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Nucleic acid extraction pretreatment of milk

A pretreatment and milk technology, which is applied in the field of nucleic acid extraction and pretreatment of milk, can solve the problems of loss of nucleic acid, unwelcome by users, and toxicity, etc., and achieve the effects of reducing fat content, improving nucleic acid extraction efficiency, and easy extraction

Pending Publication Date: 2022-04-29
SHENZHEN ANIEASY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to fat removal, organic solvents such as n-hexane are added for extraction, but part of the nucleic acid will be lost, and organic solvents such as n-hexane are toxic and are not welcomed by users.

Method used

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  • Nucleic acid extraction pretreatment of milk

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This embodiment shows a nucleic acid extraction pretreatment of milk, which specifically includes the following steps: take 1 mL of raw milk, put it into a 1.5 mL centrifuge tube, add 0.1 oz. g. Shake vigorously (speed ≥ 1500 rpm) on a mixer for 3 minutes; centrifuge at 4000 rpm for 1 minute, and transfer the supernatant to another 1.5mL centrifuge tube. Add 400uL pH8.0 10mM TNCa buffer containing 0.25mg / mL proteinase K, incubate at 55°C and vortex for 5min, then centrifuge at 7500rpm for 3min, and remove the supernatant. Add 350 μL of 0.35 mg / mL lysozyme, vortex evenly, and incubate at 37°C for 10 minutes to complete the pretreatment.

Embodiment 2

[0030]This embodiment shows a nucleic acid extraction pretreatment of milk, which specifically includes the following steps: take 1mL of milk, put it into a 1.5mL centrifuge tube, add 0.05g each of alumina and zirconia microbeads with a particle diameter of about 0.2mm , shake vigorously (speed ≥ 1500 rpm) on a mixer for 3 minutes; centrifuge at 4000 rpm for 1 minute, and transfer the supernatant to another 1.5mL centrifuge tube. Add 400uL pH8.0 10mM TNCa buffer containing 0.25mg / mL proteinase K, incubate at 55°C and vortex for 5min, then centrifuge at 7500rpm for 3min, and remove the supernatant. Add 350 μL of 0.35 mg / mL lysozyme, vortex evenly, and incubate at 37°C for 10 minutes to complete the pretreatment.

Embodiment 3

[0032] This embodiment shows a nucleic acid extraction pretreatment of milk, which specifically includes the following steps: take 1mL of milk, put it into a 1.5mL centrifuge tube, add 0.15g each of alumina and zirconia microbeads with a particle diameter of about 0.3mm , shake vigorously (speed ≥ 1500 rpm) on a mixer for 3 minutes; centrifuge at 4000 rpm for 1 minute, and transfer the supernatant to another 1.5mL centrifuge tube. Add 400uL pH8.0 10mM TNCa buffer containing 0.25mg / mL proteinase K, incubate at 55°C and vortex for 5min, then centrifuge at 7500rpm for 3min, and remove the supernatant. Add 350 μL of 0.35 mg / mL lysozyme, vortex evenly, and incubate at 37°C for 10 minutes to complete the pretreatment.

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Abstract

The invention discloses nucleic acid extraction pretreatment of milk, which comprises the following steps: adding aluminum oxide and zirconium oxide microspheres into a milk sample to be treated, oscillating for a period of time, and then centrifuging to take supernate. According to the method disclosed by the invention, the milk sample is subjected to better pretreatment (homogenization and fat removal) by using a method of adding aluminum oxide and zirconium oxide microspheres to carry out sample oscillation centrifugation, the oscillation homogenization is beneficial to more complete release of microorganisms from wrapped fat and protein particles, and the nucleic acid extraction efficiency is further improved; fat is effectively adsorbed through zirconium oxide and aluminum oxide microbeads, meanwhile, separation is simple, the efficiency and quality of extracting nucleic acid from a sample are effectively improved, and amplification is more effective.

Description

technical field [0001] The invention relates to the field of biology, in particular to a nucleic acid extraction pretreatment of milk. Background technique [0002] In molecular biology experiments, nucleic acid extraction is the most basic and commonly used technique. Before the usual nucleic acid extraction method starts, a certain sample pretreatment process needs to be carried out according to the characteristics of the sample, so that the efficiency of nucleic acid extraction can be improved. [0003] There are some differences between milk samples and other samples. Milk samples have high fat and protein content, and usually form particles in a single or combined form, which has the characteristics of high viscosity and difficult release of microorganisms. In particular, samples with excessive microorganisms are more viscous, and the nucleic acid extraction process is more difficult to handle. [0004] Currently, the commonly used pretreatment methods generally use p...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006C12Q2521/537
Inventor 邓炀马红圳何洁严义勇周巧妮韦家波
Owner SHENZHEN ANIEASY BIOTECHNOLOGY CO LTD