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

A technology of short-bodied puncture nematodes and ring isothermal amplification, which is applied in the direction of DNA/RNA fragments and recombinant DNA technology, can solve the problems of unfavorable promotion and use of grassroots detection units, high technical requirements for operators, and achieve visualization and good sensitivity , good detection effect

Inactive Publication Date: 2016-02-24
林康艺
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, foreign countries have reported the method of detecting and quantifying the short-bodied puncture nematode 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, It is not conducive to the promotion and use in grassroots testing units

Method used

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  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus penetrans and application thereof
  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus penetrans and application thereof
  • Loop-mediated isothermal amplification (LAMP) primers for rapidly detecting pratylenchus penetrans 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 peF31 (as shown in SEQ ID NO.1)

[0031] 5'-CCGGATTGGAGGAATGTTGT-3'

[0032] Outer primer peB31 (as shown in SEQ ID NO.2)

[0033] 5'-GCGCACACGATAAACTCCTT-3'

[0034] Internal primer peFIP1 (as shown in SEQ ID NO.3):

[0035] 5'-CACATGTTGCATGCAACTGCCAaaaaTTTTGGATGTGAATGGGGGA-3'

[0036] Internal primer peBIP1 (as shown in SEQ ID NO.4):

[0037] 5'-TTCTGCCAATTCGGTCCTGTGCttttttGTTTCAAGACGGGTCGGAT-3'

[0038] (2) Primer set 2

[0039] Outer primer peF32 (as shown in SEQ ID NO.5)

[0040] 5'-TCGAGTTGGTGTGGGGTG-3'

[0041] Outer primer peB32 (as shown in SEQ ID NO.6)

[0042] 5'-GGCAATCGTGATTGCTCAG...

Embodiment 2

[0048]The specific detection of embodiment 2LAMP reaction

[0049] 1. In order to verify the effectiveness and specificity of the primer set and LAMP reaction system of the present invention, we simultaneously used the DNA of a plurality of different populations of Brachyphyll puncture nematodes and other non-target nematodes as templates for detection. The specific nematode species are shown in the table 1.

[0050] Table 1 The population of nematodes used in the experiment and the corresponding visual detection results

[0051]

[0052]

[0053] 2. Extract the DNA of each nematode in Table 1, respectively, and use primer set 1 and primer set 2 to carry out LAMP amplification according to the LAMP reaction conditions described in Example 2.

[0054] 3. The results are attached figure 2 , attached image 3 and as shown in Table 1, figure 2 Among them, tubes 3, 4, 5, 6, 7, and 8 have color changes and are positive; image 3 Among them, tubes 2, 3, 4, 5, 6, 7, and 8...

Embodiment 3

[0057] Example 3 Visualization detection and sensitivity detection of LAMP reaction

[0058] 1. In summary, the ring isothermal amplification method for the rapid detection of Brachybody puncture nematode established by the present invention is as follows:

[0059] The PCR reaction system is: 1 μL DNA, primers PeFIP1 / PeBIP1 each 1.6 μM, primers PeF31 / PeB31 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.

[0060] Reaction conditions: react at 65°C for 60 minutes.

[0061] 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.

[0062] 3. Car...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) primer group for rapidly detecting pratylenchus penetrans. The primer group comprises an outer primer pair peF31 / peB31 and an inner primer pair peFIP1 / peBIP1, 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 penetrans, especially pratylenchus penetrans 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 loop isothermal amplification primer for rapid detection of Brachybody puncture nematode 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. Short-bodied nematodes are one of the three plant nematodes that cause the most crop losses, and Pratylenchus penetrans is one of the most important short-bodied nematodes. They can infect more than 3,000 different species of plants and are widely distributed. However, different types o...

Claims

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Application Information

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