Magnetic Immuno Capture PCR Assay(MIPA)for Rapid Detection of Acute Hepatopancreatic Necrosis Diseas

来源 :中国生态学学会微生物生态专业委员会2016年学术年会 | 被引量 : 0次 | 上传用户:kuwaning
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  Acute hepatopancreatic necrosis disease(AHPND)is a severe,newly emergent penaeid shrimp disease and caused by a unique strain of Vibrio parahaemolyticus that has a plasmid harboring virulent genes.In this study,magnetic immuno capture PCR assay(MIPA)was established for the detection of AHPND.The outer membrane protein,OmpU was used as the immuno capture target to separate the AHPND pathogens,and followed by PCR assay base on specific primers of two AHPND virulent genes,pirA and pirB.Magnetic beads were coated by antisera against the purified recombinant OmpU at 4 ℃ for 18 h,then captured at 37 ℃ for 2 h,leading to a capture rate of more than 85%.The detection sensitivity of AHPND by MIPA was 101 CFU/mL,while the direct PCR showed a detection limit of 104 CFU/mL.The MIPA system was stabled at pH between 3 to 9 and salt concentration between 1%and 5%.The detection sensitivity had no significant difference while non-AHPND bacteria existed.Furthermore,the detection sensitivity can achieve the same 101 CFU/mL in the simulation testing system of water,sediment,and shrimp sample.Therefore,this method could be used at the detect of AHPND for the prevention and surveillance of this infectious disease in aquaculture.
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