急性肝胰腺坏死病重组酶介导恒温荧光快速检测方法的建立及在环境样品中的应用

Establishment of a Recombinase Aided Isothermal Fluorescencerapid Detection Method for Acute Hepatopancreatic Necrosis and Its Application in Environmental Samples

为建立急性肝胰腺坏死病(Acute Hepato Pancrentic Necrosis Disease,AHPND)实时荧光重组酶介导扩增(Recombinase Aided Amplification,RAA)快速检测方法,根据NCBI中致急性肝胰腺坏死病的副溶血性弧菌(Vibrio parahaemolyticus,Vp_(AHPND))中PVA1质粒的保守序列设计了3对引物和1条探针,通过荧光值和出峰时间,筛选引物、优化反应温度、评估方法的敏感性和特异性,同时把该方法应用于临床模拟环境DNA(Environmental DNA,e DNA)样品检测。结果显示,筛选出的Vp_(AHPND)的F2/R2引物组特异性强,仅能扩增Vp_(AHPND)阳性核酸,与常见的对虾病原肝肠胞虫(Enterocytozoon Hepato Penaei,EHP)、白斑综合征病毒(White Spot Syndrome Virus,WSSV)、传染性皮下和造血器官坏死病毒(Infectious Hypodermal and Haematopoietic Necrosis Virus,IHHNV)、细菌性肝胰腺坏死(Infection with Hepatobacter Penaei,NHPB)、十足目虹彩病毒1型感染(Decapod Iridescent Virus 1,DIV1)、副溶血性弧菌(Vibrio Parahaemolyticus,VP)均无交叉反应;检测方法灵敏度高,检测限低至1.4×10^(2)copies/μL,并且在20 min内快速完成了Vp_(AHPND)的检测。采用实时荧光RAA法,在64份临床模拟环境DNA(e DNA)样品中检出Vp_(AHPND)阳性8份,检测结果与荧光定量PCR检测方法一致。研究表明e DNA采样策略与Vp_(AHPND)的实时荧光RAA检测方法相结合,能大大提升临床诊断速度,可为急性肝胰腺坏死病快速检测提供重要的技术依据。

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(Vp_(AHPND))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 Vp_(AHPND) in clinical simulated environment DNA(eDNA)samples.The results showed that the F2/R2 primer set of Vp_(AHPND) 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 1.4×10^(2) copies/μL.The detection of Vp_(AHPND) was completed within 20 minutes.Using the real-time fluorescence RAA method,Vp_(AHPND) 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 Vp_(AHPND) significantly improves the clinical diagnosis speed of shrimp,providing a rapid and reliable diagnostic method for customs and aquaculture.