CN108998569A - A kind of hepatitis type B virus serum sample saves dilution and preparation method thereof - Google Patents
A kind of hepatitis type B virus serum sample saves dilution and preparation method thereof Download PDFInfo
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- CN108998569A CN108998569A CN201810912948.2A CN201810912948A CN108998569A CN 108998569 A CN108998569 A CN 108998569A CN 201810912948 A CN201810912948 A CN 201810912948A CN 108998569 A CN108998569 A CN 108998569A
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- dilution
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a kind of hepatitis type B virus serum samples to save dilution, and ingredient includes Trise-HCL, NaCL, BSA, gentamicin sulphate, P-300, sunset yellow, lemon yellow and deionized purified water；In terms of 1L deionized purified water, each component content is Trise-HCL 0.01mol, NaCL8.8g, BSA20g, gentamicin sulphate 0.68ml, P-3002.0ml, sunset yellow 0.01g, lemon yellow 0.01g.The advantage of the invention is that ingredient is few; preparation method is simple; preparing the finished product preservation dilution completed can protect the viral nucleic acid structure of matter not to be damaged; inhibit DNase activity; the DNA of effective protection virus is from degradation; have many advantages, such as stabilization, easy to maintain, convenient, preservation and dilution suitable for hepatitis type B virus (HBV DNA) serum sample.Test proves, after the preservation dilution that hepatitis type B virus (HBV DNA) serum sample is added that the present invention prepares, DNA, RNA completely good can be saved 1 week at 37 DEG C, be saved 1 month at 20-25 DEG C, it is saved 6 months at 4 DEG C, the long-term preservation at -20 DEG C or -80 DEG C.
Description
Technical field
The present invention relates to biological products technologies, save dilution more particularly, to a kind of hepatitis type B virus serum sample,
The invention further relates to the preparation methods of the preservation dilution.
Background technique
Serum of hepatitis B diluted sample is chiefly used in kit Quality Control and third party's Quality Control.Hepatitis type B virus abbreviation hepatitis B
Virus is a kind of dsDNA virus, belongs to Hepadnaviridae.The genome (DNA) of hepatitis B is by the dna of two spirals
The ring structure that chain surrounds.Wherein a longer minus strand has formed complete ring-type；The shorter normal chain of another length,
In semicircular.The substance for leading to DNA degradation includes water and the nuclease that can digest DNA.This different substance in nature nowhere
Do not exist.Cause the factor of DNA degradation very much, such as extraneous physical factor, chemical factor, biological factor.
Conventional serum of hepatitis B, which saves dilution, mainly to be had: 1, mankind's negative serum, blood plasma；2, DEPC water,
DNAsin etc.；3, with liquid nitrogen or cryo-conservation.Its disadvantage are as follows: 1, preservation effect it is poor, easy bleeding degradation；2, sample needs after diluting
Cryo-conservation is carried out immediately.Therefore, a kind of guarantor for keeping serum of hepatitis B sample stable of similar human serum state is developed
Dilution is deposited, is had great importance.
Summary of the invention
The purpose of the present invention is to provide it is a kind of can effectively protect the hepatitis B virus nucleic acid structure of matter be not damaged it is B-mode
Hepatitis virus serum sample saves dilution, and the present invention also provides the preparation methods of the preservation dilution.
To achieve the above object, the present invention can take following technical proposals:
Hepatitis type B virus (HBV DNA) serum sample of the present invention saves dilution, ingredient include Trise-HCL,
NaCL, BSA, gentamicin sulphate, P-300, sunset yellow, lemon yellow and deionized purified water；In terms of 1L deionized purified water, respectively
Component content is Trise-HCL 0.01mol, NaCL8.8g, BSA20g, gentamicin sulphate 0.68ml, P-3002.0ml, day
Fall yellow 0.01g, lemon yellow 0.01g.
Hepatitis type B virus serum sample of the present invention save dilution the preparation method comprises the following steps:
The first step measures suitable deionized purified water；
Second step, by the Trise-HCL, NaCL accurately measured, BSA, gentamicin sulphate, P-300, sunset yellow, lemon yellow according to
It is stirred evenly in secondary addition deionized purified water；
Third step is settled to 1L after adjusting the pH to 7.8 ~ 8.2 of solution；
4th step, high pressure sterilization (121 DEG C, 15min) after sealing, then saves backup for 2 ~ 8 DEG C.
The advantage of the invention is that ingredient is few, preparation method is simple, and the finished product for preparing completion, which saves dilution, can protect disease
Malicious nucleic acid substances structure is not damaged, and inhibits DNase activity, and the DNA of effective protection virus has stable, Yi Bao from degradation
The advantages that depositing, is convenient is suitable for the preservation and dilution of hepatitis type B virus (HBV DNA) serum sample.Test proves, B-mode
After the preservation dilution that the present invention prepares is added in hepatitis virus (HBV DNA) serum sample, DNA, RNA can be completely good 37
It saves 1 week at DEG C, is saved 1 month at 20-25 DEG C, saved 6 months at 4 DEG C, the long-term preservation at -20 DEG C or -80 DEG C.
Detailed description of the invention
Fig. 1 shows control group PCR amplification detection figure.
Fig. 2 shows experimental group PCR amplification detection figure.
Fig. 3 shows the diluted HBV serum clinical sample PCR amplification detection figure of negative serum in 37 DEG C of environment.
Fig. 4 shows the HBV serum clinical sample PCR amplification detection figure of diluted in 37 DEG C of environment.
Fig. 5 shows the diluted HBV serum clinical sample PCR amplification detection figure of negative serum in room temperature environment.
Fig. 6 shows that the HBV serum clinical sample PCR amplification of diluted in room temperature environment detects figure.
Fig. 7 shows the diluted HBV serum clinical sample PCR amplification detection figure of negative serum in 2-8 DEG C of environment.
Fig. 8 shows that the HBV serum clinical sample PCR amplification of diluted in 2-8 DEG C of environment detects figure.
Fig. 9 shows the diluted HBV serum clinical sample PCR amplification detection figure of negative serum in -20 DEG C of environment.
Figure 10 shows the HBV serum clinical sample PCR amplification detection of diluted in -20 DEG C of environment.
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment.Wherein technological means used in embodiment
If it is no it is special indicate, be conventional method；If the reagent being related to can be obtained with consumptive material without specified otherwise by commercial sources
It takes.
Embodiment 1 prepares hepatitis type B virus serum sample and saves dilution
The first step measures suitable deionized purified water；
Second step, by the Trise-HCL0.01mol/L, NaCL8.8g accurately measured, BSA20g, gentamicin sulphate 0.68ml,
P-300 2.0ml, sunset yellow 0.01g, lemon yellow 0.01g are sequentially added in deionized purified water and are stirred evenly；
Third step adds deionized purified water to be settled to 1L after adjusting the pH to 7.8 ~ 8.2 of solution；
4th step, high pressure sterilization (121 DEG C, 15min) after sealing, then 2 ~ 8 DEG C of preservations.
Embodiment 2 dilutes clinical sample experiment
1, instrument, reagent and sample
Key instrument: dry type constant-temperature metal bath, FLEX extraction apparatus, Biohazard Safety Equipment, pipettor, macro stone real-time fluorescence quantitative PCR
Instrument etc..
Main agents: the hbv nucleic acid assay kit of Da'an Gene Company, Zhongshan University
(PCR- fluorescence probe method).
Sample: fresh HBV positive clinical sample.
2, embodiment
With embodiment 1 prepare serum virus Sample preservation dilution by HBV positive sample dilute 5 times, 50 times, 500 times,
5000 times.Design control combination two kinds of dilutions of experimental group, with the hepatitis B of Da'an Gene Company, Zhongshan University
Viral nucleic acid assay kit (PCR- fluorescence probe method) extracts amplification to two groups of diluted samples of different diluent.
Control group: negative serum dilution and HBV positive sample.
Experimental group: serum virus Sample preservation dilution of the present invention and HBV positive sample.
3, experimental result
PCR amplification detection figure is shown in Fig. 1, Fig. 2.
The testing result of 1 HBV positive sample of table
The experimental results showed that the HBV positive sample for the serum virus Sample preservation diluted prepared with the present invention, amplification is bent
Line is in typical S curve, and diluted concentration gradient interval is preferable, the dilution suitable for clinical serum plasma sample.
The test of 3 storage stability of embodiment
1, instrument, reagent and sample
Key instrument: dry type constant-temperature metal bath, FLEX extraction apparatus, Biohazard Safety Equipment, pipettor, macro stone real-time fluorescence quantitative PCR
Instrument etc..
Main agents: the hbv nucleic acid assay kit of Da'an Gene Company, Zhongshan University
(PCR- fluorescence probe method)
Sample: fresh HCV pseudovirus sample
2, experimental program
Hepatitis type B virus (HBV DNA) serum sample prepared with embodiment 1 saves dilution and dilutes HBV positive sample
100 times, 1000 times are respectively labeled as P1, P2 as in, low concentration test sample.Respectively in -20 ± 5 DEG C of environment, 2-8 DEG C
It in environment, places in room temperature (20-25 DEG C) environment and in 37 DEG C of environment, this experimental design is as follows: the sample after dilution is carried out
Label, be respectively put into designated environment, test by the specified period, it is specific as follows: 1. in -20 ± 5 DEG C of environment, 3 months or
Half a year detection is primary；2. in 2-8 DEG C of environment, detection in every 7 days is primary, detect 35 days；In room temperature 3. (20-25 DEG C) environment, every 3 days
Detection is primary, detects 10 days；4. in 37 DEG C of environment, every other day detection is primary, detect 7 days.It is extracted with kit.
Control group: placing the diluted sample of negative serum in -80 DEG C of environment, and it is dilute to place present invention preservation in -80 DEG C of environment
Release the diluted sample of liquid.
Experimental group: hepatitis type B virus (HBV DNA) the nucleic acid serum sample that the present invention prepares saves diluted
Sample is respectively in the environment of placement -20-20 ± 5 DEG C, in 2-8 DEG C of environment, in room temperature (20-25 DEG C) environment and in 37 DEG C of environment.
3, experimental result
The Detection of Stability result (see figure 3) of negative serum diluted sample in 2-20 ± 5 DEG C of environment of table
Conclusion: from Fig. 3 and table 2 it can be seen that in -20 ± 5 DEG C of environment negative serum diluted sample place 24 months two it is dense
It spends concentration of specimens value and Linear Stability is more stable.
The Detection of Stability result (see figure 4) of diluted sample in 3-20 ± 5 DEG C of environment of table
The Detection of Stability result (see figure 5) of negative serum diluted sample in the 2-8 DEG C of environment of table 4
Conclusion: from Fig. 5 and table 4 it can be seen that negative serum diluted sample places 35 days two concentration samples in 2-8 DEG C of environment
Concentration value and Linear Stability are more stable.
The Detection of Stability result (see figure 6) of diluted sample in the 2-8 DEG C of environment of table 5
Conclusion: from Fig. 6 and table 5 it can be seen that 35 days two concentration samples of diluted sample placement are dense in 2-8 DEG C of environment
Angle value and Linear Stability are preferable.
The Detection of Stability result (see figure 7) of negative serum diluted sample in table 6 (20-25 DEG C) room temperature environment
Conclusion: from Fig. 7 and table 6 it can be seen that in (20-25 DEG C) room temperature environment negative serum diluted sample place 10 days two
Concentration samples concentration value and Linear Stability are more stable.
The Detection of Stability result (see figure 8) of diluted sample in table 7 (20-25 DEG C) room temperature environment
Conclusion: from Fig. 8 and table 7 it can be seen that in (20-25 DEG C) room temperature environment diluted sample place 10 days two it is dense
It spends concentration of specimens value and Linear Stability is more stable.
The Detection of Stability result (see figure 9) of negative serum diluted sample in 8 37 DEG C of environment of table
Conclusion: from Fig. 9 and table 8 it can be seen that from Fig. 9 and table 8 it can be seen that negative serum diluted sample is put in 37 DEG C of environment
It sets 7 days two concentration samples concentration values and Linear Stability is more stable.
The Detection of Stability result (see figure 10) of diluted sample in 9 37 DEG C of environment of table
Conclusion: from Figure 10 and table 9 it can be seen that 7 days two concentration samples of diluted sample placement are dense in 37 DEG C of environment
Angle value and Linear Stability are more stable.
By being tested above it can be seen that being saved with hepatitis type B virus prepared by the present invention (HBV DNA) serum sample
The HBV Almost Sure Sample Stability that dilution saves is preferable, can substitute negative serum dilution hepatitis type B virus (HBV DNA) blood completely
Clear Sample preservation.
Claims (2)
1. a kind of hepatitis type B virus serum sample saves dilution, it is characterised in that: its ingredient include Trise-HCL, NaCL,
BSA, gentamicin sulphate, P-300, sunset yellow, lemon yellow and deionized purified water；In terms of 1L deionized purified water, each ingredient
Content is Trise-HCL 0.01mol, NaCL8.8g, BSA20g, gentamicin sulphate 0.68ml, P-3002.0ml, sunset yellow
0.01g, lemon yellow 0.01g.
2. the preparation method of the preservation dilution of hepatitis type B virus serum sample described in claim 1, it is characterised in that: including under
State step:
The first step measures suitable deionized purified water；
Second step, by the Trise-HCL, NaCL accurately measured, BSA, gentamicin sulphate, P-300, sunset yellow, lemon yellow according to
It is stirred evenly in secondary addition deionized purified water；
Third step is settled to 1L after adjusting the pH to 7.8 ~ 8.2 of solution；
Then 4th step, high pressure sterilization after sealing save backup for 2 ~ 8 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810912948.2A CN108998569A (en) | 2018-08-13 | 2018-08-13 | A kind of hepatitis type B virus serum sample saves dilution and preparation method thereof |
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| CN201810912948.2A CN108998569A (en) | 2018-08-13 | 2018-08-13 | A kind of hepatitis type B virus serum sample saves dilution and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684869A (en) * | 2019-11-07 | 2020-01-14 | 郑州安图生物工程股份有限公司 | Nucleic acid preserving fluid, application thereof and quality control product containing nucleic acid preserving fluid |
Citations (5)
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| WO1990010060A1 (en) * | 1989-02-24 | 1990-09-07 | Southwest Foundation For Biomedical Research | Non-a, non-b hepatitis hepatocyte cell culture |
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Patent Citations (5)
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|---|---|---|---|---|
| WO1990010060A1 (en) * | 1989-02-24 | 1990-09-07 | Southwest Foundation For Biomedical Research | Non-a, non-b hepatitis hepatocyte cell culture |
| CN103323308A (en) * | 2013-05-23 | 2013-09-25 | 北京博晖创新光电技术股份有限公司 | Reagent for processing trace whole blood and application thereof |
| CN204882568U (en) * | 2015-07-02 | 2015-12-16 | 深圳市绿诗源生物技术有限公司 | ELISA detect reagent box of pig O type foot and mouth disease virus antibody |
| CN105524916A (en) * | 2016-01-01 | 2016-04-27 | 广州邦德盛生物科技有限公司 | Solution for preserving and diluting DNA samples and preparation of solution |
| CN107102134A (en) * | 2017-03-02 | 2017-08-29 | 江苏华冠生物技术股份有限公司 | A kind of sample diluting liquid suitable for peripheral blood detection |
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| 齐玉杰 等: "乳制品中三聚氰胺的免疫亲和凝胶柱检测方法", 《中国乳品工业》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684869A (en) * | 2019-11-07 | 2020-01-14 | 郑州安图生物工程股份有限公司 | Nucleic acid preserving fluid, application thereof and quality control product containing nucleic acid preserving fluid |
| CN110684869B (en) * | 2019-11-07 | 2023-05-23 | 郑州安图生物工程股份有限公司 | Nucleic acid preservation solution and application thereof, and quality control product containing nucleic acid preservation solution |
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Application publication date: 20181214 |