适用于RPA-LFD技术检测对虾肝胰腺DNA样品的快速制备试剂研究  

Rapid Preparation Reagent for RPA-LFD Detection of DNA Samples from the Shrimp Hepatopancreas

作　　者：姚梦丽 白昌明[1] 王崇明[1] 辛鲁生 YAO Mengli;BAI Changming;WANG Chongming;XIN Lusheng(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Affairs,Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity,Qingdao 266071,China;Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology,Yulin 537000,China;College of Fisheries,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛海洋科技中心海洋渔业科学与食物产出过程功能实验室,农业农村部海水养殖病害防治重点实验室,青岛市海水养殖流行病学与生物安保重点实验室,山东青岛266071 [2]广西农产资源化学与生物技术重点实验室,广西玉林537000 [3]中国海洋大学水产学院,山东青岛266003

出　　处：《渔业科学进展》2024年第4期166-174,共9页Progress in Fishery Sciences

基　　金：国家自然科学基金(31902400);广西农产资源化学与生物技术重点实验室开放基金(2022KF07)共同资助。

摘　　要：为摆脱常规核酸样品提取步骤繁琐、耗时等问题,实现真正的现场快速检测,本研究致力于研制和优化一种对虾肝胰腺中DNA样品的核酸快速制备试剂,即核酸释放剂,适用于重组酶聚合酶扩增技术(recombinasepolymeraseamplication,RPA)与侧向流层析试纸条技术(lateralflow dipstick, LFD)结合的RPA-LFD技术检测。实验优选20~100 mmol/L Tris-HCl、50~250 mmol/L KCl、0.01%~0.10%十二烷基硫酸锂(LDS)、0.5%~2.0%聚乙二醇辛基苯基醚(TritonX-100)、1~5mmol/L乙二胺四乙酸二钠(EGTA2Na)、0.5~5.0 mmol/L牛血清白蛋白(BSA)、1~5 mg/mL明胶、0.01%~0.10%海藻糖、1%~5%甜菜碱等配制成核酸释放剂。以对虾肝肠孢虫(Enterocytozoonhepatopenaei,EHP)阳性样本和阴性样本对核酸释放剂各组分间配比进行优化,采集绿豆大小的对虾肝胰腺组织加入100μL核酸释放剂,100℃加热3 min,取上清液进行RPA-LFD反应,测试各组分不同浓度配比;并以对虾急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)阳性样品及阴性样品作为检测模板对优化后核酸释放剂再次验证。结果显示,核酸释放剂的各组分最佳配比为100 mmol/L Tris-HCl、100 mmol/L KCl、0.02%LDS、0.5%Triton X-100、1 mmol/L EGTA2Na、0.05%海藻糖、1 mg/mL明胶、0.5 mmol/L BSA、2%甜菜碱。RPA-LFD方法检测可显著区分阳性和阴性样品。本研究优化的核酸释放剂,适用于RPA-LFD检测对虾肝胰腺病原DNA样品的制备,有效避免了常规DNA样品繁琐、耗时的制备步骤,极大地提高了核酸水平病原检测效率。This study is committed to developing and optimizing a rapid nucleic acid preparation reagent(nucleic acid release agent)that is suitable for recombinase polymerase amplification with lateral flow dipstick(RPA-LFD)technology to detect DNA nucleic acid samples in shrimp hepatopancreas to realize on-site rapid detection and remove the cumbersome and time-consuming steps of conventional nucleic acid sample extraction.The optimum compositions of nucleic acid release agent are 20–100 mmol/L Tris-HCl,50–250 mmol/L KCl,0.01%–0.10%(W/V)lithium dodecyl sulfate(LDS),0.5%–2.0%(V/V)Triton X-100,1–5 mmol/L ethylenediaminetetraacetic acid disodium salt(EGTA2Na),0.5–5.0 mmol/L bovine serum albumin(BSA),1–5 mg/ml gelatin,0.01%–0.10%(W/V)trehalose,and 1%–5%(W/V)betaine.The proportion of each component of nucleic acid release agent was optimized using positive and negative samples of shrimp Enterocytozoon hepatopenaei(EHP).The RPA-LFD reaction initially involved collecting shrimp hepatopancreas(the size of a mung bean),adding 100μL nucleic acid release agent,heating for 3 min at 100 and℃using the supernatant for analysis.The optimal ratio of each component of nucleic acid releaser was determined to be 100 mmol/L Tris-HCl,100 mmol/L KCl,0.02%LDS,0.5%Triton X-100,1 mmol/L EGTA2Na,0.05%trehalose,1 mg/mL gelatin,0.5 mmol/L BSA,and 2%betaine.The positive and negative samples of acute hepatopancreatic necrosis disease(AHPND)were used as detection templates to verify the optimized nucleic acid release agent.The optimized nucleic acid release agent could be used to prepare nucleic acid samples,and the prepared samples were available for RPA-LFD method testing.The optimized nucleic acid release agent in this study can be used to prepare DNA nucleic acid samples of the shrimp hepatopancreas for RPA-LFD detection.It avoids the tedious and time-consuming preparation steps of conventional DNA nucleic acid samples and greatly improves the efficiency of nucleic acid level pathogen detection.Disease is the bottleneck that res

关 键 词：核酸释放剂 重组酶聚合酶扩增(RPA) 侧流层析试纸条(LFD) 虾肝胰腺 病原快速检测 

分 类 号：S945.46[农业科学—水产养殖]