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A detection kit for the visual detection of D. rottennsis and its application

A potato and stem nematode technology, applied in the field of detection kits for visual detection, can solve the problems of difficulty in widespread use and high price, and achieve the effects of convenient and timely guidance, low equipment cost, and increased stability

Active Publication Date: 2022-07-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, PCR gene amplification technology is widely used because of its simplicity and ease, but it needs to rely on expensive heating and cooling thermal cyclers, which will be limited by the environment and equipment, and it is difficult to promote it on a large scale.

Method used

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  • A detection kit for the visual detection of D. rottennsis and its application
  • A detection kit for the visual detection of D. rottennsis and its application
  • A detection kit for the visual detection of D. rottennsis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Design and screening of primers and probes amplified by recombinase polymerase to identify D. rottennsis

[0062] 1. Design of primers and probes for recombinase polymerase amplification

[0063] D. potato destructor Types A-G, M. incognita, K. elegans, C. beet, C. soy, C. Phillips, C. gramineatum, C. Scribner via NCBI download The gene sequences of nematodes such as nematodes , Caffrea short-body nematodes, pine wood nematodes, D. dwarf nematodes, double-tailed nematodes, oat nematodes and other nematodes were compared to determine the specificity of the ITS2 sequence conserved regions of D. potato rot. Targets D. rot, but not R. incognita, weevil root-knot nematode, beet cyst nematode, soybean cyst nematode, Philip cyst nematode, graminearum, potato golden nematode, Scree The nematodes such as Brachiomorpha bunner, Brachyodon caffeine, B. xylophilus, D. dwarf, and S. oat nematodes can be used for the specific detection of D. rotten nematodes.

[0064] Des...

Embodiment 2

[0113] Embodiment 2. Sensitivity test for identifying D. rottennsis of potato

[0114] Take gDNA from a single nematode and dilute it to 1 / 5, 1 / 5 of the original concentration 2 , 1 / 5 3 , 1 / 5 4 , 1 / 5 5 , 1 / 5 6 , 1 / 5 7 A total of 8 concentrations were used to evaluate the sensitivity of the recombinase polymerase amplification reaction of the present invention.

[0115] use kits and kit for amplification.

[0116] The primers of the kit are the first set of special primers DdNFO-F2 and DdNFO-R2 of the present invention, and the probe is DdITS2-NFO; The primers of the kit are the second group of dedicated primers DdITS2-F2 and DdITS2-R2 of the present invention.

[0117] 1. Extract the DNA of a single nematode to be tested

[0118] The method is the same as in Example 1, and the DNA of D. rottennsis (concentration is 0.1 head / μL) is serially diluted by 5 times, and the DNA of different concentrations after the dilution is used as the template to carry out the recomb...

Embodiment 3

[0127] Example 3. Recombinase polymerase amplification specificity test for identifying D. rottennsis of potato

[0128] The A-C type and other nematodes of different geographical populations of D. rottenta were used as test materials (Table 3) to detect the recombinase polymerase amplification technology of the present invention, agarose gel electrophoresis detection method and lateral flow chromatography test strip detection Specificity after method binding. use kits and kit for amplification. The primers of the kit are the first group of special primers DdNFO-F2 and DdNFO-R2 of the present invention, and the probe is Dd-NFO; The primers of the kit are the second group of dedicated primers DdITS2-F2 and DdITS2-R2 of the present invention.

[0129] 1. Extracting a single DNA of the nematode to be tested: the method is the same as that of Example 1, wherein the nematode to be tested is shown in Table 3.

[0130] Table 3 shows the species or populations of nematodes us...

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Abstract

The invention discloses a detection kit for visual and rapid detection of D. rottennsis of potato and its application. The present invention provides a primer for identifying D. rottennsis, which consists of primer F and primer R; the primer F is a single-stranded DNA molecule shown in sequence 1; the primer R is a single-stranded DNA molecule shown in sequence 2 DNA molecule. The invention provides a detection kit for visual and rapid detection of D. rottennsis of potato and its application, and provides technical support for field detection and field diagnosis. The amplification technology of the present invention is easy to operate, does not require special learning, and after being combined with the colloidal gold chromatography test strip method, the detection results can be directly observed with the naked eye, which can assist in solving the urgent problems in current plant quarantine and plant protection.

Description

technical field [0001] The invention belongs to the technical field of detection of plant parasitic nematodes, in particular to a detection kit for visual detection of D. rotten nematodes and applications thereof. Background technique [0002] Ditylenchus destructor (Throne, 1945) is a global quarantine pest with more than 100 host species, and potato is its main host, causing potato dry rot. D. destructor was introduced to northern my country from Japan in 1937, and now occurs in Hebei, Inner Mongolia, Shaanxi, Jilin, Liaoning, Shandong, Henan, Anhui and other provinces and cities, which can seriously harm the entire growth period and storage of potatoes and sweet potatoes. Expect. The newly revised "National List of Agricultural Phytosanitary Pests" in November 2020 lists three quarantine plant nematodes, D. rot is one of them. In 2019, my country's potato planting area reached more than 86 million mu, ranking first in the world in terms of planting area and yield. D. rot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11G01N33/543G01N33/558
CPCC12Q1/6888G01N33/54313G01N33/558
Inventor 刘倩陈潇威简恒
Owner CHINA AGRI UNIV
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