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Specific primers used for PCR detection of Aphelenchoides besseyi and application method thereof

A technology of S. bescii and detection methods, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. demanding effects

Inactive Publication Date: 2016-05-25
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional method of identifying S. bessieensis is mainly based on morphological characteristics. Due to the wide variety of S. A. ritzemabosi ), Strawberry slippery nematode ( A. fragariae ) morphological characteristics are very similar, and the development of morphological identification requires solid classification skills, which has certain limitations

Method used

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  • Specific primers used for PCR detection of Aphelenchoides besseyi and application method thereof
  • Specific primers used for PCR detection of Aphelenchoides besseyi and application method thereof
  • Specific primers used for PCR detection of Aphelenchoides besseyi and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, single-plex PCR detection method specificity test

[0032] 1. Single Nematode DNA Extraction

[0033] Wash the nematodes with double-distilled water, pick a single nematode and put it in 8 μL ddH 2 In a PCR tube of O, use a small scalpel to cut the nematodes into 2-3 segments, centrifuge briefly, freeze and thaw 2 times (liquid nitrogen treatment for 1 min, 65°C water bath for 2 min), and add 2 μL of lysis solution (the lysis solution is 10×PCRBuffer, a mixture of double-distilled water and 20mg / mL proteinase K (volume ratio 10:9:1), 10×PCRBuffer and 20mg / mL proteinase K were purchased from TAKARA Company), incubated at 56°C for 1 h, and heated at 95°C 10min, and the supernatant after transient centrifugation can be used for subsequent molecular detection.

[0034] singleplex PCR amplification

[0035] PCR reaction system 50 μL: 10 μL nematode DNA template, 10×PCRBuffer (Mg 2+ ) 5μL, dNTP 2μL, primers (GU-F / GU-R) 1μL each, r Taq Enzyme 0.4μL, ddH ...

Embodiment 2

[0040] Embodiment 2. Double PCR detection method specificity test

[0041] 1. Single Nematode DNA Extraction

[0042] Same as Example 1.

[0043] double PCR amplification

[0044] PCR reaction system 50 μL: 10 μL nematode DNA template, 10×PCRBuffer (Mg 2+ ) 5μL, dNTP 2μL, internal standard primers F194 / AB28 1μL each, specific primers GU-F / GU-R 1μL each, r Taq Enzyme 0.4 μL, ddH 2 O28.6 μL. PCR reaction program: pre-denaturation at 95°C for 5min; 39 cycle reactions: denaturation at 95°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 1.5min; final incubation at 72°C for 10min

[0045] 3. Electrophoresis detection

[0046] With embodiment 1.

[0047] Duplex PCR Assay Specificity Test Results

[0048] In the S. basie-specific PCR reaction system, the universal primer F194 / AB28 was added as an internal standard to construct a one-step double PCR detection method. the result shows( figure 2 ), two clear and stable bands were amplified in both S. ba...

Embodiment 3

[0049] Embodiment 3, double PCR detection method sensitivity test

[0050] 1. Single Nematode DNA Extraction

[0051] With embodiment 1.

[0052] double PCR amplification

[0053] With embodiment 2.

[0054] Electrophoresis detection

[0055] With embodiment 1.

[0056] Sensitivity test results of the double PCR detection method

[0057] The sensitivity test results of a single nematode DNA undiluted, 5× diluted, 10× diluted, 20× diluted, 40× diluted, 80× diluted, 160× diluted, and 320× diluted as the gradient concentration showed that a single S. The specific bands were clear and bright, and when diluted to 10×, the bands of specific products and internal standard primers were clearly visible, indicating that the sensitivity of the double PCR detection method established in this study was 1 / 10 nematode DNA, which fully satisfied Sensitivity requirements.

[0058]

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Abstract

The invention provides a pair of specific primers used for PCR detection of Aphelenchoides besseyi and an application method thereof. The critical innovation of the method lies in that the newly-designed specific primers for Aphelenchoides besseyi can be combined with reported universal primers for ITS of ribosome of plant-parasitic nematodes so as to establish a novel duplex PCR detection method for rapid identification of Aphelenchoides besseyi. The method provided by the invention is highly efficient and stable, reaches a sensitivity level of 1 / 10 nematode DNAs and can be promoted and applied in quarantine and identification of ports and agricultural and forestry departments in China.

Description

technical field [0001] The present invention relates to the field of quarantine and identification of plant nematodes, and provides a pair of specific primers for PCR detection of S. Specific primers and general amplification primers for the ribosomal ITS region of plant parasitic nematodes have been reported, and a new double PCR detection method for the detection of S. It is suitable for the application of quarantine and identification laboratories related to ports and agriculture and forestry. Background technique [0002] Bessie worm ( Aphelenchoides besseyi Christie, 1942) is an important migratory plant ectoparasitic nematode. Its host range involves more than 35 genera of higher plants. It mainly infects economic crops such as rice, millet, and strawberry, resulting in the decline of crop quality and large-scale yield reduction. The nematode has strong stress resistance and can stay latent in the propagation material for a long time, and spreads mainly through the t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2600/166C12Q2545/101C12Q2537/143
Inventor 林宇王金成张裕君刘跃庭冯洁顾建锋陈先锋
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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