Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

Pamela Nicholson; Pattarasuda Rawiwan; Win Surachetpong

doi:10.3791/58596

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Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

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10:55 min

November 10, 2018

DOI: 10.3791/58596-v

Pamela Nicholson¹, Pattarasuda Rawiwan², Win Surachetpong²

¹Institute of Veterinary Bacteriology Vetsuisse Faculty,University of Bern, ²Department of Veterinary Microbiology and Immunology and Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies,Kasetsart University

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Overview

This protocol details the diagnosis of Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. It encompasses the entire process from tissue dissection to RNA extraction, cDNA synthesis, and TiLV detection through conventional or quantitative PCR.

Key Study Components

Area of Science

Veterinary Microbiology
Virology
Fish Health Management

Background

Tilapia Lake Virus causes significant mortality in tilapia, impacting food security.
Tilapia is the second most important fish species globally.
Rapid and accurate detection methods are essential for managing outbreaks.
This protocol aims to provide a reliable method for TiLV detection.

Purpose of Study

To develop a highly sensitive method for detecting TiLV in fish tissues.
To assist farmers and organizations in controlling TiLV spread.
To provide a step-by-step guide for researchers and practitioners.

Methods Used

Sample collection and euthanization of fish using cloth oil.
RNA extraction from liver tissue using a laminar flow hood.
cDNA synthesis followed by real-time PCR for TiLV detection.
Analysis of amplification curves and CT values to quantify TiLV load.

Main Results

Successful extraction and quantification of RNA from tilapia tissues.
Detection of TiLV using real-time PCR with specific conditions.
Establishment of a standard curve for virus quantification.
Identification of TiLV through melting peak analysis.

Conclusions

The protocol provides a reliable method for TiLV detection.
It can aid in managing outbreaks and ensuring food security.
Future applications may enhance viral disease control in aquaculture.

Frequently Asked Questions

What is Tilapia Lake Virus?

Tilapia Lake Virus is a viral pathogen that causes high mortality rates in tilapia, affecting aquaculture and food security.

Why is rapid detection of TiLV important?

Rapid detection allows for timely intervention to control outbreaks and minimize economic losses in aquaculture.

What methods are used in this protocol?

The protocol includes RNA extraction, cDNA synthesis, and real-time PCR for detecting TiLV.

How does real-time PCR work for TiLV detection?

Real-time PCR amplifies specific DNA sequences, allowing for quantification of the virus based on CT values.

What are the implications of this research?

This research provides a framework for controlling TiLV outbreaks, which is crucial for the sustainability of tilapia farming.

Can this method be applied to other fish species?

While this protocol is specific to tilapia, similar methods may be adapted for other fish species affected by viral diseases.

该协议使用 rt-pcr 方法诊断罗非鱼组织中的罗非鱼病毒 (tilv)。整个方法描述了从组织解剖到总 rna 提取, 然后是 cdna 合成和检测 tilv 的常规 pcr 或定量 pcr 使用 dsdna 结合染料。

欢迎大家我叫温·苏拉切蓬。我是泰国卡塞特斯塔特大学兽医微生物学和免疫学系助理教授。

我和我的团队专门从事罗非鱼中新出现的病毒性疾病。我们现在正在研究罗非鱼湖病毒，这种病毒导致罗非鱼的高死亡率。由于罗非鱼是全世界第二大养殖鱼类，它为一百万人提供蛋白质来源，对粮食安全起着重要作用。

因此，最近暴发的罗非鱼湖病毒引起了社会经济的影响，导致许多科学家试图找到解决这个问题的方法。到目前为止，我们需要一个非常敏感，快速和准确的方法来检测病毒。在此视频中，我们将向您展示从样本采集、样本群和实时 PCR 分析开始检测鱼组织中的罗非鱼湖病毒。

首先，将水中过量的布油溶解，使鱼安乐死。将死鱼放在托盘上，使用 95% 乙醇对设备进行消毒，然后用酒精燃烧器燃烧。慢慢切开鱼从下腹部向上，收集肝脏。

将肝脏的一部分收集到1.5毫升的离心管中。RNA提取的第一部分应在拉米纳尔流罩中进行，并穿戴防护设备。将一毫升RNA提取试剂加入管中。

使用组织害虫将肝脏粉化成均匀。在管中加入200微升氯仿。然后，通过反转管几次轻轻混合。

在室温下留出三分钟。在12，000 RCF的4摄氏度下离心15分钟。小心地从离心机中收集管子。

轻轻将无色上水相的 500 微升转移到新管中。向管中加入一卷异丙醇。在零下20摄氏度下加热两个小时或长达一夜，以沉淀RNA。

孵育后，在相同的离心机条件下将溶液离心。从离心机收集管子，然后丢弃上流液。RNA 颗粒可被箭头视为指向。

将一毫升75%乙醇加入管中清洗RNA颗粒，并反转数次。在摄氏4度，在10，000 RCF下离心15分钟。从离心机收集管子，然后丢弃上流液。

使用自动移液器抽出剩余的乙醇，在室温下空气干燥5至10分钟。加入30至60微升RNase自由水，预热至55至60摄氏度，使RNA颗粒溶解。使用一至两微升RNA溶液使用微体积分光光度计测量浓度。

结果将显示在屏幕上。使用无 RNase 水将浓度稀释到每微升 200 纳克。使用以下列出的化学品和条件进行 cDNA 合成。

使用热循环器将RNA转化为cDNA，并符合建议的条件。以下是使用实时 PCR 确定 TiLV 负载的化学品和条件。将 qPCR 主混合物分配到每瓶白色 qPCR 条管中，将六毫升混合。

随后，将四毫升的cDNA样品加载到井中。在此阶段，必须在每一个井中更换新的管尖，以避免样品受到污染。用透明 qPCR 条盖紧密密封 qPCR 条带。

通过轻拂条带的尖端，轻轻混合溶液。然后使用微型离心机向下旋转，收集容器底部的所有液体。使用视频中显示的两步条件，在 96 井板中选择要使用的井。

选择网络绿色作为染料的植物。选择未知作为示例类型，并将名称插入示例名称框中。打开实时 PCR 机器的盖子，将 qPCR 条带放入分配的井中。

然后合上盖子。使用所选条件运行机器。盖达到所需温度后，机器将启动并运行。

qPCR 条件结束后，显示放大曲线。此处，速率箭头指出阈值水平在指数相位和线性相位之间被切断，从而产生 CT 值。然后将 CT 值外推到病毒日志副本编号中，以计算使用标准曲线保留的病毒数量。

融化峰值还显示，以确定 qPCR 检测到的病毒是否是 TiLV，其中熔融峰值一般在 79.5 至 80.5 摄氏度之间。因此，我们希望这种方法将成为世界各地的农民和组织的重要工具，他们可能需要实施控制并限制TiLV感染的传播。

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