Kit for extracting nucleic acid from sputum and extraction method

A technology for extracting nucleic acid and an extraction method, which is applied in the field of biology, can solve the problems of difficult separation of cell components, abnormal cell shape variation, and difficulty in extracting high-purity DNA, and achieve simple and convenient operation steps, stable quality, and high nucleic acid purity. Effect

Inactive Publication Date: 2014-12-24
宁波有成生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sputum exfoliation cytology examination has the following disadvantages: fresh sputum needs to be used, and the characteristic part of it should be selected for examination; tumor cells have their specific developmental evolution process and morphological characteristics, and some cases are often found in the absence of typical tumor cells. Morphological changes of abnormal cells appear before the tumor cells. When judging whether they are tumor cells, it is necessary to rely on the professional level, prudence and sense of responsibility of the examiner, and there are subjective factors.
[0005] Due to the large amount of mucus in the sputum, the cell components in it are not easy to separate, and there are certain difficulties in the extraction of high-purity and relatively large amounts of DNA.

Method used

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  • Kit for extracting nucleic acid from sputum and extraction method
  • Kit for extracting nucleic acid from sputum and extraction method
  • Kit for extracting nucleic acid from sputum and extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Take 1 portion of sputum from a patient with tonsillitis, add 2 times the amount of digestive juice KB, shake and mix evenly. Add 50 μL proteinase K solution, 55 ° C water bath for 2 h. Add the same amount of binding solution BS as the sputum, shake and mix evenly, and then centrifuge the whole solution to pass through the nucleic acid adsorption column (pass through the column in batches, 700 μL each time). Add 500 μL SWB1 to the adsorption column, centrifuge at 12000 rpm for 1 min, then add 500 μL SWB2, centrifuge at 12000 rpm for 1 min, and finally elute with 50 μL eluent to obtain a DNA solution.

[0036] Take 5 μL of DNA solution and mix it evenly with 6×DNA Loading Buffer, add it to the sample hole of 0.7% agarose gel, electrophoresis at 5V / cm for 30 minutes, and observe the electrophoresis results under the gel imager, see figure 2 . The bands extracted from sputum can be seen in the electrophoresis diagram, and the molecular weight is greater than 23Kb.

Embodiment 2

[0038] Take the sputum of a patient diagnosed with lung cancer (the sputum sample coughed up from the lungs by the patient), add 3 times the amount of digestive juice KB, and shake and mix well. Add 100 μL of proteinase K and bathe in water at 55°C for 3h. Add the same amount of binding solution BS as the sputum, shake and mix evenly, and then centrifuge the whole solution to pass through the nucleic acid adsorption column (pass through the column in batches, 700 μL each time). Add 500 μL SWB1 to the adsorption column, centrifuge at 12000 rpm for 1 min, then add 500 μL SWB2, centrifuge at 12000 rpm for 1 min, and finally elute with 50 μL eluent to obtain a DNA solution.

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Abstract

The invention relates to the field of biology, and relates to a method of extracting a nucleic acid by taking sputum as a sample and application of the extraction method. The invention provides a dedicated kit for extracting a nucleic acid from sputum and an extraction method. The specific extraction method comprises the following steps: adding a digestive juice KB which is 1-5 times of the sputum into the sputum, vibrating and uniformly mixing the liquid, adding protease to the liquid, keeping warm at the temperature of 37-65 DEG C for 1-5 hours, adding a binding solution BS which is 0.5-3 times of the sputum sample capacity, after vibrating and uniformly mixing, enabling the complete solution to pass through a nucleic acid adsorption column, allowing the nucleic acid in the sample to be bound with a silica membrane of the adsorption column, conducting steps of washing, eluting and the like, and extracting the nucleic acid from the sputum. The nucleic acid extracted by the kit provided by the invention is high in yield and purity, and can be directly used for molecular biological detection.

Description

technical field [0001] The invention relates to a method for extracting nucleic acid (DNA and RNA) in the field of biology, in particular to a method for extracting nucleic acid from human sputum and its application. technical background [0002] Lung cancer is one of the common malignant tumors that seriously threaten the health of our people. According to statistics, 600,000 patients die from lung cancer every year in my country, and the rate of growth is 25% per year. Basic research on kylation changes and tumors[J]. Marker immunoassay and clinical. 2012,27(3):122-314). Among lung cancer patients, up to 2 / 3 of the cases are already in the advanced stage when they are diagnosed (Spiro SG, Silvestri GA. The treatment of advanced non-small cell Jung cancer[J]. Curr Opin Pulm Med, 2005,11:287 -291). Although the early diagnosis rate of lung cancer is only 15%, the 5-year survival rate of these patients can reach 60%-90%. Therefore, early diagnosis of high-risk and suspecte...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 邱英华周细武唐涛
Owner 宁波有成生物医药科技有限公司
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