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Method for extracting and transforming excrement DNA in quantity

A feces, a large number of technologies, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of difficult DNA, high price, and less DNA in feces

Active Publication Date: 2019-03-19
杭州艾维克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many difficulties to be solved in clinical diagnosis, such as the degradation of DNase in the collection and separation process, and the existence of various inhibitors, which lead to a small amount of human fecal DNA extracted from feces or the extracted DNA is difficult to use. amplified by PCR
[0005] At present, there are related fecal DNA extraction methods on the market, but the amount of fecal DNA extracted is small or the DNA is difficult to use for PCR amplification, and the operation is cumbersome and expensive; there are also related fecal DNA conversion methods, but there are transformation effects Instability, cumbersome operation, and high price; in addition, the traditional method is completed in two stages for fecal DNA extraction and transformation, which has many shortcomings such as large DNA loss, low utilization rate, cumbersome operation, and high price, which is difficult to meet Downstream molecular diagnostic needs

Method used

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  • Method for extracting and transforming excrement DNA in quantity
  • Method for extracting and transforming excrement DNA in quantity
  • Method for extracting and transforming excrement DNA in quantity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The control kit used the QIAamp Fast DNA Stool Mini Kit to extract a total of 8 stool samples. The extraction steps are as follows:

[0089] 1. Take 10mL InhibitEx Buffer in a centrifuge tube, then take 1g of stool sample, vortex for 1min until the sample is fully homogenized;

[0090] 2. Take 2mL of the suspension into a centrifuge tube and centrifuge at full speed for 1min;

[0091] 3. Take 600 μL supernatant to a 2 mL centrifuge tube, add 25 μL Proteinase K and 600 μL Buffer AL respectively, shake to mix and vortex for 15 seconds;

[0092] 4. Heat and crack at 70°C for 10 minutes;

[0093] 5. Take out and add 600 μL of absolute ethanol, shake and mix;

[0094] 6. Add the mixed liquid to the purification column, centrifuge at 12000g for 30s, and pour off the liquid in the collection tube;

[0095] 7. Transfer all the remaining liquid to the purification column, centrifuge at 12000g for 30s, and pour off the liquid in the collection tube;

[0096] 8. Use 500μL Buff...

Embodiment 2

[0118] The control kit used EpiTect Fast BisμLfite Conversion Kits, and a total of 5 cases of fecal nucleic acid samples were converted. The conversion steps are as follows:

[0119] 1. Add 20 μL DNA sample to the PCR tube, add 85 μL Bisulfite Solution and 35 μL DNAProtect Buffer;

[0120] 2. After mixing, place the centrifuge tube on the amplification instrument, and transform according to the instructions;

[0121] 3. After transformation, transfer the above 140μL liquid into a clean 1.5mL centrifuge tube;

[0122] 4. Add 310 μL Buffer BL and 250 μL absolute ethanol, mix well and centrifuge briefly;

[0123] 5. Add the mixture to the spin column, centrifuge at 12000g for 1min, and discard the liquid in the collection tube;

[0124] 6. Add 500μL Buffer BW to the purification column, centrifuge at 12000g for 1min, and discard the liquid in the collection tube;

[0125] 7. Add 500 μL Buffer BD to the purification column, let stand at room temperature for 15 minutes, centrifu...

Embodiment 3

[0150] According to the present invention, a total of 6 cases of stool samples were extracted and transformed, and the extraction and transformation steps were as follows:

[0151] 1. Take 6mL Buffer SPB (50mM EDTA, 50mM Tris, 100mM NaCl, 30% absolute ethanol) into a centrifuge tube, then take 1g of feces sample into the tube, shake and mix well;

[0152] 2. First add 500 μL Buffer SLS (50mM EDTA, 50mM Tris, 20% SDS), mix by inverting, then add 50 μL of proteinase K, mix by inverting, and place in a constant temperature water bath at 70°C for 10 minutes;

[0153] 3. Transfer the centrifuge tube to a water bath at room temperature for 3 minutes, and centrifuge at 10,000g for 1 minute;

[0154] 4. Take 5mL supernatant into a new centrifuge tube, add 1mL Buffer SDL (50mM EDTA, 50mM Tris, 2% PVP10 and 1M sodium acetate) and mix upside down, centrifuge at 10000g for 2min;

[0155] 5. Take all the supernatant in a new centrifuge tube, add 3mL of isopropanol, mix upside down, centri...

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Abstract

The invention discloses a method for extracting and transforming excrement DNA in quantity. The method comprises the steps of collecting 1g-5g of excrement into a tube containing protecting liquid, adding cracking liquid for adequately cracking so as to remove an inhibitor, precipitating nucleic acid by virtue of an organic solvent, resuspending precipitates so as to obtain nucleic acid for nucleic acid extraction or nucleic acid transformation, carrying out a series of combined washing steps, and finally carrying out elution, so as to obtain excrement DNA. The method comprises the steps of extraction and transform of excrement DNA and combination of extraction and transformation. The method has the characteristics of low cost, high utilization rate, good repeatability, simplicity and rapidness; a large number of excrement DNA or transformed excrement DNA can be obtained, and more possibilities can be provided for the transformation or the downstream detection.

Description

technical field [0001] The invention relates to a method for extracting and transforming stool DNA in large quantities. Background technique [0002] Colorectal cancer, including colon cancer and rectal cancer, is a common malignant tumor of the digestive tract. The incidence rate is second only to gastric cancer and esophageal cancer. According to data, the 5-year survival rate of early colorectal cancer is as high as 90%, while the 5-year survival rate of advanced colorectal cancer is only 10%. Therefore, early screening of colorectal cancer is particularly important. At present, early screening measures mainly include fecal occult blood test, barium enema examination, colonoscopy, etc., but there are no obvious symptoms in the early stage of intestinal cancer, and there is a lack of early diagnosis techniques with high sensitivity and strong specificity. Most patients are diagnosed with intestinal cancer In the middle and advanced stage, the best treatment opportunity is...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2521/537
Inventor 林喜建宋庆涛廖乃淞郑立谋
Owner 杭州艾维克生物科技有限公司
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