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Method of applying RPA technology for detecting suginami root-knot nematode, RPA primer and kit

A root-knot nematode and technical detection technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of difficult application of rapid diagnosis, high professional quality requirements, and long time consumption, so as to protect the safety of agricultural and forestry production in our country , high sensitivity, short time-consuming effect

Active Publication Date: 2019-03-19
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To prevent its invasion, accurate identification must first be carried out. Traditionally, the detection and identification of root-knot nematodes is mainly based on morphology, isoenzyme technology and PCR technology. These technologies require high professional quality of operators and require advanced microscopes and PCR instruments. Such expensive instruments are time-consuming and difficult to apply to rapid diagnosis at ports

Method used

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  • Method of applying RPA technology for detecting suginami root-knot nematode, RPA primer and kit
  • Method of applying RPA technology for detecting suginami root-knot nematode, RPA primer and kit
  • Method of applying RPA technology for detecting suginami root-knot nematode, RPA primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, DNA extraction method

[0041] Pick a single nematode and put it into a 200 μL PCR tube [with 10 μL double-distilled water and 5 μL 10×PCR buffer (without Mg2+)], place it in liquid nitrogen for 1 min (or place it at -70 degrees for 0.5 h), and immediately take it out for 85 ℃ heating 2min. Then add 1 μL of 1 mg / mL proteinase K to the PCR tube, heat at 56°C for 30 minutes, and heat at 95°C for 10 minutes, and microcentrifuge the PCR tube in a microcentrifuge to obtain a DNA template.

Embodiment 2

[0042] Embodiment 2, RPA detection method

[0043] RPA React uses Basic kit, the total reaction system is 50 μL, first add 29.5 μL of reaction buffer, 2.4 μL of 10 μM upstream and downstream primers, 1 μL of DNA template, and ddH in a 0.2 ml PCR tube 2 O 12.2 μL, after mixing, add to the RPA reaction tube containing RPA freeze-dried enzyme powder and mix evenly, add 2.5 μL of 280mM MgAc solution, place the RPA reaction tube in a metal bath, and react at 38°C for 30 minutes, the reaction product detected using gel electrophoresis. Electrophoresis: 5μL RPA product was separated by 10g / L agarose gel electrophoresis, stained with DNA dye and visualized under the ultraviolet gel imaging system. If a specific fragment with a size of 145bp appeared, it was root-knot nematode. The sample without a band at 145bp is not M. suginifera.

Embodiment 3

[0044] Embodiment 3, accuracy and reliability test---the specific amplification of root-knot nematode suginami

[0045] Using the extraction method of Example 1 and the RPA detection method of Example 2, DNA was extracted from the root-knot nematode population in Table 1 respectively, and RPA detection was carried out with DNA as a template. As a result, only root-knot nematode suginami was specifically amplified products, other root-knot nematode populations had no bands. partial results such as figure 1 As shown, each lane is respectively: M: DL100 molecular marker (its bands are 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom); 1: Apple Root-knot nematode strain M1; 2: Root-knot nematode strain C1 of camellia; 3: Root-knot nematode strain MIYN1; 4: Root-knot nematode strain MHSD1; 5: Root-knot nematode MAJS2; 6: Root-knot nematode java Nematode MJFJ1; 7: Meloidogyne elegans MEHN2; 8: Meloidogyne hispanica MSHM1; 9: Meloidog...

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Abstract

The invention provides a RPA primer of applying RPA technology for detecting suginami root-knot nematode and a RPA kit comprising the RPA primer. Sequences of the RPA primer are as follows: upstream primer is MSRPAF:5'-TCCCTTCTCATGGGCTCATTAAGTCTTAAACCG-3', and downstream primer MSRPAR:5'-ACTTTGGTCGTGTAACGGCTAACGCTGGTGTCT-3'. The invention further provides a method utilizing the RPA kit to detect suginami root-knot nematode. The method can detect single or mixed DNA samples of suginami root-knot nematode , is accurate in detection, high in sensitivity, simple in operation, short in time consumption and high in repeatability, can accurately identify suginami root-knot nematode in seedlings coming in border and is of important significance in preventing invasion of non-Chinese species of root-knot nematode and protecting agriculture and forestry production safety of China.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a set of primers and a method for detecting root-knot nematode firmilax by using a recombinase polymerase isothermal amplification technology. technical background [0002] Root-knot nematode (Meloidogyne Goeldi, 1887) is an obligate endoparasitic nematode of plant roots. It is one of the most diverse, widely distributed and most harmful groups of plant pathogenic nematodes, and has very important economic significance. In 2007, my country included root-knot nematode (non-Chinese species) in the "List of Imported Plant Quarantine Pests of the People's Republic of China", and implemented strict quarantine at ports to protect my country's agricultural and ecological safety. [0003] my country's port entry-exit quarantine laboratories or other relevant departments are faced with the problem of rapid and accurate identification of root-knot nematodes. In recent years, altho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888
CPCC12Q1/6888C12Q2600/124
Inventor 方亦午顾建锋刘乐乐陈先锋
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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