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PCR detection primer and method for anoectochilus roxburghii colletotrichum orbiculare

A technology for anthracnose bacteria and detection primers, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, inability to achieve rapid inspection, etc., and achieves simple, fast and practical operation. Good and practical effect

Active Publication Date: 2016-01-20
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to quickly detect and identify different types of anaesthesive soil organisms called Aeoctorchus catheteratus that cause disease in vegetables grown underground or crops raised indoor during their production cycle. It also provides useful tools for studying plant growth mechanics such as rooting, nutrients uptake, water supply, temperature regulation, etc., which could help predict future health issues associated with these microorganism populations.

Problems solved by technology

The patented technique described in the patents involves identifying diseases caused by certain types of microorganisms like Bacillaria spp., Anthraxoninians or Micrococcaceae spiroformulas. These organism require specialized tools to identify them quickly and easily during their natural cycle. Traditional methods involve culturing these organisms until they reach sufficient size and become infectious enough to cause damage. To address this issue, researchers started exploring new ways to improve upon current techniques involving culture and observation.

Method used

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  • PCR detection primer and method for anoectochilus roxburghii colletotrichum orbiculare
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  • PCR detection primer and method for anoectochilus roxburghii colletotrichum orbiculare

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Primers specific amplification of Clematis anthracnose bacteria

[0031] 1. Specific Detection of Anthracnose Bacteria in Clematis aureus

[0032] PCR reaction system 20.0μL, including 2× Taq PCRMasterMix 10.0 μL, primers CR1 and CF1 5 μM each, template DNA 50-100 μg, d.d.H 2O to make up to 20.0 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 30 sec, a total of 35 cycles, and extension at 72°C for 10 min.

[0033] 2. Test results

[0034] Specificity of detection: Except for the 326bp product that can be specifically amplified from the DNA of Clematis anthracnose, the DNA of other 21 different fungi failed to amplify any product, which has strong specificity.

Embodiment 2

[0035] Embodiment 2: Sensitivity detection of primers to Clematis anthracnose bacteria

[0036] 1. Dilution of DNA concentration: the extracted genome DNA of Clematis anthracis was measured by a spectrophotometer, and then diluted in series.

[0037] 2. Sensitivity Detection of Anthracnose Bacteria from Clematis aureus

[0038] PCR reaction system 20.0μL, including 2× Taq PCRMasterMix 10.0 μL, primers CR1 and CF1 5 μM each, template DNA 50-100 μg, d.d.H 2 O to make up to 20.0 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 30 sec, a total of 35 cycles, and extension at 72°C for 10 min.

[0039] 3. Detection results: In the 20.0μL reaction system, the genomic DNA of Clematis anthracis can obtain obvious amplification bands, and the detection sensitivity can reach 11.2×10 -5 ng / μL.

Embodiment 3

[0040] Example 3: Detection of Clematis anthracnose in diseased plant samples.

[0041] 1. Sample collection: Clematis plant tissue samples were collected from the Clematis production base in Yongtai County, Fuzhou City, Fujian Province.

[0042] 2. DNA extraction and detection

[0043] DNA was extracted from diseased plant tissue using the NaoH rapid lysis method, and PCR amplification was performed according to the above method. The PCR reaction system was 20.0 μL, including 2× Taq PCRMasterMix 10.0 μL, primers CR1 and CF1 5 μM each, template DNA 50-100 μg, d.d.H 2 O to make up to 20.0 μL. The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 30 sec, a total of 35 cycles, and extension at 72°C for 10 min.

[0044] 3. Test results

[0045] see results image 3 , if the 326bp product can be specifically amplified, it can be judged that there is Clematis anthracis in ...

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Abstract

The invention discloses a PCR detection primer and method for anoectochilus roxburghii colletotrichum orbiculare. According to the method, a pair of specific primers (CF1:5'-CGGAGGATAACCAAACTCTG-3' and CR1:5'-CGAGACGTAAAGTTACTACGC-3') for anoectochilus roxburghii colletotrichum orbiculare are mainly designed and adopted and are subjected to PCR amplification and agarose gel electrophoresis to specifically form specifically-amplified products with the fragment length of 326bp in pure DNA of anoectochilus roxburghii colletotrichum orbiculare and bacteria-bearing plants. The specific molecular detection primer and the use method thereof can be applied to the rapid, sensitive and specific detection of colletotrichum orbiculare in plants infected with anoectochilus roxburghii colletotrichum orbiculare in production practice and can be simultaneously applied to early diagnosis and pathogen monitoring and authentication of diseases in the planting of medicinal plants, thereby providing reliable theoretical and technical supports to the prevention and the control of diseases caused by anoectochilus roxburghii colletotrichum orbiculare.

Description

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Claims

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Application Information

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Owner INST OF PLANT PROTECTION FAAS
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