Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detection of turnip mosaic virus using reverse transcription loop-mediated isothermal amplification technique

A turnip mosaic virus and loop-mediated isothermal technology, applied in the field of plant virology, can solve the problems of low accuracy, long test period and low sensitivity, achieve high specificity, reduce detection cost, and improve detection efficiency. Effect

Inactive Publication Date: 2016-08-24
NORTHWEST A & F UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods have different disadvantages, for example, the required test period is long, the operation is cumbersome, the sensitivity is not high, the specificity is not strong, the accuracy is low, and it is easy to be limited by the detection materials, etc.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detection of turnip mosaic virus using reverse transcription loop-mediated isothermal amplification technique
  • Method for detection of turnip mosaic virus using reverse transcription loop-mediated isothermal amplification technique
  • Method for detection of turnip mosaic virus using reverse transcription loop-mediated isothermal amplification technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Primer Design

[0052] First, the inventor downloaded the whole genome sequence of TUMBV from NCBI, and designed multiple sets of primers using Primer4.0 (http: / / primerexplorer.jp / e / v4_manual / index.html), and then according to the The primers were selected based on comprehensive factors such as the conservation of the sequence region, the hairpin structure of the primer, the GC content of the dimer and the Tm value. The primer set has 4 primers, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP). The primers refer to SEQ ID NO.1-4. Primers for RT-PCR, the present invention uses the primers of Zhuang Mu et al., refer to SEQ ID NO.5-6. See Table 1 for information on all primers.

[0053] The information of the primer sequence SEQ ID NO.1-4 in table 1:

[0054]

[0055] 2. RNA extraction

[0056] Using the polysaccharide polyphenol plant total RNA rapid extraction kit (centrifugal column) (Bioteke), according to the operating instructions, e...

Embodiment 2

[0060] Example 2 , RT-PCR and RT-LAMP detection sensitivity comparison:

[0061] In order to compare the sensitivity of RT-LAMP and RT-PCR, the present invention uses a pair of specific primers containing TuMVCP gene by Zhuang Mu et al.

[0062] The RT-PCR process is as follows:

[0063] The RT-PCR reaction system is 12.5 μl, including 3.5 μl DEPC-treated water, 0.5 μl downstream primers, and 1 μl ASGV RNA, then bathed in 70°C water for 5 minutes, then placed on ice for 5 minutes, then added 1.75 μl DEPC-treated water, 2.5 μl dNTPs (10mM) , 2.5μl 5×M-MLV buffer (50mM Tris-HCL, 75mM KCL, 3mM MgCl 2 , 10 mM DTT), 0.5 μl M-MLV reverse transcriptase, 0.25 μl RNase inhibitor, then react at 42° C. for 1 h, and then react at 95° C. for 5 minutes. Take 2 μl of the reaction product for PCR reaction. For the operation method, refer to the method of Zhuang Mu et al. [1] .

[0064] Then the RT-PCR amplified product was recovered by Bioteke Gel Extraction Kit and connected to pMD18-T...

Embodiment 3

[0068] Example 3 , RT-PCR and RT-LAMP to detect field samples:

[0069] Several field samples were detected by RT-PCR and RT-LAMP respectively, and the results of the two methods were consistent.

[0070] The RT-LAMP detection method does not require expensive instruments and equipment, the operation procedure is simple, and it has the characteristics of fast, efficient, high specificity, and high sensitivity. Its application range is becoming wider and wider, and it has played a great role in the detection of pathogenic microorganisms. Advantages, and successfully applied in the related research of tumor genes. The RT-LAMP method described in this experiment and the RT-PCR method were used to simultaneously detect multiple samples of diseased plants such as rapeseed, Chinese cabbage, and cauliflower, and the results were consistent. It is obvious that the RT-LAMP method can save a lot of time, and easy to use.

[0071] In a word, the present invention establishes a set of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for detecting a turnip mosaic virus (TuMV) by adopting a reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology. The method comprises the following steps: performing total RNA extraction, RT-LAMP reaction, real-time turbidity detection and electrophoresis detection on the TuMV, and determining whether the TuMV is existent in a sample. RT-LAMP primers are designed according to a conservation region of the TuMV gene, and the method for quickly, sensitively and highly specifically detecting the TuMV is created by adopting the one-step RT-LAMP technology. A novel means is provided for quickly detecting the TuMV.

Description

technical field [0001] The invention belongs to the technical field of plant virology, and in particular relates to a method for detecting turnip mosaic virus by adopting reverse transcription loop-mediated isothermal amplification technology. Background technique [0002] Turnip mosaic virus is one of the members of the genus Potyvirus. It has a wide range of hosts and can infect more than 300 kinds of plants belonging to 156 genera in 43 families, including important vegetables such as rapeseed, cabbage, cabbage, and cauliflower. It can cause a 5-10% reduction in vegetable production every year. The disease is serious and difficult to control, and has always been an important problem in vegetable production. To prevent and treat viral diseases, we must first prescribe the right medicine. Regarding the detection methods of the virus, the detection methods commonly used at present mainly include indicator plant method, electron microscope technology, serological technology...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 赵磊郝兴安吴云锋
Owner NORTHWEST A & F UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com