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Feces phage DNA extracting method, kit for extracting feces phage DNA and application of kit

An extraction method and phage technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of high equipment requirements, long centrifugation time, time-consuming, etc., and achieve the effect of shortening extraction time, concentration and purity.

Pending Publication Date: 2018-08-17
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among the above two methods, the CsCl density gradient centrifugation method has high requirements on equipment and long centrifugation time; the formamide / CTAB method and phenol / chloroform extraction method are also time-consuming to extract DNA, and formamide, CTAB (hexadecyl Alkyltrimethylammonium bromide) and phenol / chloroform are both harmful solvents

Method used

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  • Feces phage DNA extracting method, kit for extracting feces phage DNA and application of kit
  • Feces phage DNA extracting method, kit for extracting feces phage DNA and application of kit
  • Feces phage DNA extracting method, kit for extracting feces phage DNA and application of kit

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Embodiment 1

[0036] The purpose of this embodiment is to provide a method for extracting fecal phage DNA, comprising the steps of:

[0037] (1) take by weighing chicken manure 3g, described chicken manure is derived from the fresh chicken manure of 25 day-old Roman pink laying hens, is collected from the breeding farm that raises scale and is 50,000 laying hens, closed chicken coop; Put in aseptic Add 40mL of SM buffer into a 50mL tube, place it on a vortex shaker and shake it for 30min, so that the phage attached to the feces can be fully released into the buffer; then filter it with a 100μm sieve to remove larger particles of impurities; the filtrate Centrifuge at 2,500 g for 5 min, followed by 10,000 g for 10 min, and then use 0.45 and 0.22 μm filters to remove bacteria and eukaryotic cells to obtain a filtrate.

[0038] (2) With reference to the extraction method adopted by Quirós and Colomer-Lluch et al., the filtrate was directly concentrated and purified using a 100-kDa ultrafiltrat...

Embodiment 2

[0051] The purpose of this example is to compare the difference in the extraction effect between a method for extracting fecal phage DNA provided by the present invention and other DNA extraction methods.

[0052] The excrement used in this embodiment comes from the fresh chicken manure of 25-day-old Roman pink laying hens, collected from a farm with a breeding scale of 50,000 laying hens, and a closed chicken house;

[0053] Reagents used in this example: DNase I and proteinase K were purchased from Chengdu Yimeng Technology Co., Ltd., 2×Taq PCRMasterMix, GoldView dye, agarose, DNA Marker 2000plμs were purchased from Chengdu Qingke Zixi Biotechnology Co., Ltd.

[0054] The preparation of other reagents used in this embodiment:

[0055] (1) SM buffer solution (50mmol / L Tris (pH 7.5), 100mmol / L NaCl, 8mmol / L MgSO4, 0.002% gelatin): weigh 5.8g of NaCl, MgSO 4 ·7H 2 O 2g, 1mol / L Tris 50mL, 2% by mass gelatin aqueous solution 5mL, distilled water to 1L, autoclave at 121°C for 20...

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Abstract

The invention belongs to the technical field of biology and discloses a feces phage DNA extracting method. According to the method, an ultrafiltration concentration method and a silica gel adsorptioncolumn method are combined; namely, an ultrafiltration concentrator is utilized to concentrate and purify phage at first, and then the silica gel adsorption column method is utilized to extract the phage DNA. The invention further discloses a kit for utilizing the feces phage DNA extracting method to extract the feces phage DNA and application of the kit. The method disclosed by the invention hasthe advantages that by improving the prior art, a 100-kDa ultrafiltration concentrator and a silica gel adsorption column are combined, and the feces phage DNA with a concentration about 53.47ng / mu Lcan be obtained within 5h. The feces phage DNA extracting method disclosed by the invention shortens extracting time, and the extracted DNA has better concentration and purity and can meet later qPRCand metagenome sequencing requirements.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting fecal phage DNA, a kit for extracting fecal phage DNA and applications of the kit. Background technique [0002] The intestinal microbial community is a natural reservoir of drug resistance genes, and animal manure contains abundant drug resistance genes. Bacteriophage is a kind of virus with bacteria as the host, and is an important carrier for the horizontal transfer of drug-resistant genes. Therefore, the extraction of phage DNA is the key to study the types and abundance of drug-resistant genes in fecal DNA. The method of extracting phage DNA from feces samples is introduced in detail in Nature protocols. The phage stock solution obtained from feces is purified by CsCl density gradient centrifugation, and then the phage DNA is extracted by the formamide / CTAB method [Thurber, R.V.H., Matthew Breitbart, Mya Wegley, Linda Rohwer, Forest, Laborato...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101
Inventor 张安云杨艳鲜王红宁雷昌伟杨鑫
Owner SICHUAN UNIV
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