Kit for detection of D. destructor in sweet potato based on loop-mediated isothermal amplification and its application

A ring-mediated isothermal and stem nematode technology, applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of avoiding time loss, high positive rate, and low technical dependence

Active Publication Date: 2011-11-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There is no kit and method for detection of D. destructor in sweet potato by the loop-mediated isothermal amplification method

Method used

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  • Kit for detection of D. destructor in sweet potato based on loop-mediated isothermal amplification and its application
  • Kit for detection of D. destructor in sweet potato based on loop-mediated isothermal amplification and its application
  • Kit for detection of D. destructor in sweet potato based on loop-mediated isothermal amplification and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the composition of kit

[0055] 1. Preparation of each component of the kit

[0056] 1. Preparation of primers

[0057] According to the rDNA-ITS region of D. destructor in sweet potato, the specific oligonucleotide primers were designed in the section where interspecific polymorphism existed. Three sets of primers were synthesized. The first set of primers consisted of universal outer primers (DdU-F3 and DdU-B3) and universal inner primers (DdU-FIP and DdU-BIP), and the second set of primers consisted of D. sweetpotato nematode-specific outer primers (DdA-F3 and DdA-B3), type A D. sweetpotato-specific inner primers (DdA-FIP and DdA-BIP) and type A D. sweetpotato-specific loop primers (DdA-LP and DdA-BP), the third group The primers consisted of type B D. sweetpotato nematode specific outer primers (DdB-F3 and DdB-B3) and type B D. sweet potato specific inner primers (DdB-FIP and DdB-BIP).

[0058] DdU-F3: 5'-AGTTGTATGCTTCTTTTTGTCC-3' (sequence 1 of the...

Embodiment 2

[0106] Embodiment 2, the application of kit

[0107] 1. Genomic DNA extraction

[0108] Genomic DNA of a single nematode was extracted from seven nematode experimental samples as follows:

[0109] 1. Add 20 μl of sample pretreatment solution to the cover glass, pick a single nematode in the sample pretreatment solution, squeeze the cover glass to rupture the worm body, and obtain the body fluid mixture; draw 10 μl of the body fluid mixture, and transfer it to a pre-installed 10 μl nematode. in a 1.5ml centrifuge tube of bacterial water.

[0110] 2. Quick freezing in liquid nitrogen for 1 minute, 65°C water bath for 20 minutes, and 95°C water bath for 5 minutes.

[0111] 3. Centrifuge at 12,000 rpm for 5 minutes, and take 5 μl of the supernatant as a reaction template (genomic DNA).

[0112] 2. Identify whether the sample to be tested is D. destructor of sweet potato

[0113] Genomic DNA extracted from 7 kinds of nematodes were subjected to LAMP reaction respectively.

[0...

Embodiment 3

[0125] Embodiment 3, the sensitivity detection of kit

[0126] 1. A small amount of genomic DNA extraction

[0127] Carry out the following steps with type A sweet potato D. destructor and B type D. rot:

[0128] 1. Collect freshly isolated D. destructor from sweet potato, centrifuge and wash in a 1.5ml centrifuge tube to form 10-20μL nematode pellet.

[0129] 2. Use tweezers to freeze the tube and pointed glass rod in liquid nitrogen for 30 seconds, and grind it into powder vigorously.

[0130] 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.

[0131] 4. Put the tube in a 50°C water bath for 4-5h.

[0132] 5. Next, extract once with equal volumes of Tris saturated phenol and chloroform / isoamyl alcohol, and centrifuge at 12,000 rpm for 10 min.

[0133] 6. Take the supernatant into a new tube, add 0.1 times the volume of 3M NaAc (pH5.2) aqueous solution and 2.5 ti...

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Abstract

The invention discloses a kit for detecting Ditylenchus destructor based on loop-mediated isothermal amplification and application thereof. The invention provides a special primer for assistant identification of Ditylenchus destructor and consists of DNAs shown in sequences 1-14 in a sequence table. The kit provided by the invention comprises the special primer. According to the invention, the kit for assistant identification of Ditylenchus destructor is provided through designing the specific primer and optimizing reaction conditions; and through applying the kit, an interspecies level and an infraspecies level of detection can be achieved, and the problems that the routine molecule detection method has the defects of high cost, low efficiency and slow speed and can not meet the requirements of current plant quarantine and plant protection are solved.

Description

technical field [0001] The invention belongs to the technical field of plant parasitic nematode detection, in particular to a kit for detecting sweet potato rot nematode (Ditylenchus destructor) based on loop-mediated isothermal amplification and an application thereof. Background technique [0002] Ditylenchus destructor, also known as Ditylenchus destructor or Ditylenchus destructor, was first found on potatoes and causes potato rot, so it is also called Ditylenchus rot nematode (Potato rot nematode). D. destructor is a migratory endoparasitic nematode that mainly damages the underground parts of plants, especially tubers and bulbs. D. destructor has a very wide range of hosts. There are more than 120 known hosts, and tuber crops such as potatoes and sweet potatoes and some flower bulbs suffer the most. D. destructor of sweet potato is an important plant parasitic nematode in agricultural production, and it is also a quarantine dangerous nematode in my country and many ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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