| 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(recombinase polymerase amplication, RPA)与侧向流层析试纸条技术(lateral flow 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(Triton X-100)、1~5 mmol/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(Enterocytozoon hepatopenaei, 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100 μL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。 |
| 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 |
| DOI：10.19663/j.issn2095-9869.20230209001 |
| 分类号: |
| 基金项目: |
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| Rapid preparation reagent for RPA-LFD detection of DNA samples from the shrimp hepatopancreas |
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YAO Mengli,BAI Changming,WANG Chongming,XIN Lusheng
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1.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;2.College of Fisheries, Ocean University of China, Qingdao 266003, China;3.Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin 537000, China
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| Abstract: |
| 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 100 μL 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 restricts the green development of the shrimp breeding industry, which can cause huge annual economic losses. Detection and monitoring of shrimp related pathogens are effective to solve this problem. Enterocytozoon hepatopopenaei (EHP) and Vibrio parahaemolyticus (VP) are two common pathogenic microorganisms in shrimp culturing that cause AHPND. E. hepatopopenaei is an obligate intracellular parasite that mainly parasitizes in the hepatopancreas of shrimp. Severe EHP infections lead to atrophy of the hepatopancreas of shrimp. E. hepatopopenaei has a wide range of hosts, including Penaeus vannamei, P. monodon, and Palaemon carincauda. AHPND is an aquatic disease caused by Vibrio parahemolvticus with the virulent plasmid PirA/B. It was first found in China and Vietnam in 2010, then in Malaysia, Thailand, Mexico, the Philippines, and other countries. P. monodon, P. vannamei, P. chinensis, and P. japonicus are susceptible hosts. The distribution of pathogenic vibrio can be detected in the hepatopancreas tissue of diseased shrimp, and it can cause hepatopancreas atrophy. Broadly, most shrimp pathogens (such as DIV1, WSSV, TSV, and IHHNV) infect the hepatopancreas of shrimp. DIV1 infections cause pale atrophy of the hepatopancreas. Histopathological examination identified eosinophils and dark eosinophilic inclusions in the cytoplasm of the hepatopancreas of shrimp infected with DIV1. Some of them were coated or contained slight basophilic staining, and hemocytes were pyknotic. WSSV and TSV can cause pale atrophy symptoms of the hepatopancreas, while IHHNV can also cause hepatopancreas enlargement of shrimp. Therefore, the nucleic acid samples of the hepatopancreas of shrimp can be widely used as templates for detection of most shrimp pathogens.
Isothermal nucleic acid amplification technology removes the dependence on conventional nucleic acid temperature change amplification using the PCR amplification instrument, and is widely used for rapid detection. Currently, isothermal amplification technology covers loop mediated isothermal amplification (LAMP), strand displacement amplification (SDA) and recombinase polymerase amplification (RPA), and so on. Among them, RPA technology was developed in recent years using recombinases to promote the insertion and binding of oligonucleotide primers in DNA double strand complementary sequences, and realize exponential amplification of specific DNA regions under the action of Bsu DNA polymerase. It has a short reaction time, strong specificity, high sensitivity, and operates at a constant temperature of 37–42 ℃. It is suitable for on-site detection and analysis. The combination of RPA with the lateral flow dipstick (LFD) to read the detection results is simple to operate, fast, with high specificity and sensitivity. It is a new method suitable for rapid detection in the field. The combination of RPA-LFD technology and a simplified nucleic acid extraction method can help to realize portable on-site rapid nucleic acid detection. |
| Key words: Nucleic acid releasing reagent Recombinase polymerase amplification Lateral flow dipstick Shrimp hepatopancreas Pathogen rapid detection |
