Ring isothermal amplification primers capable of fast detecting pratylenchus neglectus and application thereof

A technique for isothermal amplification of nematodes and loops, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve good repeatability, good detection effect, and ensure safety.

Inactive Publication Date: 2016-02-17
林康艺
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the deficiencies in the existing detection technology of brevity nematodes, and provide a set of primers and detection methods for the rapid detection of brevity nematodes

Method used

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  • Ring isothermal amplification primers capable of fast detecting pratylenchus neglectus and application thereof
  • Ring isothermal amplification primers capable of fast detecting pratylenchus neglectus and application thereof
  • Ring isothermal amplification primers capable of fast detecting pratylenchus neglectus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Primer Design

[0029] 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:

[0030] (1) Primer set 1:

[0031] Outer primer pnF31 (as shown in SEQ ID NO.1):

[0032] 5'-ACGGATAGAGCCAGCGTA-3'.

[0033] Outer primer pnB31 (as shown in SEQ ID NO.2):

[0034] 5'-TCGGGTTCCAGCAAGCT-3'.

[0035] Internal primer pnFIP1 (as shown in SEQ ID NO.3):

[0036] 5'-TCCGTCCCAATCTGGAGAGCGGttttttTCGGGCCAGCATTCATCTG-3'

[0037] Internal primer pnBIP1 (as shown in SEQ ID NO.4):

[0038] 5'-GTTTGTACCCGGCCGAGCGttttttGCAAAAGCAGGTTCACACC-3'

[0039] (2) Primer set 2

[0040] Outer primer pnF32 (as shown in SEQ ID NO.5)

[0041] 5'-ACGTGAACCGGTGAGGTG-3'

[0042] Outer primer pnB32 (as shown in SEQ ID NO.6)

[0043] 5'-TCGGGTTCCAGCAAGCT-3' ...

Embodiment 4

[0060] The specific detection of embodiment 4LAMP reaction

[0061] 1. In order to verify the effectiveness and specificity of the primers and the LAMP reaction system of the present invention, we simultaneously used the DNA of a plurality of different populations of the selected short-body nematodes and other non-target nematodes as templates for detection. The specific nematode species are shown in Table 1 shown.

[0062] Table 1 The population of nematodes used in the experiment and the corresponding visual detection results

[0063]

[0064]

[0065] 2. Extract the DNA of each nematode in Table 1, and use primer set 1, primer set 2, and primer set 3 respectively to perform LAMP amplification according to the LAMP reaction conditions described in Example 2.

[0066] 3. The results are attached figure 2 , attached image 3 , attached Figure 4 and as shown in Table 1, figure 2 (Primer set 1) Tubes 1, 2, 3, 4, and 7 had color changes and were positive; image 3 ...

Embodiment 3

[0069] Example 3 Visualization detection and sensitivity detection of LAMP reaction

[0070] 1. In summary, the method of ring isothermal amplification for the rapid detection of the rejected short-bodied nematode established by the present invention is as follows:

[0071] The PCR reaction system is: 1 μL DNA, primers pnFIP2 / pnBIP2 each 1.6 μM, primers pnF32 / pnB32 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.

[0072] Reaction conditions: react at 65°C for 60 minutes.

[0073] 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.

[007...

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Abstract

The invention discloses a ring isothermal amplification primer group capable of fast detecting pratylenchus neglectus. The primer group comprises an outer side primer pair pnF32 / pnB32 and an inner side primer pair of pnFIP2 / pnBIP2. The sequences of primers are shown as the SEQ ID NO.5-8. A ring isothermal amplification method is established according to the primer group, ring isothermal amplification is performed with sample DNA as a template, a result can be adjudged in two modes after reaction ends, according to one mode, electrophoresis is performed on amplification products, and samples with a specific ladder-like strip are considered as positive, and according to the other mode, SYBR green I is added into a reaction system, and samples where color changes are observed through naked eyes are considered as positive. The ring isothermal amplification method is low in requirement for instruments and equipment, fast, safe, good in specificity and high in sensitivity, the method provides technical support for detection of pratylenchus neglectus, particularly, a basic layer quarantine unit provides technical support for fast detection work of pratylenchus neglectus, and good application and popularization value is achieved.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a loop isothermal amplification primer for rapid detection of unselected short-body nematodes and its application. Background technique [0002] Brachybody nematodes, also known as root-rot nematodes, are an important migratory endoparasitic nematode consisting of 68 valid species. Root rot nematodes move, puncture and feed in the root of the plant, which 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 neglectus is one of the three plant nematodes that cause the greatest economic losses, and Pratylenchus neglectus is one of the most important nematodes. Not only are they widely distributed, but they can also infect a variety of important crops. However, diff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2563/107C12Q2565/125
Inventor 林康艺
Owner 林康艺
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