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LAMP rapid detection method for Heterodera filipjevi and its application

A detection method, the technology of Philips spore, is applied in the directions of biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of limiting the popularization and application of PCR detection methods, and achieve the effects of short detection time, direct results and simple operation steps.

Inactive Publication Date: 2013-05-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR detection requires expensive professional instruments such as PCR instrument, gel electrophoresis and imaging system (ultraviolet instrument) and molecular biology reagents, and requires professional laboratory personnel in molecular biology to operate. The above detection can only be detected under laboratory conditions. , It takes a long time, which limits the popularization and application of PCR detection method in production. In the investigation of the occurrence and distribution of cereal cyst nematodes and the rapid diagnosis in the field, there is an urgent need for a simple and fast detection method

Method used

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  • LAMP rapid detection method for Heterodera filipjevi and its application
  • LAMP rapid detection method for Heterodera filipjevi and its application
  • LAMP rapid detection method for Heterodera filipjevi and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Extraction, RAPD amplification and sequence analysis of Phillips cyst nematode DNA

[0079] 1.1 Extraction of Phillips cyst nematode DNA

[0080] Pick a single Phillips cyst and place it in a medium containing 10 μl ddH 2 In a 0.2mL centrifuge tube of O, freeze with liquid nitrogen, take it out and put it on ice, turn it in the centrifuge tube with a sterile glass rod until the ice melts, break the cysts, release the eggs, add 8 μl of LB solution, 2 μl of 600 μg / ml proteinase K solution was then frozen at -80°C for 30 minutes. The centrifuge tube was taken out, incubated at 65°C for 90min, and reacted at 95°C for 10min. After treatment, the supernatant was directly used as a nematode DNA template for LAMP and PCR reactions.

[0081] 1.2 Amplification and sequence analysis of SCAR fragments of Phillips cyst nematode

[0082] SCAR marker primers OPK16-HfF2 (5`-CAGGACGAAACTCATTCAACCAA-3`) and OPK16-HfR2 (5`-AGGGCGAACAGGAGAAGATTAGA-3`) were used to amplify the ...

Embodiment 2

[0083] Example 2 Establishment of LAMP Technology Detection Method for Philip's Cyst Nematode

[0084] 2.1 LAMP primer design

[0085] According to the sequencing results of the SACR-specific fragments of Phillipe cyst nematode, the following LAMP primers were designed and screened (see figure 1 ), the primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd. The primer sequences are as follows:

[0086] ①HF11-F3: 5`-GGCAGCGATCAAAAGACT-3`;

[0087] ②HF11-B3: 5`-AAATGTGATGTTCCCAAGTG-3`;

[0088] ③HF11-BIP: 5`-GAGTCCTTTTGTTTAGCATGGGTTGGAGCCATGTTATTT TGTTGA-3`;

[0089] ④HF11-FIP: 5`-TCTTGGTGCCCAAACTTCCCGCATCTAACATTCTCAATA ATTGTC-3`;

[0090] ⑤HF11-LF: 5`-GGGGCCAAAGAATGTGTAAATCAAT-3`;

[0091] ⑥HF11-LB: 5`-TTTAGCGCCCATTTAAGCGT-3`;

[0092] 2.2 LAMP reaction system configuration:

[0093] Primer mixture: 0.2 μmol / L for outer primers HF11-F3 and HF11-B3, 1.4 μmol / L for inner primers HF11-FIP and HF11BIP, and 0.4 μmol / L for loop primers HF11-LB and HF1...

Embodiment 3

[0096] Example 3 Phillips cyst nematode LAMP specific detection

[0097] Collect cereal cyst nematodes, Phillips cyst nematodes, soybean cyst nematodes, upland rice cyst nematodes, pea cyst nematodes, barley cyst nematodes, Javan root-knot nematodes, peanut root-knot nematodes, banana perforator nematodes, and elephant ear bean roots For Knot nematodes (see Table 1), the DNA was extracted as a template to perform LAMP detection together with the Phillips cyst nematode DNA template to test the specificity of the LAMP detection method for Phillips cyst nematode.

[0098] Table 1 Sample codes and sources of other plant nematode populations tested

[0099]

[0100] After the above primer mixture and reaction buffer mixture are mixed evenly, add 1 μl of template DNA and proceed according to the reaction conditions in 2.3. After the reaction is completed, add 1 μl of the prepared chromogen and mix well, and observe the color change. The second tube is Phillips spp. Cyst DNA, gre...

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Abstract

The invention relates to an LAMP (loop-mediated isothermal amplification) rapid detection method for Heterodera filipjevi and its application. The method comprises: according to an RAPD specific fragment sequence of Heterodera filipjevi, designing and screening 6 LAMP primers, i.e. HF11-F3, HF11-B3, HF11-FIP, HF11-BIP, HF11-LB and HF11-LF, extracting the genome DNA of Heterodera filipjevi, establishing a loop-mediated isothermal amplification system and optimizing it, subjecting an amplification product to color development and observing it, thus rapidly detecting and identifying Heterodera filipjevi. The detection method provided in the invention has the advantages of strong specificity, high sensitivity, simple operation, high efficiency and fastness, as well as visually observable results, and has very high application value in rapid detection of Heterodera filipjevi and the early and field diagnosis aspect of Heterodera filipjevi disease.

Description

technical field [0001] The invention relates to a rapid detection method and application of LAMP of Philip's cyst nematode, belonging to the field of biotechnology. Background technique [0002] Cereal cyst nematode (CCN) is an important pathogenic nematode of cereal crops distributed worldwide. Since the nematode was discovered in Germany in 1874, it has occurred and harmed more than 40 countries around the world. Among them, cereal cyst nematodes in China, Australia, Europe, India, the Middle East and other regions have suffered serious damage and suffered huge losses. economic loss. At present, the wheat cyst nematode group (Heterodera avenae group) includes 12 species of cyst nematodes, among which cereal cyst nematodes (H. avenae), Philip cyst nematodes (H. filipjevi) and barley cyst nematodes (H. latipons ) are the most dangerous. [0003] In my country, cereal cyst nematode (Heterodera avenae wollenweber) was first discovered in Tianmen County, Hubei Province in 19...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 彭德良徐小琴彭焕黄文坤亓晓莉姜道宏
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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