Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method

A technology of cereal cyst nematode and detection method, which is applied in the field of rapid detection of cereal cyst nematode LAMP, can solve the problems of limiting the popularization and application of PCR detection method and long time, and achieves short detection time, simple operation steps and obvious results Effect

Inactive Publication Date: 2013-04-17
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
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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 methods in production. In the investigation of the occurrence and distribution of cereal cyst nematodes, there is an urgent need for a simple and fast detection method

Method used

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  • Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method
  • Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method
  • Rapid detection method of heterodera avenae wollenweber LAMP and application of detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Extraction, RAPD amplification and sequence analysis of cereal cyst nematode DNA

[0072] 1.1 Extraction of cereal cyst nematode DNA

[0073] Pick a single cereal cyst and put it in a container containing 10 μ lddH 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.

[0074] 1.2 RAPD amplification and sequence analysis of cereal cyst nematode

[0075] Cereal cyst nematode-specific SCAR fragments were amplified using SCAR marker primers OPD13-HaF1 (5`-TGACGAGAACATATGATGGGGAT-GAT-3`) and OPD13-HaR1 (5`-GAG...

Embodiment 2

[0076] Example 2 Establishment of the method for detection of cereal cyst nematodes by LAMP technology

[0077] 2.1 LAMP primer design

[0078] According to the results of RAPD amplification sequence sequencing of cereal 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:

[0079] ①HA5-F3: 5`-TGATAGGGAAAAAATTCTCAA-3`;

[0080] ②HA5-B3: 5`-GTCGGCAATGGTCAACAA-3`;

[0081] ③HA5-BIP: 5`-TTGTGGTGCCGTAGTGTTAGAAATGC TTCATTTGAGAACAATG--3`;

[0082] ④HA5-FIP: 5`-ACACAACGCTTAGTCCGGTCCCAATGGCA TGGCAAAGA--3`;

[0083] ⑤HA5-LB: 5`-CGGGGATGAAAGAAGGCAAAGTG--3`;

[0084] 2.2 LAMP reaction system configuration:

[0085] Primer mixture: outer primer HA5-F3 and HA5-B3 each 0.2 μmol / L, inner primer HA5-FIP and HA5BIP each 1.4 μmol / L, loop primer HA5-LB 0.4 μmol / L;

[0086] Reaction mixture: 3.2mmol / L dNTP, 20mmol / L Tris-HCl (pH8.8), 10...

Embodiment 3

[0088] Example 3 Cereal Cyst Nematode LAMP Specific Detection

[0089] Collection of cereal cyst nematode, Phillips cyst nematode, soybean cyst nematode, upland rice cyst nematode, pea cyst nematode, barley cyst nematode, root-knot nematode incognita, root-knot nematode java, root-knot nematode peanut, banana perforator (see Table 1), the DNA was extracted as a template for LAMP detection together with the cereal cyst nematode DNA template to test the specificity of the cereal cyst nematode LAMP detection method.

[0090] Table 1 Sample codes and sources of other plant nematode populations tested

[0091]

[0092] 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, add 1 μl of the prepared chromogen and mix well, and observe the color change. The first tube is grain Cyst DNA, green fluorescence can be observed, other tubes of nematodes and negative...

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Abstract

The invention relates to a rapid detection method of heterodera avenae wollenweber LAMP and application of the detection method. Five LAMP primers, namely HA5-F3, HA5-B3, HA5-FIP, HA5-BIP and HA5-LB are designed and screened according to a heterodera avenae wollenweber random amplified polymorphic dna (RAPD) sequence obtained by cloning; and an LAMP reaction system is configured and optimized. By performing DNA extraction, loop-mediated isothermal amplification, amplified product developing and observation on the heterodera avenae wollenweber, the heterodera avenae wollenweber can be rapidly detected. The detection method is high in specificity, low in cost and convenient to operate, and has high application values on rapid detection of the heterodera avenae wollenweber and early and field diagnosis of the heterodera avenae wollenweber diseases.

Description

technical field [0001] The invention relates to a rapid detection method and application of cereal cyst nematode LAMP, belonging to the field of biotechnology. Background technique [0002] Cereal cyst nematode (Heterodera avenae Wollenweber, 1 924), Cereal cyst nematode (CCN for short, also known as oat cyst nematode), is an important pathogenic nematode of cereal crops distributed all over the world. 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. For example, the damage area of ​​cereal cyst nematode in Australia has reached 2 million hectares, the general output loss is 23-50%, and the loss is 73-89% when serious, and the annual economic loss is about 72 million Australian dollars (Brennan J P, Murray G M.Aust ralian w heat d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 彭德良徐小琴彭焕亓晓莉黄文坤贺文婷姜道宏
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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