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RPA primer, kit and detection method for detecting four common root-knot nematodes

A root-knot nematode and detection method technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as root-knot nematode detection that has not yet been seen, and achieve low requirements for equipment and high detection The effect of high sensitivity and detection efficiency

Active Publication Date: 2018-09-21
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, RPA technology has been widely used in the rapid detection of viruses, bacteria, mycoplasma and animal parasitic nematodes, but there is no report on the detection of root-knot nematodes.

Method used

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  • RPA primer, kit and detection method for detecting four common root-knot nematodes
  • RPA primer, kit and detection method for detecting four common root-knot nematodes
  • RPA primer, kit and detection method for detecting four common root-knot nematodes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Design and screening of four common root-knot nematode RPA primers

[0043] Taking four reported common root-knot nematode SCAR sequences as target genes, and according to the RPA primer design principles, RPA primers that can be used in the RPA kit (TwistAmp Basic) were designed. Four common root-knot nematode DNAs were used for the screening of RPA primers, and four sets of RPA primer pairs with high amplification efficiency, sensitivity and specificity were obtained. Their sequences are as follows:

[0044] RPA primer pairs for detection of M. incognita:

[0045] Upstream primer Mi-RPA-F (SEQ ID No.1): 5'-GTATCATTATGAAGCTAAGACTTTGGGCTACAA-3';

[0046] Downstream primer Mi-RPA-R (SEQ ID No.2): 5'-TCATCACAACAACCAATACAAACATCCCGCTA-3';

[0047] RPA primer pairs for detection of M. javanica:

[0048] Upstream primer Mj-RPA-F (SEQ ID No.3): 5'-GTGCGCGATTGAACTGAGCCCAGACTGAAACGA-3';

[0049] Downstream primer Mj-RPA-R (SEQ ID No.4): 5'-TATTTTTCAATTGGCCTCCTTGAGTGGGCTTAT-3';...

Embodiment 2

[0057] Establishment of four detection methods for common root-knot nematode RPA

[0058] The detection method for detecting four kinds of common root-knot nematodes of the present embodiment comprises the following steps:

[0059] (1) DNA extraction from the sample: pick a single root-knot nematode 2nd instar larvae, place it in a 200 μL PCR tube containing 20 μL lysis buffer, then freeze in liquid nitrogen for 1 min, and then repeat 3 times in a water bath at 95 °C for 2 min, and finally Incubate at 65°C for 1.5h, process at 95°C for 10min, centrifuge at 14000rpm for 1min, take the supernatant and store it at -20°C for later use; Peanut root-knot nematode and elephant ear root-knot nematode;

[0060] (2) using the DNA extracted in step (1) as the DNA template to be detected, adopt the RPA primer described in Example 1, and carry out the RPA amplification reaction in the RPA reaction tube;

[0061] The reaction conditions of the RPA amplification reaction in step (2) are: r...

Embodiment 3

[0068] Specific detection of four common root-knot nematode RPA primers

[0069] The detection method that is used to detect four kinds of common root-knot nematodes of the present embodiment is the same as embodiment 2, the difference is:

[0070] The samples are root-knot nematode incognita, root-knot nematode java, root-knot nematode peanut, root-knot nematode elephant ear, root-knot nematode northern, Ditynematode rot and soybean cyst nematode respectively.

[0071] Such as figure 2 as shown, figure 2 The labels "1", "2", "3", "4", "5", and "6" refer to the detection results of 6 groups of M. incognita. figure 2 The labels "7", "8", "9", "10", and "11" refer to the detection results of 5 populations of root-knot nematode javanica, figure 2 The labels "12", "13", "14", "15", and "16" refer to the detection results of 5 populations of peanut root-knot nematode, figure 2 The labels "17", "18", "19" and "20" in the table refer to the detection results of 4 populations...

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Abstract

The invention discloses an RPA primer, a kit and a detection method for detecting four common root-knot nematodes, and belongs to the technical field of biosafety. The RPA primer comprises an RPA primer pair for detecting Meloidogyne incognita, an RPA primer pair for detecting Meloidogyne javanica, an RPA primer pair for detecting Meloidogyne arenria and an RPA primer pair for detecting Meloidogyne enterolobii. The invention also provides the kit containing the RPA primer. The invention further provides the detection method based on the RPA primer. The method adopts DNA extracted from a sampleas a template to perform an RPA amplification reaction. An RPA technique is applied to the molecular detection of four common root-knot nematodes for the first time. The RPA primer, the kit and the detection method have the characteristics of strong specificity, high sensitivity, high detection efficiency and low requirements on instruments and devices, and are of great significance for on-site detection, early diagnosis and guided scientific pesticide application of root-knot nematode diseases.

Description

technical field [0001] The invention belongs to the technical field of biological safety, and in particular relates to an RPA primer, a kit and a detection method for detecting four common root-knot nematodes. Background technique [0002] Root-knot nematode (Meloidogyne spp.) is a kind of plant nematode that seriously endangers agricultural production. It can parasitize almost all vascular plants such as vegetables, fruit trees, flowers and weeds, and causes agricultural losses of up to tens of billions of dollars worldwide every year. At present, more than 100 species of root-knot nematodes have been reported, among which, the more serious ones are M.incognita, M.javanica, M.arenaria, etc. ), root-knot nematode (M.hapla) and root-knot nematode (M.enterolobii). In recent years, with the adjustment of my country's planting structure and the expansion of facility vegetable planting scale, especially the phenomenon of continuous cropping in greenhouses and repeated cropping i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2521/507C12Q2522/101C12Q2565/125
Inventor 鞠玉亮操海群潘月敏林宇吴慧平
Owner ANHUI AGRICULTURAL UNIVERSITY
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