方法文章

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

DOI：

10.3791/61025

⸱

2020年5月18日

 ,  ,  ,  , 
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)

本文内容

摘要

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We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

摘要

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Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

引言

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Tilapia lake virus disease (TiLVD) is a viral disease in tilapia (Oreochromis spp.) that reportedly causes tilapia deaths in many regions of the world, including Asia1,2, Africa, and America. The disease was first recognized during the mass mortality of tilapia in 2009 in Israel, where the number of wild tilapia in Lake Kinneret plummeted dramatically from 257 to 8 tons per year2. The disease is caused by the tilapia lake virus (TiLV), which has been assigned to the family Amnoonviridae as a new genus Tilapinevirus and a new species Tilapia tilapinevirus

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方案

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This experiment, which involved the use of animal tissue, was approved by the Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (protocol number ACKU61-VET-009).

1. Tissue collection

  1. Euthanize tilapia fish using an overdose of clove oil (i.e., more than 3 mL/L). Tricaine methanesulfonate can be used as an alternative to clove oil.
  2. Use sterile mayo scissors and forceps to cut open the abdomen of the postmortem tilapia and excise approximately 30–50 mg of liver tissue, or using a microscope cover glass, collect 100 µL of mucus by scraping the fish skin layer longitudin....

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结果

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In this study, an RT-LAMP assay was developed to detect TiLV infection in tilapia. The tested samples were collected from 14 farms located in different parts of Thailand between 2015 and 2016. The infected and uninfected fish were primarily grouped based on physical diagnoses and the appearances of symptomatic TiLVD. TiLV infection was subsequently confirmed using RT-PCR after the collection process. Agarose gel electrophoresis and the detection of a luminescent green color were selected as the evaluation methods of the LA.......

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讨论

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The aquaculture industry is continuously threatened by viral infections that cause substantial economic losses9,23,28. For instance, the emerging TiLV poses a major threat to tilapia-producing countries in many parts of the world1,6,9. Until now, there have been no specific therapeutics available to prevent TiLVD. While the development .......

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披露

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The authors have nothing to disclose.

致谢

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The project is financially funded by Thailand Research Fund (TRF) grant number RDG6050078 and the Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand under the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Ministry of Education, Thailand. The research is supported in part by the Graduate Program Scholarship from the Graduate School, Kasetsart University. The authors would like to thank Dr. Kwanrawee Sirikanchana for the narrative speaking of the video and Piyawatchara Sikarin for editing the video.

....

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材料

本文使用的材料清单
姓名公司目录编号评论
<强>组织采集：
丁香油Better PharmaN/A
甲磺酸三卡因Sigma-AldrichE10521丁香油的替代选择
RNA 提取：
酸性胍苯酚基试剂（TRIzol 试剂）ThermoFisher Scientific Corp.
酸性胍苯酚类试剂（GENEzol 试剂）GeneaidGZR100
Direct-zol RNA 试剂盒：Zymo ResearchR2071
- Direct-zol RNA PreWash
- RNA 洗涤缓冲液
- 不含 DNase/RNase 的水
- Zymo-spin IIICG 色谱柱
- 收集管
RT-LAMP：
1x SD II 反应缓冲液BiotechrabbitBR1101301
镁 （MgSO4）Sigma-Aldrich7487-88-9
dNTP 套装BiolineBIO-39053
甜菜碱Sigma-AldrichB2629
钙黄绿素混合物Merck1461-15-0
Bst DNA 聚合酶BiotechrabbitBR1101301
AMV 逆转录酶PromegaM510A
无核酸酶水Invitrogen10320995
Elite 干浴培养箱，单单元主要科学EL-01-220
凝胶电泳：
琼脂糖Vivantis TechnologiesPC0701-500G
Tris-硼酸盐-EDTA （TBE） 缓冲液Sigma-AldrichSRE0062
Tris-乙酸-EDTA （TAE） 缓冲液：
- TrisVivantis TechnologiesPR0612-1KG
- 醋酸（冰），EMSUREMerck Millipore1000632500
-乙二胺四乙酸二钠 （EDTA），VetecSigma-AldrichV800170-500G
Neogreen NeoScience Co.， Ltd.GR107
DNA 凝胶上样染料 （6X）ThermoFisher Scientific Corp.R0611
DNA 分子量标准和标记Vivantis TechnologiesPC701-100G
Mini Ready Sub-Cell GT（水平电泳系统）Bio-Rad1704487
PowerPac HC 电源Bio-Rad1645052
Gel Doc EZ 系统Bio-Rad1708270
UV 样品盘Bio-Rad1708271
N&α;BI 成像仪Neogene Science
cDNA 合成：
ReverTra Ace qPCR RT 试剂盒ToyoboFSQ-101
Viva cDNA 合成试剂盒Vivantis TechnologiescDSK01cDNA 合成的替代选项
NanoDrop2000（微量分光光度计）ThermoFisher Scientific Corp.ND-2000
T100 热循环仪Bio-Rad1861096
RT-qPCR：
iTaq 通用型 SYBR Green 超混合液Bio-Rad1725120
无核酸酶的水，无菌水MultiCell809-115-CL
8 管 PCR 联排，白色Bio-RadTLS0851
扁平 PCR 管 8 联排管，光学Bio-RadTCS0803
CFX96 Touch 热循环仪Bio-Rad1855196<
strong>通用设备和材料：
Mayo 剪刀N/A
镊子N/A
Vortex Genie 2（涡旋混合器）科学行业
微量离心机 LM-60LioFugeCM610
康宁 LSE 微型微量离心机康宁6765
移液器RaininPipete-Lite XLS
QSP 过滤移液器吸头Quality Scientific PlasticsTF 系列
康宁 Isotip 过滤吸头MerckCLS series
无核酸酶 1.5 mL 微量离心管，NEST无锡 NEST Biotechnology615601
15596026硫酸物

参考文献

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  1. Surachetpong, W., et al. Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015-2016. Emerging Infectious Diseases. 23 (6), 1031-1033 (2017).
  2. Eyngor, M., et al. Identification of a novel RNA virus lethal to tilapia. Journal of Clinic....

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RNA

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