反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测
1Department of Biotechnology, Faculty of Applied Science,King Mongkut’s University of Technology North Bangkok (KMUTNB), 2Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine,Kasetsart University, 3Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies,Kasetsart University, 4Department of Chemical and Process Engineering, The Sirindhorn Thai-German International Graduate School of Engineering (TGGS),King Mongkut’s University of Technology North Bangkok (KMUTNB)
Summary
与传统RT-PCR技术相比，我们提出了一种RT-LAMP测定方法，用于在相对较短的时间内使用简单的仪器检测罗非鱼中的TiLV。该议定书可能有助于控制TiLVD的流行传播，特别是在发展中国家。
Abstract
罗非鱼湖病毒病（TiLVD）是罗非鱼湖病毒（TiLV）引起的罗非鱼病毒病，是水产养殖业的一个长期挑战，导致世界许多地区罗非鱼的大规模发病率和死亡率。因此，有必要对TiLV感染进行有效、快速和准确的诊断检测，以检测最初的感染并防止该病在水产养殖中传播。本研究提出了一种高度灵敏、实用的逆转录环介导的等温放大（RT-LAMP）方法，以检测鱼组织中的罗非鱼湖病毒。对受感染样本的RT-qPCR和RT-LAMP检测的比较显示，63（100%）有阳性结果和 51 （80.95%）样本。此外，对未感染样本的分析表明，所有63个未受感染组织均对RT-qPCR和RT-LAMP检测均产生阴性结果。使用RT-LAMP评估了罗非鱼中5种病原体的交叉反应，所有测试均显示阴性结果。从受感染的鱼获得的肝脏和粘液样本均显示使用RT-LAMP方法的可比结果，表明粘液可用于RT-LAMP作为非致命性检测，以避免杀死鱼。结果表明，该检测结果表明，在1小时内对罗非鱼组织进行TiLV检测提供了一种有效的方法。因此，该方法推荐作为农场的筛选工具，用于快速诊断TiLV。
Introduction
罗非鱼湖病毒病（TiLVD）是罗非鱼病毒性疾病，据说在世界许多地区，包括亚洲1、2、2非洲和美国，导致罗非鱼死亡。1该病于2009年在以色列罗非鱼大规模死亡期间首次得到确认，金内雷特湖的野生罗非鱼数量从每年257吨急剧下降到8吨。这种疾病是由罗非鱼湖病毒（TiLV）引起的，该病毒已被分配给安农韦里达家族作为一种新的属罗拉平病毒和新物种罗非鱼罗拉平病毒3。TiLV的基因特征表明，该病毒是一种新型的包络、负感、单链RNA病毒，具有10个片段编码10个蛋白质1,1、2、4。,4各种品种的罗非鱼属萨罗罗罗登，奥利奥米米，和提拉平和其他温水鱼（如，巨型葫三（奥斯罗莫斯戈拉米）已被证明易受TiLV22，5。5目前，这种病毒继续在全球传播，可能通过受感染的活鱼66，77的运动，而通过冷冻罗非鱼或其产品传播病毒的风险是有限的8。由于TiLV感染造成的大量死亡率有可能对罗非鱼行业产生重大有害的经济影响。例如，埃及夏季死亡率综合征与TiLV感染有关的经济影响计算为1亿美元。因此，必须开发一种快速和适当的诊断方法，以促进在养鱼场控制这种疾病。
到目前为止，TiLVD的诊断一直基于分子检测、病毒隔离和组织病理学。不同的PCR协议和引素已经开发为TiLV诊断10，11。10,例如，一种基于SYBR的绿色反向转录定量PCR（RT-qPCR）方法，其检测灵敏度为仅两个拷贝/μL，用于TiLV检测10。其他用于TiLV检测的PCR方法包括TaqMan定量PCR11、RT-PCR2、嵌套RT-PCR12和半嵌套11RT-PCR13。1213然而，这些方法需要先进的实验室设备和相对较长的时间来产生结果，由于反应的复杂性，这使得它们不适合现场应用。
循环介导的等温放大（LAMP）测定是一种快速、简单、实用的场内应用14、15。14,该技术采用绞线位移反应原理，而扩增反应在等温条件下运行，没有复杂和昂贵的热循环器14，15。14,因此，放大的LAMP产品或RT-LAMP产品在阶梯状带中进行分析，使用加糖凝胶电泳，带有荧光染色，以安全可视化DNA或RNA14，或用肉眼观察是否存在浊度或白色沉淀物16、17、18。,17,18由于这些原因，该技术已用于现场检测不同的鱼类病原体17、18、19、20、21、22、23、24、25、26、27。17,18,19,20,21,22,23,24,25,26,27本研究的目的是建立一个快速，敏感和准确的RT-LAMP检测的TiLV检测。RT-LAMP测定在30分钟内提供鱼样中TiLV的筛选。该技术可应用于TiLVD的诊断和监测。
Protocol
这项涉及使用动物组织的实验，得到了泰国曼谷卡塞萨特大学机构动物护理和使用委员会的批准（协议号为ACKU61-VET-009）。 1. 组织收集 使用过量的丁香油（即超过3 mL/升）对罗非鱼进行安乐死。三氯苯甲烷硫化剂可用作丁香油的替代品。 使用无菌的梅奥剪刀和钳子切开死后罗非鱼的腹部，切除约30-50毫克的肝脏组织，或使用显微镜盖玻璃，通过纵向刮鱼皮层（?…
Representative Results
在这项研究中，开发了RT-LAMP检测，以检测罗非鱼的TiLV感染。2015年至2016年间，从泰国不同地区的14个农场采集了经测试的样本。受感染和未感染的鱼主要根据身体诊断和有症状的TiLVD的出现进行分组。TiLV感染随后在收集过程后使用RT-PCR得到确认。选择阿加罗斯凝胶电泳和发光绿色检测作为LAMP安培的评估方法（图2）。受感染和未感染的罗非鱼的肝脏和粘液的特点是TiLV疾病症?…
Discussion
水产养殖业不断受到病毒感染的威胁，造成直接经济损失99、23、28。23,28例如，世界上许多地区的罗非鱼生产国对罗非鱼生产国构成重大1,6,威胁。到目前为止，还没有特定的治疗药物可用于预防TiLVD。虽然疫苗的开发正在进行中，但高效疫苗在可用于商业目…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该项目由泰国研究基金（TRF）赠款号RDG6050078和泰国曼谷卡塞萨特大学高级研究所农业和食品高级研究中心资助，泰国高等教育研究促进和国立研究大学项目，高等教育委员会办公室，泰国教育部。这项研究部分由卡塞萨特大学研究生院研究生课程奖学金提供支持。作者要感谢KwanraweeeSirikanchana博士对这段视频的叙述和对皮亚瓦萨拉·西卡林编辑视频的叙述。
Materials
| Tissue collection: | |||
| Clove oil | Better Pharma | N/A | |
| Tricaine methanesulfonate | Sigma-Aldrich | E10521 | An alternative option to clove oil |
| RNA extraction: | |||
| Acid guanidinium-phenol based reagent (TRIzol reagent) | ThermoFisher Scientific Corp. | 15596026 | |
| Acid guanidinium-phenol based reagent (GENEzol reagent) | Geneaid | GZR100 | |
| Direct-zol RNA Kit: | Zymo Research | R2071 | |
| – Direct-zol RNA PreWash | |||
| – RNA Wash Buffer | |||
| – DNase/RNase-free water | |||
| – Zymo-spin IIICG columns | |||
| – Collection Tubes | |||
| RT-LAMP: | |||
| 1x SD II reaction buffer | Biotechrabbit | BR1101301 | |
| Magnesium sulfate (MgSO4) | Sigma-Aldrich | 7487-88-9 | |
| dNTP set | Bioline | BIO-39053 | |
| Betaine | Sigma-Aldrich | B2629 | |
| Calcein mixture | Merck | 1461-15-0 | |
| Bst DNA polymerase | Biotechrabbit | BR1101301 | |
| AMV reverse transcriptase | Promega | M510A | |
| Nuclease-free water | Invitrogen | 10320995 | |
| Elite dry bath incubator, single unit | Major Science | EL-01-220 | |
| Gel electrophoresis: | |||
| Agarose | Vivantis Technologies | PC0701-500G | |
| Tris-borate-EDTA (TBE) buffer | Sigma-Aldrich | SRE0062 | |
| Tris-acetic-EDTA (TAE) buffer: | |||
| – Tris | Vivantis Technologies | PR0612-1KG | |
| – Acetic acid (glacial), EMSURE | Merck Millipore | 1000632500 | |
| – Disodium Ethylenediaminetetraacetate dihydrate (EDTA), Vetec | Sigma-Aldrich | V800170-500G | |
| Neogreen | NeoScience Co., Ltd. | GR107 | |
| DNA gel loading dye (6X) | ThermoFisher Scientific Corp. | R0611 | |
| DNA ladder and markers | Vivantis Technologies | PC701-100G | |
| Mini Ready Sub-Cell GT (Horizontal electrophoresis system) | Bio-Rad | 1704487 | |
| PowerPac HC power supply | Bio-Rad | 1645052 | |
| Gel Doc EZ System | Bio-Rad | 1708270 | |
| UV sample tray | Bio-Rad | 1708271 | |
| NαBI imager | Neogene Science | ||
| cDNA synthesis: | |||
| ReverTra Ace qPCR RT Kit | Toyobo | FSQ-101 | |
| Viva cDNA Synthesis Kit | Vivantis Technologies | cDSK01 | An alternative option for cDNA synthesis |
| NanoDrop2000 (microvolume spectrophotometer) | ThermoFisher Scientific Corp. | ND-2000 | |
| T100 Thermal Cycler | Bio-Rad | 1861096 | |
| RT-qPCR: | |||
| iTaq Universal SYBR Green Supermix | Bio-Rad | 1725120 | |
| Nuclease-free water, sterile water | MultiCell | 809-115-CL | |
| 8-tube PCR strips, white | Bio-Rad | TLS0851 | |
| Flat PCR tube 8-cap strips, optical | Bio-Rad | TCS0803 | |
| CFX96 Touch Thermal Cycler | Bio-Rad | 1855196 | |
| General equipment and materials: | |||
| Mayo scissors | N/A | ||
| Forceps | N/A | ||
| Vortex Genie 2 (vortex mixer) | Scientific Industries | ||
| Microcentrifuge LM-60 | LioFuge | CM610 | |
| Corning LSE mini microcentrifuge | Corning | 6765 | |
| Pipettes | Rainin | Pipete-Lite XLS | |
| QSP filtered pipette tips | Quality Scientific Plastics | TF series | |
| Corning Isotip filtered tips | Merck | CLS series | |
| Nuclease-free 1.5 mL microcentrifuge tubes, NEST | Wuxi NEST Biotechnology | 615601 |
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Citazione di questo articolo
Phusantisampan, T., Rawiwan, P., Roy, S. R. K., Sriariyanun, M., Surachetpong, W. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus. J. Vis. Exp. (159), e61025, doi:10.3791/61025 (2020).