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Reagent, kit and extraction method for extracting DNA (deoxyribonucleic acid) of fungus attached to grain seeds

A kit and seed technology, applied in the biological field, can solve the problems that affect the simplicity, timeliness and automation of the extraction process, and achieve the effects of rapid destruction, high extraction efficiency, and improved accuracy

Pending Publication Date: 2022-04-12
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, in order to ensure the cleavage effect, the cleavage process of the prior art generally uses biologically active enzymes such as proteinase K and assists heat treatment at 56°C to 65°C, which directly affects the simplicity, timeliness and automation of the entire extraction process.

Method used

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  • Reagent, kit and extraction method for extracting DNA (deoxyribonucleic acid) of fungus attached to grain seeds
  • Reagent, kit and extraction method for extracting DNA (deoxyribonucleic acid) of fungus attached to grain seeds
  • Reagent, kit and extraction method for extracting DNA (deoxyribonucleic acid) of fungus attached to grain seeds

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Experimental program
Comparison scheme
Effect test

Embodiment 1-12

[0041] Present embodiment 1-12 is aimed at wheat grain attachment fungus DNA extraction method, and it comprises the following steps:

[0042] Step 1. Collect bacteria

[0043] Weigh 6g of wheat grain powder sample into a 50mL centrifuge tube, add 6g of quartz sand, 30mL of normal saline and 10μL of Tween 80, vortex at high speed for 4min, let stand at room temperature for 20min, transfer 20mL of the supernatant to a clean 50mL centrifuge tube , centrifuge at 8000rpm / min for 15min, take the cell pellet, and discard the supernatant.

[0044] Step two, cracking

[0045] Transfer the bacteria obtained in step 1 to the broken tube containing the grinding aid particles, add 1mL of lysate (the composition of the lysate is shown in Table 1), and place it in an MP FastPrep-24 homogenizer at 6m / s for 2× 30s, with an interval of 120s, then centrifuge at 12000rpm / s for 5min, and take the supernatant.

[0046] Step three, combine

[0047] Transfer the lysed supernatant obtained in ste...

Embodiment 13-21

[0063] The present embodiment 13-21 is aimed at the DNA extraction method of corn grain attachment fungus, which comprises the following steps:

[0064] Weigh the corn grain powder sample, mix it with quartz sand, normal saline and Tween 80 according to the ratio of 3g:3g:15mL:5μL, vortex at high speed for 4min, let stand at room temperature for 20min, transfer 20mL supernatant to a clean 50mL centrifuge tube Centrifuge at 8000rpm / min for 15min, and discard the supernatant. Transfer the bacteria to a broken tube containing grinding aid particles, add 1mL of lysate (the composition of the lysate is shown in Table 4), and place it in an MP FastPrep-24 homogenizer at 6m / s for 2×30s with an interval of 120s , and then centrifuged at 12000rpm / s for 5min. Such as figure 1 As shown, transfer the supernatant to the 1# well of the disposable purification strip, and then add an equal volume of binding solution (the composition of the binding solution is shown in Table 4); add the magn...

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Abstract

The invention discloses a reagent, a kit and an extraction method for DNA (deoxyribonucleic acid) extraction of fungi attached to grain seeds. A reagent for DNA extraction comprises a lysis solution, and the lysis solution comprises NaCl, EDTA (Ethylene Diamine Tetraacetic Acid) disodium, Tris-HCl, N-sodium lauroyl sarcosinate and epsilon-polylysine. According to the DNA extraction reagent for the fungi attached to the grain seeds, epsilon-polylysine and N-lauroyl sodium sarcosinate with a certain concentration ratio are added into a lysis solution system according to the structural characteristics of fungal cells, and the epsilon-polylysine and the N-lauroyl sodium sarcosinate have a synergistic effect in an optimized conventional buffer system of NaCl, disodium EDTA and Tris-HCl, so that the extraction efficiency of the fungi attached to the grain seeds is improved; the fungal cell structure can be fully, efficiently and quickly destroyed without heating treatment, so that protein is fully denatured and effectively separated from DNA, and high extraction efficiency is realized by simple components.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reagent, a kit and an extraction method for extracting the DNA of grain-adhered fungi. Background technique [0002] In recent years, with the development of molecular biology, technologies such as quantitative detection, diversity or functional analysis of microorganisms based on substrates (soil, plants, animals, water, air, etc.) attached to microbial deoxyribonucleic acid (DNA) have been continuously improved. , including pathogenic bacteria, toxin-producing bacteria, spoilage bacteria and other microorganisms real-time fluorescence quantitative nucleic acid amplification detection method (qPCR), digital PCR (dPCR), sequencing and other technologies, in food safety, clinical diagnosis, environmental monitoring, plant protection, biological It has a wide range of applications in prevention and control and other fields. In the grain industry, through molecular biological analysi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
Inventor 崔华王松雪王松山陈梦泽李森叶金
Owner ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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