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A kit for the detection of root-knot nematode Elephant ear based on loop-mediated isothermal amplification and its application

A loop-mediated isothermal, root-knot nematode technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to avoid time loss, less technology dependence, and low cost

Active Publication Date: 2011-12-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no kit and method for detection of root-knot nematode by loop-mediated isothermal amplification method

Method used

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  • A kit for the detection of root-knot nematode Elephant ear based on loop-mediated isothermal amplification and its application
  • A kit for the detection of root-knot nematode Elephant ear based on loop-mediated isothermal amplification and its application
  • A kit for the detection of root-knot nematode Elephant ear based on loop-mediated isothermal amplification and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the composition of kit

[0049] 1. Preparation of each component of the kit

[0050] 1. Preparation of primers

[0051] Specific oligonucleotide primers were designed according to the 5S rDNA gene and IGS2 region of Meloidogyne elegans, where there were polymorphic segments among species. A set of dedicated primers was synthesized, consisting of outer primers (Me-F3 and Me-B3, inner primers (Me-FIP: and Me-BIP) and loop primers (Me-LP and Me-BP).

[0052] Me-F3: 5'-CCAAGTACTAAGGAAGCCC-3' (sequence 1 of the sequence listing);

[0053] Me-B3: 5'-ATCCTAATTTTCCTCCCACACA-3' (sequence 2 of the sequence listing);

[0054] The 5' end Biotin mark of Me-FIP, nucleotide sequence is as follows (sequence 3 of sequence listing):

[0055] 5'-ACAGTGATTACGACCATACCGCGTTAACGTTGCTTAACTTGCCAGA-3';

[0056] Me-BIP: 5'-TCTAAGGCAAAGTGGGCGGAGCTCTTTTTGCCTTAAACCATTCCC-3' (SEQ ID NO: 4 of the Sequence Listing);

[0057] Me-LP: 5'-AAGCACGCCATCCCGTC-3' (sequence 5 of the sequence...

Embodiment 2

[0097] Embodiment 2, the application of kit

Embodiment 3

[0117] Embodiment 3, the sensitivity detection of kit

[0118] 1. A small amount of genomic DNA extraction

[0119] 1. Collect freshly hatched 2nd instar root-knot nematode larvae, centrifuge and wash in a 1.5ml centrifuge tube to form 10-20 μL nematode pellet.

[0120] 2. Use tweezers to freeze the tube and pointed glass rod in liquid nitrogen for 30 seconds, and grind it into powder vigorously.

[0121] 3. Add 700 μL of DNA extraction buffer (containing 50 mM Tris-HCl, pH 7.5, 50 mmol / L NaCl, 5 mM EDTA, 0.5% SDS) and 7 μL of 20 mg / mL proteinase K, and mix gently.

[0122] 4. Put the tube in a 50°C water bath for 4-5h.

[0123] 5. Next, extract once with equal volumes of Tris saturated phenol and chloroform / isoamyl alcohol, and centrifuge at 12,000 rpm for 10 min.

[0124]6. Take the supernatant into a new tube, add 0.1 times the volume of 3M NaAc (pH5.2) aqueous solution and 2.5 times the volume of pre-cooled absolute ethanol, and precipitate at -20°C for 20 minutes.

[...

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PUM

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Abstract

The invention discloses a Meloidogyne enterolobii detection kit based on loop-mediated isothermal amplification and application thereof. The invention provides special primers for assisted identification of Meloidogyne enterolobii, which are composed of DNAs (deoxyribonucleic acids) disclosed as Sequences 1 to 6 in the sequence table. The kit disclosed by the invention contains the special primers. By designing specific primers and optimizing reaction conditions, the invention provides a kit and method for qualitatively detecting Meloidogyne enterolobii 5S rDNA-IGS2 region. The invention solves the problems of high cost, low efficiency, low speed, and incapability of satisfying demands for plant quarantine and plant protection in the conventional molecular detection.

Description

technical field [0001] The invention relates to a kit for detecting root-knot nematode Elephantia stifolium based on loop-mediated isothermal amplification and an application thereof. Background technique [0002] Meloidogyne enterolobii is a highly pathogenic and widely harmful quarantine plant root-knot nematode, which can parasitize in Fabaceae, Cucurbitaceae, and Solanaceae. , Myrtaceae (Myrtaceae), Annonaceae (Annonaceae) and arrowroot (Marantaceae) and other plants. Like other root-knot nematodes, root-knot nematodes cause root nodules and plant decay, and are highly infective. In addition, the virulence exhibited by M. elegans can overcome several sources of resistance of other common root-knot nematodes, such as the resistance of the tomato cultivar resistance gene Mi-1. [0003] At present, root-knot nematode has occurred in many countries in Asia, Europe, Africa and North and South America. It poses a huge potential threat to agricultural production and environme...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 简恒牛俊海郭全新刘倩陈昌龙
Owner CHINA AGRI UNIV
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