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Species-specific primer pair for detecting glomus versiforme

A kind of Glomus mutans, species-specific technology, applied in the field of primer pairs for detection of Glomus mutans

Active Publication Date: 2012-10-31
北京中科卓明生物医学研究所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problem that the existing primer pairs cannot directly detect the partial sequence of the 25S rDNA D1 / D2 variable region with the genetic characteristics of Glomus versiforme (Glomus versiforme) from samples containing a variety of AM fungi. The primer pair LR1 / G.ver also cannot specifically amplify the corresponding specific DNA fragments from mixed samples, and a species-specific primer pair for detecting Glomus mutans is provided

Method used

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  • Species-specific primer pair for detecting glomus versiforme
  • Species-specific primer pair for detecting glomus versiforme
  • Species-specific primer pair for detecting glomus versiforme

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specific Embodiment approach 1

[0010] Specific embodiment 1: The species-specific primer pair used to detect Glomus mutans in this embodiment is named G.ver F / G.verR, and consists of a forward primer G.ver F and a reverse primer G.ver R, The base sequence of the forward primer G.ver F is 5'-GCATCTCCCAGGGTCTATAACA-3', and the base sequence of the reverse primer G.verR is 5'-GAGTGAAGAGGGAAAAGCTCAA-3'.

[0011] In this embodiment, the PCR amplification system for detecting the species-specific primer pair of Glomus mutans is 50 μL, consisting of 1.0 μL of template DNA, 4.0 μL of dNTP with a concentration of 2.5 mmol / L, and 5.0 μL of 10× buffer solution (containing Mg 2+ ), 5.0 μL of primers with a concentration of 10 pmol / L (final concentration of G.ver F and G.ver R), 0.2 μL of Taq DNA polymerase with a concentration of 5 U / μL, and the remaining sterile double-distilled water.

[0012] The PCR amplification reaction conditions of the species-specific primer pair used to detect Glomus mutans in this embodiment...

specific Embodiment approach 2

[0014] Embodiment 2: In this embodiment, the existing primer pair LR1 / G.ver is used for PCR amplification of Glomus mutans. The base sequence of the forward primer LR1 of the primer pair LR1 / G.ver is 5′-GCATATCAATAAGCGGAGGA-3′, and the base sequence of the reverse primer G.ver of the primer pair LR1 / G.ver is 5′-GTCTATAACACTCTCCCGAAG-3 '.

[0015] In this embodiment, the PCR amplification system for detecting the species-specific primer pair of Glomus mutans is 50 μL, consisting of 1.0 μL of template DNA, 4.0 μL of dNTP with a concentration of 2.5 mmol / L, and 5.0 μL of 10× buffer solution (containing Mg 2+ ), 5.0 μL of primers with a concentration of 10 pmol / L (the final concentration of LR1 and G.ver), 0.2 μL of Taq DNA polymerase with a concentration of 5 U / μL, and the remaining sterile double-distilled water.

[0016] PCR amplification reaction conditions: pre-denaturation at 95°C for 3min, denaturation at 93°C for 1min, annealing at 58°C for 1min, extension at 72°C for 1mi...

specific Embodiment approach 3

[0018] Specific embodiment three: In this embodiment, Glomus proteus is artificially added to the soil sample containing various AM fungi, and then the primer pair LR1 / G.ver and the primer pair G.ver F / G.ver R are used to carry out PCR amplification.

[0019] The PCR amplification of the primer pair G.ver F / G.ver R adopts the method of Embodiment 1, and the PCR amplification of the primer pair LR1 / G.ver adopts the method of Embodiment 2.

[0020] The soil samples containing multiple AM ​​fungi in this embodiment were taken from the forest farm of Northeast Forestry University in September 2011.

[0021] The tested soil samples contained at least three kinds of AM fungi, Glomus intraradices, Glomus mosseae and Scutellospora calospora.

[0022] After PCR amplification, agarose gel electrophoresis was carried out for detection, and the detection results were as follows: image 3 shown. image 3 The middle primer pair LR1 / G.ver amplified two bands; while the primer pair G.ver F...

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Abstract

A species-specific primer pair for detecting glomus versiforme relates to a primer pair for detecting glomus versiforme, and is used for solving the problems that the existing primer pair cannot accurately and directly detect the partial sequence of a 25S rDNA (recombinant deoxyribonucleic acid) D1 / D2 variable region with the glomus versiforme hereditary property in a sample which contains active microorganism (AM) fungi, and the existing primer pair LR1 / G.ver cannot specifically amplify the corresponding specific DNA fragment from a hybrid sample. The species-specific primer pair for detecting glomus versiforme is named as G.ver F / G.ver R, and is composed by a forward primer G.ver F and a reverse primer G.ver R. The species-specific primer pair is suitable for detecting the glomus versiforme.

Description

technical field [0001] The invention relates to a pair of primers for detecting Glomus mutans. Background technique [0002] Glomus versiforme (Glomus versiforme) is an arbuscular mycohizal (AM) fungus. It is an oligotrophic living microorganism that can infect the roots of some terrestrial plants to form mycorrhizae. Glomus proteus can promote the absorption of various nutrient elements by plants, improve plant disease resistance and drought resistance, and play an extremely important role in maintaining plant diversity and ecosystem stability. [0003] At present, single spores of AM fungi can be obtained from plant rhizosphere soil by wet sieve decantation-sucrose centrifugation, and then their DNA is extracted, and then amplified using the eukaryotic general primer pair LR1 / NDL22. Known by sequencing its exact genetic sequence. However, from soil samples with multiple AM ​​fungi, it has not been possible to directly amplify the 25S rDNA D1 / D2 variable regions with the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 接伟光蔡柏岩白莉于春光杨明月
Owner 北京中科卓明生物医学研究所有限公司
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