Heterodera filipjevi Taqman MGB probe real-time quantitative PCR detection method and application thereof

A real-time fluorescence quantification and detection method technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of few reports of wheat cyst nematodes, achieve high sensitivity, strong specificity, and reduce misjudgment. Effect

Active Publication Date: 2015-09-23
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, this technology is widely used in the detection of vario

Method used

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  • Heterodera filipjevi Taqman MGB probe real-time quantitative PCR detection method and application thereof
  • Heterodera filipjevi Taqman MGB probe real-time quantitative PCR detection method and application thereof
  • Heterodera filipjevi Taqman MGB probe real-time quantitative PCR detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: the establishment of real-time fluorescent quantitative PCR detection method of Philip's cyst nematode Taqman MGB probe

[0042] 1.1 Extraction of Nematode DNA

[0043] Pick a single cyst, put it into a centrifuge tube containing 7 μl of 10× PCR buffer, 3 μl of proteinase K solution (600 μg / ml) and 10 μl of sterilized redistilled water, and freeze in a -20°C refrigerator for 1 hour. Glass rods sterilized with 75% alcohol were rolled until the ice melted, then frozen at -20 °C for at least 2 h. After 2 hours, the centrifuge tube was taken out of the refrigerator, incubated at 65°C for 1.5h, then at 95°C for 10 minutes, and finally centrifuged at 10,000 rpm for 1 minute, and the supernatant was stored at -20°C for later use. The extracted DNA was amplified with universal primers D2A and D3B to detect the quality of the DNA.

[0044] 1.2 Phillips cyst nematode-specific primer and probe design

[0045] According to the previously developed RAPD-SCAR specifi...

Embodiment 2

[0051] Example 2: Specificity and sensitivity detection of real-time fluorescent quantitative PCR with Taqman MGB probe of Phillips cyst nematode.

[0052] 2.1 Specific detection of Phillips cyst nematode Taqman MGB probe real-time fluorescent quantitative PCR.

[0053] The specific primers qHf-F, qHf-R and the specific probe HF-probe designed by the present invention were synthesized by Bao Biological Engineering (Dalian) Co., Ltd., PCR reaction system: 2×PCR mix 10 μl; ROX reference DyeII 0.4 μl; Primer qHF-R / qHF-F (10uM) and probe HF-probe (10uM) are both 0.4μl; 1μl template DNA; sterilized ddH 2 O to make up to 20 μl. Sterile water was used as a negative control. ABI 7500FAST fluorescence quantitative PCR instrument was used for PCR. The PCR reaction conditions were pre-denaturation at 95°C for 30 sec, 95°C for 3s, 60°C for 30s, 40 cycles, and fluorescence signals were collected at 60°C.

[0054] Specific fluorescence curves can be obtained in the 8 populations of Phill...

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Abstract

The invention provides a heterodera filipjevi Taqman MGB probe real-time quantitative PCR detection method and application thereof, belongs to the technical field of plant nematode molecular detection, and discloses a specific fluorescence probe HF-probe and a group of specific primers qHF-R and qHF-F, which are designed on the basis of a heterodera filipjevi RAPD specific sequence. The probe and the primers can be amplified in heterodera filipjevi to obtain specific fluorescence curves, so that the quick molecular detection of heterodera filipjevi is achieved. The detection method has high sensitivity and strong specificity, is fast and accurate, and has high practical applied values in the aspects of early diagnosis and field monitoring and early warning of heterodera filipjevi.

Description

technical field [0001] The invention belongs to the technical field of molecular detection of plant nematodes, and relates to a rapid molecular detection method of Philip's cyst nematode and an application thereof. Background technique [0002] Wheat 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. The wheat cyst nematode group (Heterodera avenae group) includes a total of 12 cyst nematodes, among which cereal cyst nematodes (H. avenae), Philip cyst nematodes (H. filipjevi) and broad vulva cyst nematodes (H. ) are the most dangerous. [0003] In my country, cereal cyst nematode (Heterodera avenae wollenweber) was first discovered in Tia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 彭焕彭德良贺文婷黄文坤孔令安
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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