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

Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus thoreni and application thereof

A technology for isothermal amplification of Sunny nematodes and loops, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Advanced problems, to achieve the effect of visualization, good sensitivity, and various judgment methods

Inactive Publication Date: 2016-02-24
林康艺
View PDF19 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, foreign countries have reported the method of detecting and quantifying Brachyphytes sonyi by PCR or real-time fluorescent PCR method, but this method needs to use expensive PCR instrument, moreover, also needs to carry out electrophoresis detection, higher to the technical requirement of operator , which is not conducive to the promotion and use in grassroots testing units

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
  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus thoreni and application thereof
  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus thoreni and application thereof
  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus thoreni and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Primer Design

[0028] 1、根据NCBI中多种短体线虫的28s核糖体DNA序列(基因登录号为:EU130854、JX046968、KM200579、JQ303333、JX261951、JX261960、JX261954、EU130875、EU130866、EU130873、EU130845、JN244270、EU130889、HQ662581、 JN091970, AM231916, JX261948, KF765435, DQ498832, JX047005, JQ003994, GU214114, JX144360), the designed primers are as follows:

[0029] (1) Primer set 1:

[0030] Outer primer ptF31 (as shown in SEQ ID NO.1)

[0031] 5'-TGAAACACGGACCAAGGAGT-3'

[0032] Outer primer ptB31 (as shown in SEQ ID NO.2)

[0033] 5'-CACCATCTTTCGGGTCTCAC-3'

[0034] Internal primer ptFIP1 (as shown in SEQ ID NO.3):

[0035] 5'-GCGAGGGATGCCTTCACTTTCAtttaaaTTATCGTGTGCGCAAGTCAT-3'

[0036] Internal primer ptBIP1 (as shown in SEQ ID NO.4):

[0037] 5'-GGTGTCCGAGTGCAGCATGGttttttGCGTACGCTCTTCCTCCA-3'

[0038] (2) Primer set 2

[0039] Outer primer ptF32 (as shown in SEQ ID NO.5)

[0040] 5'-TGAAACCGGTGAGGTGGAA-3'

[0041] Outer primer ptB32 (as shown in SEQ ID NO.6)

[0042] 5'-AGCTGGCCTCAAAACCAAG...

Embodiment 2

[0050] The specific detection of embodiment 2LAMP reaction

[0051] 1. In order to verify the effectiveness and specificity of the primer set and the LAMP reaction system of the present invention, we have simultaneously used the DNA of a plurality of different populations of Brachyphyll sonyi and other non-target nematodes to detect as templates, and the specific nematode species such as Table 1 shows.

[0052] Table 1 The population of nematodes used in the experiment and the corresponding visual detection results

[0053] serial number

nematode species name

population number

source

1

Pratylenchus neglectus

Pn1

Nyingchi, Tibet

2

Brachybody nematodes

Pn2

Zhanjiang, Guangdong

3

Sonny brevis (p. thoreni)

Pt1

Shihezi, Xinjiang

4

Brachybody sonny

Pt2

Lhasa, Tibet

5

Brachybody sonny

Pt3

Sichuan Zitong

6

Brachybody nematodes

Pt4

Shandong Laiyang

...

Embodiment 3

[0058] Example 3 Visualization detection and sensitivity detection of LAMP reaction

[0059] 1, in sum, the ring isothermal amplification method that the present invention establishes to detect the brevium sonyi rapidly is as follows:

[0060] The PCR reaction system is: 1 μL DNA, primers PtFIP1 / PtBIP1 each 1.6 μM, primers PtF31 / PtB31 each 0.2 μM, dNTP 0.35 μM, 2.5 μL 10×BST2.0 DNA polymerase buffer (BST2.0 DNA polymerase buffer), 0.8M betaine, 1.5 μL MgSO 4 (100mM), 1μL BST2.0DNA polymerase (BST2.0DNApolymerase), the balance ddH 2 O make up, a total of 25 μL.

[0061] Reaction conditions: react at 65°C for 60 minutes.

[0062] 2. Carry out LAMP reaction according to the above reaction system and optimized reaction conditions. After the reaction, it can not only be detected by agarose gel electrophoresis, but also can be detected visually by adding SYBRGreenI dye or calcein. The result judgment method is more flexible. more convenient.

[0063] 3. Carrying out LAMP reactio...

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 discloses a loop-mediated isothermal amplification (LAMP) primer group for rapidly detecting pratylenchus thoreni. The primer group comprises an outer primer pair ptF31 / ptB31 and an inner primer pair ptFIP1 / ptBIP1, wherein the sequences of the primers are respectively shown in SEQ ID NO.1-4. An LAMP method is established based on the primer group and is characterized by using deoxyribonucleic acid (DNA) of samples as a template to carry out LAMP. The results can be judged by the following two methods after reaction ends: one method is that amplification products undergo electrophoresis and the samples with specific ladders are judged to be positive; the other method is that the samples having color change observed with the naked eye are positive by adding SYBR green I to a reaction system. The LAMP method has low requirements for instruments and equipment, is rapid and safe, has good specificity and strong sensitivity, provides technical support for detection of pratylenchus thoreni, especially pratylenchus thoreni rapid detection work of grassroots quarantine units, and has good popularization and application values.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a ring isothermal amplification primer for rapid detection of Brachybody sonyi and its application. Background technique [0002] Brachybody nematodes, also known as root-rot nematodes, are an important migratory endoparasitic nematode. This kind of nematode can move, puncture and feed in the plant roots, and at the same time, it will cause the formation of necrotic spots and cavities in the root tissue, which will cause root tissue necrosis. Plants infested by root rot nematodes show symptoms of water and nutrient deficiencies due to root necrosis. Pratylenchus thoreni is one of the three plant nematodes that cause the most crop losses, and Pratylenchus thoreni is one of the most important short-bodied nematodes. Not only are they widely distributed, but they can also infect a variety of important crops. However, different types of crops or d...

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
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119
Inventor 林康艺
Owner 林康艺
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