阅读说明：本技术 一种宫颈细胞保存液及其制备方法和宫颈细胞保存方法 (Cervical cell preservation solution, preparation method thereof and cervical cell preservation method ) 是由 高珂 岳乾 安顺 李振红 杜美 付光宇 吴学炜 苗拥军 于 2019-11-27 设计创作，主要内容包括：本发明涉及细胞保存技术领域,特别涉及一种宫颈细胞保存液及其制备方法和宫颈细胞保存方法。该宫颈细胞保存液按如下组分及配比制备而成：甲醇：50～70g；Tris-HCl溶液：30～50mL；EDTA·2Na：0.5～1.5g；NaCl：0.5～1.5g；proclin950：0.001～0.01mL；Proclin300：0.001～0.01mL。本发明宫颈细胞保存液能够维持宫颈脱落细胞内及感染细胞的病毒核酸完整性,对核酸DNA提供有效保护,防止核酸降解,有利于HPV-DNA检测。(The invention relates to the technical field of cell preservation, in particular to cervical cell preservation solution, a preparation method thereof and a cervical cell preservation method. The cervical cell preservation solution is prepared from the following components in parts by weight: methanol: 50-70 g; Tris-HCl solution: 30-50 mL; EDTA-2 Na: 0.5-1.5 g; NaCl: 0.5-1.5 g; proclin 950: 0.001-0.01 mL; proclin 300: 0.001-0.01 mL. The cervical cell preservation solution can maintain the integrity of virus nucleic acid in cervical exfoliated cells and infected cells, effectively protect nucleic acid DNA, prevent nucleic acid degradation and facilitate HPV-DNA detection.)

1. The cervical cell preservation solution is characterized by being prepared from the following components in parts by weight:

2. the cervical cell preservation solution according to claim 1, which is prepared from the following components in parts by weight:

3. the cervical cell preservation solution according to claim 1, which is prepared from the following components in parts by weight:

4. the cervical cell preservation solution according to claim 1, wherein the concentration of the Tris-HCl solution is 1.0-2.0M.

5. The cervical cell preservation solution according to claim 1, wherein the concentration of the Tris-HCl solution is 1.5M.

6. The cervical cell preservation solution according to claim 1, wherein the Tris-HCl solution has a pH of 8.0 to 9.0.

7. The cervical cell preservation solution according to any one of claims 1 to 6, wherein the Tris-HCl solution has a pH of 8.5.

8. The method for producing the cervical cell preservation solution according to any one of claims 1 to 7, comprising: mixing Tris-HCl solution, EDTA & 2Na, NaCl, 10% TritonX-100, Proclin950 and Proclin300, dissolving, and adding methanol to prepare the cervical cell preservation solution.

9. A method for preserving cervical cells, comprising the step of storing the cervical cells in the cervical cell preserving solution according to any one of claims 1 to 7 at-80 ℃ to 30 ℃.

10. The preservation method according to claim 9, characterized in that the preservation temperature is-20 ℃ to 10 ℃.

Technical Field

The invention relates to the technical field of cell preservation, in particular to cervical cell preservation solution, a preparation method thereof and a cervical cell preservation method.

Background

Cervical cancer is one of the common malignant tumors of the female reproductive tract that pose a risk to female health, and the occurrence of cervical cancer is a long-term process, the reversible phase of premalignant lesions. A plurality of research results prove that the HPV persistent infection is closely related to cervical cancer and precancerous lesion, so that the early discovery and early prevention of HPV infection are the key points for blocking the lesion.

Although the traditional pap smear method has the characteristics of high diagnostic specificity, simplicity, practicability, cheapness and the like, the sensitivity is low, the false negative rate is high, and meanwhile, a highly trained cytopathologist is required to explain the detection result and is influenced by subjective factors of a reader. The advantages of high-sensitivity HPV-DNA/RNA detection directly aiming at the etiology in cervical lesion prevention, screening, diagnosis and follow-up are increasingly shown.

Most of the cell preservation solutions in the current market are focused on fixing cell structures, are beneficial to preservation of cells for cytological detection, but cannot be well adapted to the technical requirements of collection, storage or preparation of cell DNA samples. HPV-DNA/RNA detection puts higher requirements on cervical cell preservation solution, and not only can rapidly fix white blood cells, exfoliated epithelial cells and other cells with detection significance be required, but also mucus in a sample can be diluted, and the influence on subsequent treatment or detection is reduced. The cell preservation solution or the special cervical cell preservation solution in the prior patent publication cannot effectively protect the integrity of nucleic acid in cells. Therefore, there is a need to provide a cervical cell preservation solution that can effectively prevent nucleic acid degradation, thereby facilitating HPV-DNA/RNA detection.

Disclosure of Invention

In view of the above, the invention provides a cervical cell preservation solution, a preparation method thereof and a cervical cell preservation method. The cervical cell preservation solution can effectively prevent nucleic acid degradation and is beneficial to HPV-DNA/RNA detection.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a cervical cell preservation solution which is prepared from the following components in parts by weight:

preferably, the cervical cell preservation solution is prepared from the following components in parts by weight:

preferably, the cervical cell preservation solution is prepared from the following components in parts by weight:

the fixing agent in the components of the cell preservation solution is methanol, so that cells used for subsequent detection, such as leucocytes, exfoliated epithelial cells and the like in a sample can be rapidly fixed and preserved, and meanwhile, the complete preservation of intracellular nucleic acid is facilitated;

the osmotic pressure maintaining agent is NaCl solution;

the metal chelating agent is disodium ethylene diamine tetraacetate, and an anti-coagulation reagent is arranged in the preservation solution and is used for avoiding cell accumulation;

the pH buffer is a Tris-HCl buffer solution, and can maintain the pH environment of the cell preservation solution to be stable;

the non-ionic detergent is Tritonx-100, which is beneficial to reducing the interference caused by mucus in a sample;

the preservative is a mixture of Proclin950 and Proclin300, can effectively inhibit the growth of microorganisms such as bacteria, fungi, yeasts and the like, and is beneficial to prolonging the stability time of the preservation solution.

Preferably, the concentration of the Tris-HCl solution is 1.0-2.0M.

Preferably, the concentration of the Tris-HCl solution is 1.5M.

Preferably, the pH value of the Tris-HCl solution is 8.0-9.0.

Preferably, the pH of the Tris-HCl solution is 8.5.

The invention also provides a preparation method of the cervical cell preservation solution, which comprises the following steps: mixing Tris-HCl solution, EDTA & 2Na, NaCl, 10% TritonX-100, Proclin950 and Proclin300, dissolving, and adding methanol to prepare the cervical cell preservation solution.

The invention also provides a preservation method of cervical cells, which comprises the step of placing the cervical cells in the cervical cell preservation solution and preserving at the temperature of minus 80-30 ℃.

Preferably, the storage temperature is-20 ℃ to 10 ℃.

The invention provides cervical cell preservation solution, a preparation method thereof and a cervical cell preservation method. The cervical cell preservation solution is prepared from the following components in parts by weight: methanol: 50-70 g; Tris-HCl solution: 30-50 mL; EDTA-2 Na: 0.5-1.5 g; NaCl: 0.5-1.5 g; 10% TritonX-100: 0.1-0.3 mL; proclin 950: 0.001-0.01 mL; proclin 300: 0.001-0.01 mL. The invention has the following advantages:

1) the cervical cell preservation solution can maintain the integrity of virus nucleic acid in cervical exfoliated cells and infected cells, effectively protect nucleic acid DNA, prevent nucleic acid degradation and facilitate HPV-DNA detection.

2) The buffer solution, the anticoagulant and other components in the cell preservation solution can effectively disperse the exfoliated cervical cells to avoid agglomeration, and the liquid-based cytology smear observation is facilitated.

3) The cervical cell preservation solution can be used for preserving the cervical exfoliated cell sample for a long time at room temperature and can also be used for preserving the cervical exfoliated cell sample for a long time at room temperature.

4) The components of the cell preservation solution in the invention remove the components which have inhibition in the extraction of nucleic acid DNA, and the nonionic detergent (TritonX-100) is added, so that the viscosity of the sample is effectively reduced, the rinsing degree of the sample is enhanced, and the extraction difficulty of the DNA sample is reduced.

5) When the cell preservation solution is preserved at a low temperature of between 80 ℃ below zero and 20 ℃ below zero, the water in the system can not be frozen, and the crystallization damage can be effectively prevented.

6) The preservation solution of the invention has simple technology, easy preparation and convenient operation.

7) The cell preservation solution has simple components and low cost.

Drawings

FIG. 1 is a line graph of fluorescence-PCR amplification results before and after cell preservation using the preservation solution of comparative example 1, HPV detection;

FIG. 2 is a line graph showing fluorescence-PCR amplification results before and after the preservation of cells using the preservation solution of comparative example 2, HPV detection;

FIG. 3 is a line graph of fluorescence-PCR amplification results before and after cell preservation using the preservation solution of comparative example 3, HPV detection;

FIG. 4 is a line graph of fluorescence-PCR amplification results before and after cell preservation using the preservation solution of comparative example 4, HPV detection;

FIG. 5 is a line graph showing fluorescence-PCR amplification results before and after acceleration of cell preservation using the preservation solutions of example 1 and comparative examples 5 to 8-HPV detection.

Detailed Description

The invention discloses a cervical cell preservation solution, a preparation method thereof and a cervical cell preservation method. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

It will be understood by those skilled in the art that although the weight units of the components in the formulations of the examples are g or mL, they can also be understood as parts by weight or parts by volume, i.e. the ratio between the components is satisfied.

The cervical cell preservation solution, the preparation method thereof and the reagent or instrument used in the cervical cell preservation method can be purchased from the market.

The invention is further illustrated by the following examples: