CN109722467A - Application of the sodium bicarbonate buffer system in nucleic acid extraction - Google Patents
Application of the sodium bicarbonate buffer system in nucleic acid extraction Download PDFInfo
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- CN109722467A CN109722467A CN201910054564.6A CN201910054564A CN109722467A CN 109722467 A CN109722467 A CN 109722467A CN 201910054564 A CN201910054564 A CN 201910054564A CN 109722467 A CN109722467 A CN 109722467A
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- sodium bicarbonate
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 title claims abstract description 82
- 229910000030 sodium bicarbonate Inorganic materials 0.000 title claims abstract description 41
- 235000017557 sodium bicarbonate Nutrition 0.000 title claims abstract description 41
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 39
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 39
- 239000007853 buffer solution Substances 0.000 title claims abstract description 30
- 238000000605 extraction Methods 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 18
- 239000012149 elution buffer Substances 0.000 claims abstract description 13
- 239000006166 lysate Substances 0.000 claims abstract description 11
- 239000004475 Arginine Substances 0.000 claims description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 10
- 230000035945 sensitivity Effects 0.000 abstract description 21
- 238000004458 analytical method Methods 0.000 abstract description 14
- 239000000872 buffer Substances 0.000 abstract description 7
- 238000002123 RNA extraction Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 12
- 239000003480 eluent Substances 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 241000725303 Human immunodeficiency virus Species 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 208000002672 hepatitis B Diseases 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 241000700721 Hepatitis B virus Species 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000001139 pH measurement Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 241000711549 Hepacivirus C Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
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- 206010011409 Cross infection Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000001793 Wilcoxon signed-rank test Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
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- 230000000052 comparative effect Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000011990 functional testing Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of elution buffer for kit for detecting nucleic acid, the elution buffer is sodium bicarbonate solution, concentration 5mM, pH 6.7-7.2.The present invention also provides application of the sodium bicarbonate buffer system in nucleic acid extraction, the sodium bicarbonate buffer system is sodium bicarbonate buffer liquid, concentration 5mM, pH7.0.The present invention also provides application of the arginic lysate in kit for detecting nucleic acid is contained, in the lysate, the arginic concentration is 15mM.The present invention provides a kind of buffer systems, to improve the sensitivity for analysis of nucleic acid (predominantly DNA).The present invention increases the extraction efficiency of DNA under conditions of not changing RNA extraction efficiency, to increase the sensitivity for analysis of the DNA detection of product.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to a nucleic acid extraction technology, and particularly relates to an application of a sodium bicarbonate buffer system in nucleic acid extraction.
Background
Hepatitis B is a hepatitis disease caused by infection of hepatitis B virus (hereinafter referred to as HBV), has infectivity, can be developed into liver cancer through cirrhosis, has great harm to health, and can avoid hepatitis B virus infection in large quantities through effective diagnosis. China is a big country with hepatitis B, and currently, about 1.2 hundred million hepatitis B virus carriers exist, so that diagnosis of hepatitis B is very important. In addition, hepatitis c virus (hereinafter abbreviated as HCV) and human immunodeficiency virus (hereinafter abbreviated as HIV) pose a great threat to health. At present, the main detection means of the above viruses are immunoassay (such as enzyme-linked immunosorbent assay) and molecular biotechnology (such as PCR), the former has the main substance to be detected as protein, the latter has the main substance to be detected as nucleic acid, and the latter will become the development direction of future diagnosis due to its higher analysis sensitivity and specificity. However, it is important to further improve the analysis sensitivity of nucleic acid detection, so as to effectively detect early infected patients, and to avoid cross infection caused by the blood transfusion link, thereby effectively controlling the number of infected patients.
The company has a hepatitis B virus, hepatitis C virus and human immunodeficiency virus (type 1) nucleic acid detection kit (hereinafter referred to as the product) which can simultaneously and effectively detect HBV, HCV and HIV. The product mainly comprises two parts of nucleic acid extraction and amplification detection. In the nucleic acid extraction part, the product adopts a magnetic bead method to extract nucleic acid, and the extraction process is divided into 3 parts of nucleic acid adsorption, washing and elution. In the nucleic acid adsorption part, the product mainly separates and adsorbs nucleic acid through the silicon hydroxyl and chaotropic agent (or acid environment) under the condition of high salt (ionic strength) concentration. In the nucleic acid elution part, the product elutes nucleic acid under the conditions of alkalinity and low salt (ionic strength) concentration at high temperature, and finally, the aim of separating and purifying nucleic acid is achieved. Usually, the pH condition of elution is neutral or alkaline (pH7.0-8.5), and the elution buffer system is mainly Tris-HCl buffer system.
Currently, most nucleic acid extraction products (including the present product) employ a Tris-HCl buffer system as the elution buffer system. However, since the pH of the buffer system varies significantly with temperature (the pH decreases by about 0.3 for every 10 ℃ C. increase in temperature), the neutral Tris-HCl buffer system is susceptible to acidity at high temperature, thereby decreasing the elution efficiency of nucleic acids (mainly DNA). The usual solution is to adjust the pH of Tris-HCl to a slightly alkaline pH of 8.0-8.5, but at this pH the nucleic acid (mainly RNA) becomes unstable. Therefore, it is important to find a buffer system independent of temperature variation. It can be seen that there is room for further improvement in analytical sensitivity of existing products.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides the application of a sodium bicarbonate buffer system in nucleic acid extraction, and the application of the sodium bicarbonate buffer system in nucleic acid extraction aims to solve the technical problem that the analysis sensitivity of a nucleic acid detection kit with a Tris-HCl buffer system as an extraction buffer solution is reduced due to temperature change in the prior art.
The invention provides an elution buffer solution for a nucleic acid detection kit, wherein the elution buffer solution is a sodium bicarbonate solution, the concentration of the sodium bicarbonate solution is 5mM, and the pH value of the sodium bicarbonate solution is 6.7-7.2.
Further, the pH of the sodium bicarbonate solution is 7.0.
The invention also provides the application of the sodium bicarbonate buffer system in nucleic acid extraction, wherein the sodium bicarbonate buffer system is sodium bicarbonate solution, the concentration of the sodium bicarbonate solution is 5mM, and the pH value is 7.0.
The invention provides an elution buffer system for a nucleic acid detection kit, namely a pH7.0 sodium bicarbonate buffer system (pH6.7-7.2) with the concentration of 5mM, wherein the buffer system can be combined with other components (such as Tween and TritonX-100 with the concentration of 0.05%) to be used as an eluent for nucleic acid extraction.
The invention also provides application of the lysate containing arginine in a nucleic acid detection kit, wherein the concentration of arginine in the lysate is 15 mM.
The purpose of improving the HBV analysis sensitivity can be achieved by adding 15mM arginine (hereinafter referred to as arginine-containing lysate) into the lysate (nucleic acid adsorption part) of the nucleic acid detection kit.
The sodium bicarbonate buffer system can be used as an effective elution buffer system to improve the analysis sensitivity when nucleic acid (mainly DNA) is extracted.
Compared with the prior art, the invention has remarkable technical progress. The present invention provides a buffer system to improve the sensitivity of nucleic acid (mainly DNA) analysis. The invention increases the extraction efficiency of DNA under the condition of not changing the extraction efficiency of RNA, thereby increasing the analysis sensitivity of DNA detection of products.
Drawings
FIG. 1 is a graph showing the trend of the detection rate of each index of analytical sensitivity with the pH of sodium bicarbonate eluent.
FIG. 2 is a graph showing the behavior of an eluate at pH7.0 with respect to the number of days of standing at 37 ℃.
FIG. 3 is a graph showing a comparison of detection rates under different extraction and elution conditions.
Detailed Description
Example 1
First, research summary:
the main object of the study is the pH of the sodium bicarbonate buffer solution, and the study is divided into two parts of determination and verification of the pH of the buffer system. In the pH determination part of the buffer system, a suitable pH is found in the range of pH6.4-8.0 using a gradient search method. In the validation section of the pH of the buffer system, the thermal stability of the appropriate pH was validated to preliminarily confirm the suitability of the pH.
The main research method is a functional test (component replacement method), namely, related components in the product are replaced by components to be researched, and the components to be researched are evaluated by taking analysis sensitivity as an index. Wherein,
1.1 assay sensitivity sample concentration:
HBV：5IU/mL；HCV：50IU/mL；HIV：50IU/mL
1.2 sample size: 20
1.3 evaluation criteria: the detectable rate is more than or equal to 95 percent (19/20)
Secondly, determining the pH value of a sodium bicarbonate buffer system:
as mentioned above, we replaced the buffer system in the eluent of the kit with 5mM sodium bicarbonate buffer system (hereinafter referred to as sodium bicarbonate eluent) with different pH (pH6.4-8.0), and the other components were not changed, and the assay was performed with analytical sensitivity as the test item to screen and determine the appropriate pH.
Table 1: detection rate of various indexes of analysis sensitivity under different pH sodium bicarbonate eluates
Note:
1) the HBV detection rate # is a detection result of adding a pair of primer probes aiming at HBVS genes in the original HBV reaction liquid;
2) the data at ph7.5 are the results of the 3-day test with the eluent left at 37 ℃ (primary test results are HBV: 20/20, HCV: 20/20, HIV: 19/20), the pH was not used because the results were not acceptable when left at 37 ℃ for 3 days;
3) the extraction reagent batches are 20170102, the amplification reagent batches are 20180202, and the magnetic beads are 20 #;
4) pH test paper is adopted for pH measurement;
5) the HBV detection result at pH8.0 may be abnormal data.
Note:
1 extraction reagent batches are 20170102, amplification reagent batches are 20180202, and magnetic beads are 20#
The pH test paper is adopted for 2pH measurement
The HBV detection result of 3pH8.0 may be abnormal data
The experimental results (table 1 and fig. 1) show that the pH of the buffer solution has certain influence on HBV and HIV, the detection rate of HBV does not reach the standard under the condition of acid bias, the addition of a pair of primer probes is beneficial to stabilizing the detection rate of HBV, the detection rate of HIV does not reach the standard under the condition of alkali bias, and HCV is less influenced by pH. In combination with the above results, sodium bicarbonate buffer pH7.0 at a concentration of 5mM was able to be evaluated.
Thirdly, verifying thermal stability:
as previously described, we have verified the thermal stability (also referred to as reproducibility) of sodium bicarbonate buffers at pH6.7, 7.0 and 7.2.
Table 2: heat stability test results (detection rate of various indexes of analysis sensitivity) of sodium bicarbonate eluent with different pH values
Note:
1)1, the extraction reagent batches are 20170102, the amplification reagent batches are 20180202, and the magnetic beads are 20 #;
2)2, adopting pH test paper for pH measurement;
3)3 the eluent is placed in an air bath at 37 ℃.
Note:
1)1, 20170102 batches of extraction reagents, 20180202 batches of amplification reagents and 20# of magnetic beads.
2) The pH 2 is measured by using pH test paper.
3)3 analytical sensitivity the sample size of each test item was 20.
The experimental results (table 2 and fig. 2) show that the analytical sensitivity of the sodium bicarbonate eluent with pH7.0 does not change with the number of days (3, 5 and 7d) of standing at 37 ℃, and the detection indexes of the sodium bicarbonate buffer solutions with pH6.7, 7.0 and 7.2 reach the standards after being standing at 37 ℃ for 7 days. The pH test paper showed that the pH of each buffer was slightly increased (about 0.1-0.2) after being left at 37 ℃ for 7 days.
Comparative example
The main subject of this study was a 5mM pH7.0 sodium bicarbonate buffer, and the degree of improvement in sensitivity of DNA analysis was determined by comparison with the pH7.5 Tris-HCl elution buffer system (elution buffer system of the present product) for 5mM pH7.0 sodium bicarbonate buffer.
Note:
1) the extraction reagent batches are 20170102, the amplification reagent batches are 20180202, and the magnetic beads are 20 #.
2) The pH test paper is adopted for pH measurement.
3) Analytical sensitivity the sample size for each test item was 20.
4) Arg + represents a lysate containing arginine (lot 20180202), Arg-represents a lysate containing no arginine (lot: 20170202).
5) The lysate used with the NaHCO3 eluent is Arg-.
The results of the experiment (fig. 3) show that the test results of 5mM concentration of sodium bicarbonate eluent at ph7.0 are close to those of the lysis solution containing arginine, both being significantly higher than those of the lysis solution without arginine (Wilcoxon test, p < 0.05). It is demonstrated that the sodium bicarbonate eluent with the concentration of 5mM and the pH value of 7.0 can effectively improve the analysis sensitivity of the DNA of the product compared with the original product.
Claims (4)
1. An elution buffer for a nucleic acid detection kit, characterized in that: the elution buffer solution is sodium bicarbonate solution, the concentration of the sodium bicarbonate solution is 5mM, and the pH value is 6.7-7.2.
2. An elution buffer for a nucleic acid detection kit according to claim 1, wherein: the pH of the sodium bicarbonate solution was 7.0.
3. The application of sodium bicarbonate buffer system in nucleic acid extraction is characterized by that the sodium bicarbonate buffer system is sodium bicarbonate solution whose concentration is 5mM and pH value is 7.0.
4. The application of the lysate containing arginine in the nucleic acid detection kit is characterized in that the concentration of arginine in the lysate is 15 mM.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024017155A1 (en) * | 2022-07-22 | 2024-01-25 | 友康生物科技(北京)股份有限公司 | Lysis composition |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080076911A1 (en) * | 2006-09-26 | 2008-03-27 | Ge Healthcare Bio-Sciences Corp. | Nucleic acid purification method |
| US20110027862A1 (en) * | 2009-06-29 | 2011-02-03 | Life Technologies Corporation | Sample stabilization |
| CN104762296A (en) * | 2015-04-16 | 2015-07-08 | 乐山师范学院 | Method for extracting DNA of microorganisms on anaerobic fermentation materials of lignocelluloses and complete set of reagent |
| WO2016122058A1 (en) * | 2015-01-29 | 2016-08-04 | 인제대학교 산학협력단 | Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds |
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2019
- 2019-01-21 CN CN201910054564.6A patent/CN109722467A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080076911A1 (en) * | 2006-09-26 | 2008-03-27 | Ge Healthcare Bio-Sciences Corp. | Nucleic acid purification method |
| US20110027862A1 (en) * | 2009-06-29 | 2011-02-03 | Life Technologies Corporation | Sample stabilization |
| WO2016122058A1 (en) * | 2015-01-29 | 2016-08-04 | 인제대학교 산학협력단 | Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds |
| CN104762296A (en) * | 2015-04-16 | 2015-07-08 | 乐山师范学院 | Method for extracting DNA of microorganisms on anaerobic fermentation materials of lignocelluloses and complete set of reagent |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024017155A1 (en) * | 2022-07-22 | 2024-01-25 | 友康生物科技(北京)股份有限公司 | Lysis composition |
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Application publication date: 20190507 |
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