CN106234354B - A kind of cervix cell preservation solution - Google Patents
A kind of cervix cell preservation solution Download PDFInfo
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- CN106234354B CN106234354B CN201610875401.0A CN201610875401A CN106234354B CN 106234354 B CN106234354 B CN 106234354B CN 201610875401 A CN201610875401 A CN 201610875401A CN 106234354 B CN106234354 B CN 106234354B
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- liquid
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of cervix cell preservation solutions, including PBS buffer solution, dodecyl trimethyl ammonium chloride and Proteinase K, the dodecyl trimethyl ammonium chloride, which is first mixed into PBS buffer solution, forms first preservation liquid, and the dodecyl trimethyl ammonium chloride is 5-15% saving the mass percent concentration in liquid for the first time；It is mixed into the first preservation liquid after the Proteinase K and forms finished product preservation liquid, the Proteinase K and the first mass volume ratio concentration for saving liquid are 4-6mg/ml.This cervix cell preservation solution has easy to operate, and preparation is easy, to the treatment effect of hemorrhagic sample and mucus sample preferably and the advantages of suppression will not occur.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of cell-preservation liquid more particularly to a kind of cervix cell preservation solution.
Background technique
In existing HPV (human papilloma virus) parting detection, sample process is a key link, is directly determined final
As a result accuracy.Current cervix cell preservation solution is physiological saline, and there are following institutes for the sample process of the cell-preservation liquid
The disturbing factor stated:
1, blood sample carries out in the sample of HPV detection there are about the sample of one third being courage and uprightness, and red blood cell extracting
Main component after rupturing during DNA is hemoglobin etc., and hemoglobin can not completely remove, and directly affect subsequent
PCR (polymerase chain reaction) process generates inhibiting effect to this process, causes result false negative.
2, mucus sample, carry out HPV detection sample in there are about the samples of half to contain mucus.The presence of mucus influences sample
This suction process causes to inhale sample deficiency, causes false negative.
For this purpose, existing cervix cell preservation solution just can be carried out HPV detection after needing first to carry out elution processing, so that processing
Process becomes cumbersome, once skipping elution processing, is easy to produce erroneous judgement to have an adverse effect to patient health.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the PBS buffer solution cell-preservation liquids of the prior art to need first to be washed
The deficiency that just can be carried out HPV detection after de- processing, provides a kind of cervix cell preservation solution.
In order to solve the above technical problems, the present invention adopts the following technical scheme that:
A kind of cervix cell preservation solution, it is characterised in that: including PBS buffer solution, dodecyl trimethyl ammonium chloride and egg
White enzyme K, the dodecyl trimethyl ammonium chloride, which is first mixed into PBS buffer solution, forms first preservation liquid, the dodecyl three
Ammonio methacrylate is 5-15% saving the mass percent concentration in liquid for the first time；The first guarantor is mixed into after the Proteinase K
Liquid storage forms finished product and saves liquid, and the Proteinase K and the first mass volume ratio concentration for saving liquid are 4-6mg/ml.
The PBS buffer solution is phosphate buffer (abbreviation PBS-T) of the pH=7.4 containing 0.05% Tween-20
It is had the advantages that using above-mentioned cervix cell preservation solution
1, the trimethyl component in cervix cell preservation solution of the invention can destroy red blood cell and play haemolysis
Effect, Proteinase K can effectively decompose mucus, and PBS buffer can play the role of saving cervical cell.
2, cervix cell preservation solution of the invention can be detected directly without carrying out elution processing, and detect essence
The detection accuracy spent after eluting with existing cervix cell preservation solution is suitable, simplifies operating procedure, improves the detection of HPV viruse
Efficiency.
3, cervix cell preservation solution raw material of the invention is cheap and easily-available, easily prepared.
Detailed description of the invention
Fig. 1 is HPV.16+PCR amplification standard curve, abscissa are recurring number, and ordinate is copy number.
Fig. 2 is respectively using producer's cell-preservation liquid and cell-preservation liquid of the present invention to HPV16+Save and extracts amplification
Obtained amplification curve, abscissa are recurring number, and ordinate is copy number.
Fig. 3 is respectively using producer's cell-preservation liquid and cell-preservation liquid of the present invention to HPV18+Save and extracts amplification
Obtained amplification curve, abscissa are recurring number, and ordinate is copy number.
Fig. 4 is respectively using producer's cell-preservation liquid and cell-preservation liquid of the present invention to HPV31+Save and extracts amplification
Obtained amplification curve, abscissa are recurring number, and ordinate is copy number.
Fig. 5 is respectively using producer's cell-preservation liquid and cell-preservation liquid of the present invention to HPV58+Save and extracts amplification
Obtained amplification curve, abscissa are recurring number, and ordinate is copy number.
Fig. 6 is to carry out preservation extraction to HPVCP8304 using producer's cell-preservation liquid and cell-preservation liquid of the present invention respectively
Obtained amplification curve is expanded, abscissa is recurring number, and ordinate is copy number.
Fig. 7 shows that verify type qualification test by flow hybridization method is played the role of to verify the present invention.
Specific embodiment
Experimental example 1
The mass fraction of dodecyl trimethyl ammonium chloride in cervix cell preservation solution has been tested to erythroclasis effect
With the influence to HPV testing result:
Experimentation is as follows: being mixed into PBS buffer solution by dodecyl trimethyl ammonium chloride and forms first preservation liquid；PBS
Buffer is the phosphate buffer of the pH=7.4 containing 0.05% Tween-20.
The first preservation liquid of dodecyl trimethyl ammonium chloride containing different quality percentage is respectively placed in different anti-
It answers in container and clear marking, it is separately sampled from same cervical cell sample to be put into respective reaction vessel, in identical item
Reaction result such as following table is recorded after reacting under part；
Table 1:
Reaction result is as shown in table 1: when mass percent of the dodecyl trimethyl ammonium chloride in preservation liquid for the first time is
Hemolyzing effect is preferable when 5%, and to the testing result unrestraint phenomenon of HPV, when dodecyl trimethyl ammonium chloride is first
Complete hemolysis can be accomplished and show to the testing result unrestraint of HPV by saving when the mass percent in liquid is 10%, 15%
As when dodecyl trimethyl ammonium chloride can accomplish completely when saving the mass percent in liquid for the first time and being 20%, 25%
Haemolysis, but have part suppression to the testing result of HPV, when saving in liquid without trimethyl, absolutely not
Haemolysis and to the testing result of HPV completely inhibit, to sum up, the mass percent of preferred dodecyl trimethyl ammonium chloride is
5%-15%, the mass percent of most preferred dodecyl trimethyl ammonium chloride are 10%.
Experimental example 2
Tested same volume is the first preservation liquid of 10% dodecyl trimethyl ammonium chloride containing mass percent
In, it is separately added into the Proteinase K of different quality, the finished product for forming different quality volume by volume concentration saves liquid to the processing to mucus
Effect and influence to HPV testing result.
Experimentation is as follows: what it is in same volume is the first of 10% dodecyl trimethyl ammonium chloride containing mass percent
In secondary preservation liquid, it is separately added into the Proteinase K of different quality, forms Proteinase K and the first mass volume ratio concentration for saving liquid
Different multiple finished products save liquid, be placed in the acceptance of the bid of differential responses container remember it is clear, it is separately sampled from same sample to be put into respectively
Reaction vessel in, after being reacted under the same conditions, record reaction result such as table 2；
Table 2:
| The mass volume ratio concentration of Proteinase K | Handle the effect of mucus | Influence (Quality Control IC point) to HPV result |
| 1mg/mL | Not exclusively | Part inhibits |
| 2mg/mL | Not exclusively | Part inhibits |
| 3mg/mL | Completely | Part inhibits |
| 4mg/mL | Completely | Unrestraint phenomenon |
| 5mg/mL | Completely | Unrestraint phenomenon |
| 6mg/mL | Completely | Unrestraint phenomenon |
| 7mg/mL | Completely | Part inhibits |
| 8mg/mL | Completely | Part inhibits |
| 9mg/mL | Completely | Part inhibits |
Reaction result is as shown in table 2: when Proteinase K and the first mass volume ratio concentration for saving liquid be respectively 4mg/ml,
When 5mg/ml, 6mg/ml, mucus and the testing result unrestraint phenomenon to HPV can be handled completely；
When Proteinase K and the first mass volume ratio concentration for saving liquid are respectively 3mg/ml, 7mg/ml, 8mg/ml, 9mg/
Mucus can be handled completely when ml and has part suppression to the testing result of HPV；
When Proteinase K and the first mass volume ratio concentration for saving liquid are respectively 1mg/ml, 2mg/ml, processing mucus is simultaneously
Not exclusively and there is part inhibition to the testing result of HPV；
In conclusion preferred Proteinase K and the first mass volume ratio concentration for saving liquid are 4-6mg/ml, most preferably
Proteinase K and the first mass-volume concentration ratio for saving liquid are 5mg/ml.
Experimental example 4
Having tested cervix cell preservation solution of the invention, (dodecyl trimethyl ammonium chloride is saving the quality in liquid for the first time
Percentage is 10%, and Proteinase K is 5mg/ml with the first mass volume ratio concentration for saving liquid) it is compared in the case where not eluting
The prior art producer's cervix cell preservation solution (Chaozhou Kaipu Biochemistry Co., Ltd. production cell-preservation liquid, mainly at
Point: PBS) whether generated curve and CT value have difference in fluorescent quantitative PCR technique confirmatory experiment in the case where elution；
Before experiment, HPV.16 need to be carried out+PCR amplification obtains standard curve, as shown in Figure 1.
When experiment, with 5 cervical cell courage and uprightness samples, be respectively adopted the present invention do not elute, the elution of producer's cell-preservation liquid
Operation is not eluted using the present invention by fluorescent quantitative PCR technique verifying and is produced with the two kinds of operations of elution of producer's cell-preservation liquid
Raw curve and CT value has indifference, and experimental result is as shown in figures 2-6, in which: abscissa is recurring number, and ordinate is copy
Number；
The experimental results showed that in shown five experiments generated curve and the prior art it is substantially overlapping, illustrate this hair
It is bright effectively and will not suppression PCR process.
Experimental example 5
Having tested the present invention, (dodecyl trimethyl ammonium chloride is 10% saving the mass percent in liquid for the first time, egg
White enzyme K and the first mass volume ratio concentration for saving liquid are 5mg/ml) producer of the comparison prior art in the case where not eluting
Cervix cell preservation solution (Chaozhou Kaipu Biochemistry Co., Ltd. production cell-preservation liquid, main component: PBS) elution and
Flow hybridization method verifying type qualification test is carried out in the case where not eluting to be played the role of to verify the present invention,
As shown in fig. 7, the experimental result is indicated by HPV probe film parting schematic diagram, parting schematic diagram is arranged by four rows six
Table composition, the upper left corner of table is one immediately below Biotin point for detecting film item whether effective Biotin point
Grid is that whether there is mortifier during detecting PCR, the Quality Control IC point of amplification efficiency and DNA concentration, remaining each grid is each
Represent a kind of HPV viruse hypotype.
Experimental result will be recorded on probe film item, and probe film item is equipped with table corresponding with parting schematic diagram,
Biotin point 1 is corresponding with the table position of HPV film parting schematic diagram respectively with IC point 2, the HPV viruse hypotype institute examined
The grid at place is also corresponding with HPV probe film parting schematic diagram.
Probe film item after experiment is as shown in fig. 7, a row shares five groups of experiment sample 1001-1005, from left to right,
One to the 4th is sample 1001-1004, is to contain blood sample, and the 5th is sample 1005, is free from blood sample, three rows in Fig. 7 point
Type schematic diagram respectively corresponds three kinds of experimental conditions, and first row is the experimental conditions that cell-preservation liquid group of the invention does not elute, the
Two rows are the experimental conditions after the cell-preservation liquid group elution of the prior art, and the cell-preservation liquid group of third row's prior art is not washed
De- experimental conditions, experimental result are described as follows shown in table 3:
The results showed that after the experimental conditions of cell-preservation liquid of the invention and the cell-preservation liquid elution of the prior art
Experimental conditions it is consistent, and the cell-preservation liquid group of prior art HPV testing result in the case where not eluting can be suppressed.It is real
Test results showed that cell-preservation liquid of the invention can also possess under conditions of removing elution step from it is in the prior art
Cell-preservation liquid is by elution treated HPV detection accuracy.
Summarize above-mentioned experimental example, this cervix cell preservation solution has easy to operate, and preparation is easy, to hemorrhagic sample and mucus
The treatment effect of sample is preferably and the advantages of will not occur suppression.
Claims (4)
1. a kind of cervix cell preservation solution, it is characterised in that: including PBS buffer solution, dodecyl trimethyl ammonium chloride and albumen
Enzyme K, the dodecyl trimethyl ammonium chloride, which is first mixed into PBS buffer solution, forms first preservation liquid, the dodecyl front three
Ammonium chloride is 5-15% saving the mass percent concentration in liquid for the first time；The first preservation is mixed into after the Proteinase K
Liquid forms finished product and saves liquid, and the Proteinase K and the first mass volume ratio concentration for saving liquid are 4-6mg/ml.
2. cervix cell preservation solution according to claim 1, it is characterised in that: the PBS buffer solution is to spit containing 0.05%
The phosphate buffer of the pH=7.4 of temperature -20.
3. cervix cell preservation solution according to claim 1, it is characterised in that: the dodecyl trimethyl ammonium chloride exists
The first mass percent concentration saved in liquid is 10%.
4. according to claim 1 to cervix cell preservation solution described in 3 any claims, it is characterised in that: the Proteinase K
It is 5mg/ml with the first mass volume ratio concentration for saving liquid.
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| CN201610875401.0A CN106234354B (en) | 2016-09-30 | 2016-09-30 | A kind of cervix cell preservation solution |
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| CN106234354B true CN106234354B (en) | 2019-05-21 |
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| CN108235981B (en) | 2016-12-23 | 2021-07-23 | 西比曼生物科技（香港）有限公司 | Cell cryopreservation liquid capable of being used clinically |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363011A (en) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | Cervical exfoliated cell preservative fluid |
| CN101485303A (en) * | 2009-02-26 | 2009-07-22 | 广州鸿琪光学仪器科技有限公司 | Exfoliated cell preservative fluid |
| CN103120153A (en) * | 2012-11-26 | 2013-05-29 | 刘召宏 | Exfoliative cells preserving fluid |
| CN105092328A (en) * | 2015-07-30 | 2015-11-25 | 厦门宝太生物科技有限公司 | Erythrocyte removing treating fluid and application |
-
2016
- 2016-09-30 CN CN201610875401.0A patent/CN106234354B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363011A (en) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | Cervical exfoliated cell preservative fluid |
| CN101485303A (en) * | 2009-02-26 | 2009-07-22 | 广州鸿琪光学仪器科技有限公司 | Exfoliated cell preservative fluid |
| CN103120153A (en) * | 2012-11-26 | 2013-05-29 | 刘召宏 | Exfoliative cells preserving fluid |
| CN105092328A (en) * | 2015-07-30 | 2015-11-25 | 厦门宝太生物科技有限公司 | Erythrocyte removing treating fluid and application |
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Inventor after: Feng Jing Inventor after: Wei Quhao Inventor after: Xiao Linlin Inventor before: Xiao Linlin Inventor before: Feng Jing Inventor before: Wei Quhao |
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Effective date of registration: 20190417 Address after: No. 6600 Nanfeng Highway, Nanqiao Town, Fengxian District, Shanghai 201400 Applicant after: Shanghai Fengxian Centrl Hospital Applicant after: Xiao Linlin Address before: No. 6600 Nanfeng Highway, Nanqiao Town, Fengxian District, Shanghai 201400 Applicant before: Shanghai Fengxian Centrl Hospital |
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Effective date of registration: 20191128 Address after: 201400, Shanghai, Fengxian District South Village Road, No. 6600 Patentee after: Shanghai Fengxian Centrl Hospital Address before: 201400, Shanghai, Fengxian District South Village Road, No. 6600 Co-patentee before: Xiao Linlin Patentee before: Shanghai Fengxian Centrl Hospital |