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 expen

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

Example Embodiment

[0027] Example 1 Primer design

[0028] 1. According to the 28s ribosomal DNA sequence of various short-body nematodes in NCBI (gene accession numbers: EU130854, JX046968, KM200579, JQ303333, JX261951, JX261960, JX261954, EU130875, EU130866, EU130873, EU130845, JN244270, EU130889, HQ662581, JN091970, AM231916, JX261948, KF765435, DQ498832, JX047005, JQ003994, GU214114, JX144360), the design primers are as follows:

[0029] (1) Primer set 1:

[0030] Outer primer peF31 (as shown in SEQIDNO.1)

[0031] 5’-CCGGATTGGAGGAATGTTGT-3’

[0032] Outer primer peB31 (as shown in SEQIDNO.2)

[0033] 5’-GCGCACACGATAAACTCCTT-3’

[0034] Inner primer peFIP1 (as shown in SEQIDNO.3):

[0035] 5’-CACATGTTGCATGCAACTGCCAaaaaTTTTGGATGTGAATGGGGGA-3’

[0036] The inner 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 SEQIDNO.5)

[0040] 5’-TCGAGTTGGTGTGGGGTG-3’

[0041] Outer primer peB32 (as show...

Example Embodiment

[0048] Example 2 Specific detection of LAMP reaction

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

[0050] Table 1 Nematode populations used in the experiment and the corresponding visual detection results

[0051]

[0052]

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

[0054] 3. The results are attached figure 2 , Attached image 3 And Table 1, figure 2 Among them, the colors of tubes 3, 4, 5, 6, 7, and 8 are positive; image 3 Among them, the colors of tubes 2, 3, 4, 5, 6, 7, and 8 are positive.

[0055] The results show that in the ...

Example Embodiment

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

[0058] 1. In summary, the loop isothermal amplification method established by the present invention for rapid detection of Brachytomonas spp. is as follows:

[0059] The PCR reaction system is: 1μL DNA, primers PeFIP1 / PeBIP1 each 1.6μM, primers PeF31 / PeB31 each 0.2μM, dNTP 0.35μM, 2.5μL 10×BST2.0DNA polymerase buffer (BST2.0DNApolymerasebuffer), 0.8M betaine, 1.5μLMgSO 4 (100mM), 1μLBST2.0 DNA polymerase (BST2.0 DNA polymerase), balance ddH 2 O make up, a total of 25μL.

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

[0061] 2. Carry out the LAMP reaction according to the above reaction system and optimized reaction conditions. After the reaction, it can be detected not only by agarose gel electrophoresis, but also by adding SYBRGreenI dye or calcein for visual detection. The result judgment method is more flexible and more convenient.

[0062] 3. Using different concentrations of Brachymeria elegans DNA...

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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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