Method for simultaneously extracting compost sample PLFA and DNA

A sample and composting technology, applied in the preparation of test samples, etc., can solve the problems of cumbersome extraction steps and achieve the effects of simplifying the extraction procedure, reducing errors, and shortening the time

Inactive Publication Date: 2010-08-25
HUNAN UNIV
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Problems solved by technology

If the combined application of different methods can learn from each other and avoid the inevitable deviation caused by the principle of the method itself, the combined application of various methods has become a trend to analyze the changes in the compost microbial community structure. However, the PLFA method and nucleic acid analysis, both require the extraction of substances representing biological information from compost samples, and the extraction steps are relatively cumbersome. The combination of extraction methods provides the possibility for the combined application of methods

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  • Method for simultaneously extracting compost sample PLFA and DNA

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Embodiment Construction

[0028] 1. Extraction of PLFA (as shown in Figure 1)

[0029] (1-1) Weigh 5.00g of compost sample into a glass tube with a polytetrafluoroethylene cover, add 5mL of dichloromethane, 10mL of methanol, and 5mL of phosphate buffer solution (pH 7.4, 0.05mol / L);

[0030] (1-2) After ultrasonic homogenization for 10 minutes, stand overnight at 4°C in the dark;

[0031] (1-3) Centrifuge for 20 minutes at a speed of 8000 rpm. Transfer the liquid phase to a new glass tube, then add 5mL dichloromethane and 4mL pure water to continue extracting PLFA; store the solid phase at -20°C for DNA extraction;

[0032] (1-4) After standing still for 24 hours, transfer the aqueous phase to a new centrifuge tube a for DNA precipitation; filter the organic phase with a 4# fiber filter membrane; use a nitrogen blowing instrument (DC-12, Shanghai Anpu Science) for the filtrate Instrument Co., Ltd.) after drying, add 2 mL of n-hexane to dissolve (if the dissolution effect is not good, you can add a dro...

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Abstract

The invention provides a method for simultaneously extracting and purifying phospholipid fatty acid PLFA and deoxyribonucleic acid DNA from the same composting sample, and the extracting method is based on an extraction method of lipid, after phase separation, the lipid enters an organic phase while the DNA is left in a water phase and a solid phase to realize the simultaneous extraction of the PLFA and the DNA finally; the extracting method of the invention has the advantages of simplifying the procedures for extracting the lipid of the composting sample and the DNA, ensuring the identity ofthe information source of the sample, well reducing the error caused by composting sampling to the measuring method, and being more meaningful when the quantity of the samples is small.

Description

technical field [0001] The invention relates to a mixed extraction method of PLFA and DNA of microorganisms in compost samples. Background technique [0002] Microorganisms are an important factor in composting. In order to degrade organic matter more effectively to control the composting process and improve composting efficiency, it is necessary to study the number, composition and interaction of various microorganisms in the composting process. Since most environmental microorganisms are in a non-culturable state, it is difficult to understand the actual microbial community structure. In recent years, both nucleic acid analysis and lipid analysis have tried to overcome this difficulty. These methods all have the advantage of being able to directly extract living cell components from environmental samples for analysis. [0003] PLFA (Phospholipid fatty acid) spectrogram analysis is a representative of biochemical methods. Phospholipids are an important part of living cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/34G01N1/28
Inventor 喻曼曾光明陈耀宁张嘉超黄红丽宋琳玲
Owner HUNAN UNIV
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