重组酶聚合酶扩增技术检测南美白对虾急性肝胰腺坏死病OA北大核心CSTPCD

Acute hepatopancreatic necrosis disease(AHPND)is a newly emerged bacterial disease in shrimps(Penaeidae),causing significant economic losses in the global shrimp farming industry.AHPND is primarily caused by Vibrio spp.carrying virulent pirA and pirB.In this study,specific primers and probes were designed and screened for pirA and pirB,and a recombinase polymerase amplification(RPA)method was developed.This RPA reaction ran in 39℃for 20 min.The results revealed successful screening of primer/probe combinations for both pirA and pirB genes.Through a series of tests using the RPA method,these primer/probe combinations demonstrated specific detection of the target virulence genes,without amplification of genomic DNA from 13 microorganisms,such as Vibrio alginolyticus,V.parahaemolyticus,and Enterocytozoon hepatomegaly.The detection limit of the RPA method was 0.01 and 0.1 fg/μL of recombinant plasmid pirAB-pMD19,when the primers and probes targeting pirA or pirB were used.The primer concentrations were optimized at 0.4 µmol/L each,while the optimal concentration for the probe was 0.16 µmol/L.The RPA method was used to detect suspicious tissues of Penaeus vannamei using optimized sets of primers and probe.The results showed that the RPA method could accurately detect pirA and pirB from shrimp tissues,which was consistent with the PCR method recommended by the World Organization for Animal Health(WOAH).In summary,this PRA methods targeting pirA and pirB respectively could rapid and accurately detect ANPND-causing bacteria with easy operation and short time-consumption,making it a promising supplementary tool for routine monitoring and rapid detection of AHPND.