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Tobacco root-knot nematode disease pathogen detection kit based on loop-mediated isothermal amplification technology and application method of tobacco root-knot nematode disease pathogen detection kit

A tobacco root-knot nematode and ring-mediated isothermal technology, applied in the field of plant disease pathogen detection, can solve the problems of long time-consuming and cumbersome identification process, and achieve the effects of short time-consuming, favorable promotion and application, and wide application range

Inactive Publication Date: 2018-03-20
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the use of conventional PCR detection method, the identification process is cumbersome, time-consuming, and requires relatively expensive equipment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Preparation of LAMP detection kit for the pathogenic nematode of tobacco root-knot nematode

[0062] (1) Design of LAMP primers for the identification of three kinds of tobacco root-knot nematode pathogenic nematodes: according to the genome sequence information of root-knot nematode incognita, root-knot nematode peanut and root-knot nematode javanica published by the National Center for Biotechnology Information (NCBI) , using Primer Explorer V4 software LAMP primers. A set of LAMP primers for the identification of the above three root-knot nematodes was designed, respectively:

[0063] Meloidogyne incognita LAMP primers:

[0064] MiF3: 5'-GTGCTTCGTCTTTTGCTT-3'

[0065] MiB3: 5'-ACTTTCCTTGGAATTGGAACA-3'

[0066] MiFIP:

[0067] 5'-AGGAAGGTATACTATCCAAGACCCA-TTTAAAATCTGTTTCGGCACAC-3'

[0068] MiBIP:

[0069] 5'-TTCACAAAAAACCCAATATGTCAGC-CGATATCTAGGGGTGTTTGA-3'

[0070] Peanut root-knot nematode LAMP primers:

[0071] MaF3: 5'-TGCTTTCAACGCGGTATG-3'

[0072] MaB3:...

Embodiment 2

[0088] Extraction of DNA from tobacco root-knot nematode samples

[0089] Cut 10-20 root knots from the cleaned root samples of tobacco root-knot nematode disease, put them in a high-pressure sterilized mortar, add liquid nitrogen, grind them into powder, and transfer them to 100ng·μl pre-added with 100μl -1 Put proteinase K solution in a centrifuge tube of 1.5ml, heat at 95°C for 10min at 60°C for 1h, 12000r·min -1 Centrifuge for 1 min, and the obtained supernatant is the DNA extract for PCR detection.

Embodiment 3

[0091] Tobacco root-knot nematode pathogenic nematode LAMP detection kit was used for detection

[0092] Take 3 aseptically treated 0.2ml PCR tubes, and add 22 μl each of the I reaction solution, A reaction solution and J reaction solution included in the kit for detecting the pathogenic nematode LAMP of tobacco root-knot nematode disease; Add 2 μl of DNA extract from tobacco root-knot nematode sample and 1 μl of large fragment of Bst DNA polymerase in sequence; after mixing evenly, place the PCR tube in a constant temperature water bath at 60-65°C for 45 minutes and at 82°C for 5 minutes. After the incubation, add 2 uL 100×SYBR Green Ⅰ to each tube, and observe the color change under ultraviolet irradiation after mixing; if the I reaction solution turns green, it means that the sample to be tested contains M. incognita, and if it turns orange, it means that the sample to be tested does not contain Meloidogyne incognita; if the A reaction solution is green, it means that the s...

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Abstract

The invention discloses a tobacco root-knot nematode disease pathogen detection kit based on a loop-mediated isothermal amplification technology and an application method of the tobacco root-knot nematode disease pathogen detection kit. The tobacco root-knot nematode disease pathogen detection kit based on the loop-mediated isothermal amplification technology comprises a reaction solution I for detecting meloidogyne incognita, a reaction solution A for detecting meloidogyne arenaria, a reaction solution J for detecting meloidogyne javanica, a BstDNA polymerase klenow fragment and a 100*SYBR Green I staining solution. The tobacco root-knot nematode disease pathogen detection kit is based on the loop-mediated isothermal amplification technology, has low requirements on detection conditions,can implement the detection only with a water bath pot or a thermostat and facilitates the popularization and application. Since the kit and the method do not need gel electrophoresis, a detection result can be directly observed by naked eyes, so that the consumed time is short, and the detection can be completed in 1.5h; and the kit comprises an LAMP detection primer for detecting the meloidogyneincognita, the meloidogyne arenaria and the meloidogyne javanica, so that the three common pathogen root-knot nematode of tobacco root-knot nematode disease can be synchronously detected.

Description

technical field [0001] The invention belongs to the technical field of plant disease pathogen detection, and in particular relates to a tobacco root-knot nematode pathogen detection kit based on a loop-mediated isothermal amplification technology and a use method thereof. Background technique [0002] Tobacco root-knot nematode is a soil-borne disease that seriously affects tobacco production. In recent years, most chemical nematicides used for the control of root-knot nematode have the disadvantages of high toxicity and high residue, and have been generally banned worldwide. Therefore, it has become the general trend to adopt safe and efficient control methods for root-knot nematode disease. In the process of prevention and control of tobacco root-knot nematode, it is considered to be an economical, efficient and green measure to select appropriate disease-resistant tobacco varieties. However, there are many kinds of root-knot nematodes, and different root-knot nematodes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6888
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2563/107
Inventor 李梅云李文正吴文涛高玉龙吴兴富宋中邦薛美静王丙武王扬焦芳婵曾建敏李永平
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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