CN103271023B

CN103271023B - Cell preservation solution with liquefying function for cervical exfoliated cell sample

Info

Publication number: CN103271023B
Application number: CN201310173184.7A
Authority: CN
Inventors: 匡有吉; 赵雷; 田野; 黄明辉
Original assignee: GENETEL PHARMACEUTICALS (SHENZHEN) CO Ltd
Current assignee: GENETEL PHARMACEUTICALS (SHENZHEN) CO Ltd
Priority date: 2013-05-11
Filing date: 2013-05-11
Publication date: 2015-03-18
Anticipated expiration: 2033-05-11
Also published as: CN103271023A

Abstract

The invention discloses a cervix cell preservation solution which comprises tris(2-carboxyethyl)phosphine hydrochloride, an osmotic pressure maintaining agent and an aqueous solution of pH (Potential Of Hydrogen) buffer solution components. The invention describes a novel cervix cell preservation solution which is characterized by comprising a stable reductant at a concentration of 0.1 to 1% and a formula used for the cervix cell preservation solution; and the cervix cell preservation solution under the coordination of the DNA (Deoxyribose Nucleic Acid) direct cracking method can be used for well solving the problems of more secretion and high viscidity of a ervical exfoliated cell sample and improving the sensitivity in detection of the cervical exfoliated cell.

Description

A kind of cervical exfoliated cell sample cell-preservation liquid with liquefaction function

Technical field

The cell-preservation liquid of the present invention's generality design containing a kind of reductant, can destroy viscous protein matter secondary structure, and make containing the more cervical exfoliated cell sample liquefaction of viscous secretion, the DNA contributing to sample extracts and detects.

Background technology

When the cervical exfoliated cell of conventional cervical smear sampling carries out DNA and RNA analysis, often have sample vaginal secretion secretion many, be difficult to obtain enough cervical exfoliated cells when carrying out foranalysis of nucleic acids, sample is first carried out liquefaction process by general needs.The viscosity of secretion is very large, and the viscous protein matter mainly containing secretion is many.In mucus, moisture accounts for 95%, and large molecule mucoprotein accounts for 2% ~ 3%, and protein accounts for 0.1% ~ 0.5%, and the chemical structural formula of mucus, is attached on sugary protein side chain by disulfide bond crosslinking for main chain with a kind of protein.Conventional liquefaction principle is that secretory protein material is carried out degenerative treatments, such as, add NaOH, DTT etc.NaOH is alkaline matter, can directly by protein denaturation, but pH of sample is about the DNA direct detecting method of about 7 when needing application of sample for those, to need in sample after denaturing samples and, pH value is made to be adjusted to neutrality, carry out PCR so that follow-up or other are analyzed, and alkaline denaturation can destroy cell, thus alkaline denaturation to direct-detection DNA, RNA and need to keep some detection methods of cellular morphology inapplicable.Dithiothreitol (DTT) (Dithiothreitol, referred to as DTT) is a kind of Small molecular organic reducing agent, and chemical formula is C ₄h ₁₀o ₂s ₂.Be linear molecule under its reducing state, after oxidized, become the six-membered cyclic structure comprising disulfide bond.The name of dithiothreitol (DTT) is derived from threose (a kind of four carbon monose).DTT is also usually used to the reduction of disulfide bond in protein, can be used for stoping in the protein molecule that formed between the cysteine in protein or intermolecular disulfide bond.But DTT often cannot reduce the disulfide bond being embedded in protein structure inside (solvent is unreachable).Because DTT can go back the disulfide bond of crude protein, make protein denaturation, be usually used in the liquefaction of secretion, the liquefaction etc. of such as phlegm.Due to easy oxidation by air, the therefore less stable of DTT; But freezen protective or in inert gas process can extend its service life.Because the nucleophilicity of protonated sulphur is lower, along with the reduction of pH value, effective reproducibility of DTT also decreases; DTT also has peculiar smell, harmful.Therefore be necessary the method using the reductant of colorless and odorless, stable in properties to liquefy for the cervical cell more containing secretion, improve the efficiency that DNA extracts.

Summary of the invention

The present invention describes a kind of new cervix cell preservation solution, it is characterized in that being the stable reductant of 0.1-1% containing a kind of concentration, for the formula of cervix cell preservation solution, coordinate the method for DNA By Direct Pyrolysis, the cervical exfoliated cell sample that the more viscosity of secretion is larger can be solved preferably, add the sensitivity that cervical exfoliated cell detects.The title that the present invention relates to a kind of reductant is three (2-carboxyethyl) phosphonium salt hydrochlorate, and English name is that Tris (2-carboxyethyl) phosphine(is abbreviated as: TCEPHCl, and molecular formula is C ₉h ₁₆o ₆pCl), TCEPHCl molecular structure is as follows:

TCEPHCl is a kind of reductant of not sulfur-bearing, can reduce the disulfide bond of protein molecule, colorless and odorless, to air-stable, and the functional group reaction in discord protein, non-volatile, at room temperature can keep stable, easy to use.The present invention studies the working concentration of this reductant, use high concentration can to degrade faster mucus, but can the TAQ enzyme in PCR reactant liquor be impacted, the time that adding needs more to grow at least could degrade mucus, be 0.1-1% through repeatedly groping to obtain good working concentration with experimental verification, the sample more containing vaginal fluid that the viscosity that can liquefy in 5 minutes to 15 minutes is larger, makes DNA extract more effective.It is large and need the preservation of other cells and the liquifying method of liquefaction that the method is equally applicable to viscosity.

Simultaneously cell-preservation liquid feature includes pH buffer, and preferred buffering range is at pH6.8-pH7.4; PH buffer can be phosphate buffer (PBS), Tris/HCl or PIPES, and concentration range is at 2.5mM-10mM; Cell-preservation liquid contains osmotic pressure and maintains agent, and its component content is 0.6-9% sodium chloride.

Embodiment

Embodiment 1

The working concentration of the present embodiment research three (2-carboxyethyl) phosphine.Use formula 1(containing the formula 1 cell-preservation liquid concentrate of three (2-carboxyethyl) phosphine of 0.5%, 10mM Tris/HCl (pH7.0) solution of 0.9%NaCl.Use formula 2(containing the cell-preservation liquid of three (2-carboxyethyl) phosphine of 5%, the 10mM Tris/HCl solution of 0.9%NaCl), be mixed with formula 2 concentrating cells conserving liquid.All add concentrated cell-preservation liquid according to the ratio of 1 to 4 in use, namely get cell-preservation liquid concentrate described in 0.5ml, add the cervical cell sample that 2ml contains more secretion; Get 30 routine samples, with normal saline dilution to 6ml, sample mean is divided into 3 parts, every this 2ml of increment.Sample is divided into 3 groups, the 1st group of sample, according to the ratio of 1 to 4, namely each sample adds described formula 1 cell-preservation liquid concentrate 0.5ml respectively, is three (2-carboxyethyl) phosphine of 0.1% in sample containing final concentration; 2nd group of sample, according to the ratio of 1 to 4, each sample adds the concentrated also 0.5ml of cell-preservation liquid respectively, is three (2-carboxyethyl) phosphine of 1% in sample containing final concentration; 3rd group of sample is control group, and each sample adds respectively not containing the 10mM Tris/HCl solution 0.5ml of the 0.9%NaCl of reductant.Effect before and after inspection process.Before treatment, because viscosity is comparatively large, the amount being difficult to draw fixed volume extracts detection for follow-up DNA, and sample tube turned upside down, observing liquid in sample can not flow fast, has hysteresis phenomenon; After the concentrating cells conserving liquid adding described formula 1, process 10-15 minute and can well liquefy sample, after being disposed, sample tube is turned upside down, can free flow be observed, use pipettor to draw like a cork simultaneously; 2nd group of sample, after the ratio according to 1 to 4 adds formula 2 concentrating cells conserving liquid, eliminates the time shorten of secretion viscosity to 5-10 minute, uses pipettor to draw like a cork; The function that control group does not then liquefy, the viscosity of sample tube does not change over the course of 15 mins.

Because cell can adapt to certain osmolarity ranges, we test containing the 10 routine samples of final concentration containing three (2-carboxyethyl) phosphine of 0.6%, the cell-preservation liquid of 0.6%NaCl, 2.5mM Tris/HCl, through three (2-carboxyethyl) phosphine process 10-15 minute, the effect reducing sample viscosity can be reached equally, observe the form of cast-off cells under the microscope, can see and contain in the cell-preservation liquid of 0.9%NaCl with final concentration in the cell-preservation liquid containing 0.6%NaCl containing final concentration, cellular morphology in sample all keeps complete, and the two is significantly not different.

Embodiment 2

Get the cervical exfoliated cell swab sample (sample that cell more) of 22 examples through sequence verification, fully suspend in 2ml physiological saline, each sample is respectively got the even packing of 1ml and is put into 2 new centrifuge tubes, one group of cell sample according to 1 to 5 ratio, formula 2 cell-preservation liquid getting 0.25ml adds wherein, other one group of sample adds 0.25ml containing 0.9%NaCl, the buffer solution (pH7.0) of 10mM Tris as a control group, liquefaction process is after 10 minutes, two groups of samples are used Human infectious warts virus parting detecting reagent (gene chips) to extract DNA respectively and detect HPV result (this kit manufactures (model is HPG48) by Genetel Pharmaceuticals (Shenzhen) Co., Ltd.).Each sample duplicate detection 1 time, the judgement that target spot signal value (INS value) is greater than 11 is the HPV positive (table 1), use the cell-preservation liquid process of described formula 2, in 22 routine samples, Absorbable organic halogens detects 8 routine positive sample, and control group uses physiological saline process, stable detection can go out 4 routine positive sample, other 2 routine samples repeat experiment and only have 1 detection positive.Prove to use formula of the present invention, the detection sensitivity of HPV in the lower sample of concentration can be improved.

Table 1. contrasts described cell-preservation liquid and physiological saline process viscosity cast-off cells sample HPV testing result

Note: the numeral target spot signal value (INS value) in bracket, the judgement that INS is greater than 11 is that HPV is positive.

Above content is in conjunction with concrete preferred embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.

Claims

1. a cervix cell preservation solution, is characterized in that, comprises a kind of three (2-carboxyethyl) phosphonium salt hydrochlorate, also maintains the aqueous solution of agent, pH buffer components containing osmotic pressure; The concentration of three (2-carboxyethyl) phosphonium salt hydrochlorate is 0.1%-1%; It is 0.6-0.9% sodium-chloride water solution that described osmotic pressure maintains agent; Described pH buffer solution is Tris/HCl buffer soln, and described pH buffer solution buffering range is between pH 6.8-7.4, and the concentration range of buffer solution component is between 2.5 mM-10 mM.

2. use the method for the cervix cell preservation solution liquefaction cervical cell described in claim 1,

Comprise step:

A) the concentrated cervix cell preservation solution that three (2-carboxyethyl) phosphine content is 0.5% to 5% is prepared;

B) ratio being 1 to 4 according to concentrated cervix cell preservation solution and cervical cell sample solution is got concentrated cervix cell preservation solution and is added in cervical cell sample solution;

C) static process 5 minutes is to 15 clocks.