was completed within 20 minutes.Using the real-time fluorescence RAA method,VpAHPND was detected in 8 out of 64 clinical simulated eDNA samples,with results consistent with those obtained by fluorescence quantitative PCR.The research shows that the combination of eDNA sampling and real-time fluorescence RAA detection method of VpAHPND significantly improves the clinical diagnosis speed of shrimp,providing a rapid and reliable diagnostic method for customs and aquaculture.
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