阅读说明：本技术 一种快速的细胞超薄切片电镜检测逆转录病毒的方法 (Method for rapidly detecting retrovirus by cell ultrathin section electron microscope ) 是由 张宾 孙莉 王蕾 于 2020-05-12 设计创作，主要内容包括：本发明公开了一种快速的细胞超薄切片电镜检测逆转录病毒的方法,步骤包括：1)样品的收集与固定；2)醋酸铀块染；3)脱水、渗透和聚合；4)切片、染色后透射电镜检测。本发明与传统方法相比,极大的提升了样本制备的效率,使传统的5-7天制样周期缩短至1天,并使一种快速检测病毒的超薄切片技术成为了可能。优化后的超薄切片电镜技术检测病毒,相比传统的病毒检测方法,大幅提高了病毒检测的特异性、准确性和检测效率。(The invention discloses a method for rapidly detecting retrovirus by a cell ultrathin section electron microscope, which comprises the following steps: 1) collecting and fixing a sample; 2) uranium acetate lump dyeing; 3) dehydration, infiltration and polymerization; 4) and (5) slicing, dyeing and detecting by using a transmission electron microscope. Compared with the traditional method, the invention greatly improves the efficiency of sample preparation, shortens the traditional sample preparation period of 5-7 days to 1 day, and makes an ultrathin section technology for rapidly detecting viruses possible. Compared with the traditional virus detection method, the optimized ultrathin section electron microscope technology for detecting the virus greatly improves the specificity, the accuracy and the detection efficiency of virus detection.)

1. A method for rapidly detecting retrovirus by a cell ultrathin section electron microscope is characterized by comprising the following steps:

I. collecting samples, washing and then fixing by glutaraldehyde and osmium acid;

II, dyeing uranium acetate blocks;

III, after acetone dehydration, performing gradient permeation on the epoxy resin, and polymerizing the pure resin of the sample;

and IV, ultrathin section, detection by a transmission electron microscope after dyeing.

2. The method for rapid cell ultrathin section electron microscopy detection of the retrovirus of claim 1, wherein in step I, the sample is collected by collecting 1-2 × 10⁷And (3) a cell sample.

3. The method for rapid detection of retrovirus by cell ultrathin section electron microscopy according to claim 1, wherein in step I, the specific cleaning method is as follows: wash 3-5 times with 1 XPBS.

4. The method for rapid detection of retrovirus by cell ultrathin section electron microscopy as claimed in claim 1, wherein in step I, the specific method of double immobilization is: 5% glutaraldehyde fixation for 8-12 hours, followed by 1% osmic acid fixation for 0.5-1 hours.

5. The method for rapid detection of retroviruses by cell ultrathin section electron microscopy as claimed in claim 1, wherein in step II, the uranium acetate block is stained and placed in a refrigerator at 2-8 ℃ for 2-3 hours, so that sample shaking is reduced.

6. The method for rapid detection of retrovirus by cell ultrathin section electron microscopy as claimed in claim 1, wherein in step III, the specific method for acetone dehydration is: dehydrating with 70% acetone, 90% acetone and 100% acetone for 5 min, and shaking on a shaking table during dehydration.

7. The method for rapid detection of retrovirus by cell ultrathin section electron microscopy according to claim 1, wherein in the step III, the specific method of gradient permeation of epoxy resin is as follows: acetone: epoxy =1:1 infiltration 20 minutes later pure resin was infiltrated for 2 hours while shaking on a shaker.

8. The method for rapid cell ultrathin section electron microscopy detection of retroviruses as claimed in claim 1, wherein in step III, the specific method for polymerizing the sample pure resin is as follows: the sample was polymerized in neat resin at 60 ℃ for not less than 10 hours.

9. The method for rapid cell ultrathin section electron microscopy detection of retroviruses as claimed in claim 1, wherein in step IV, the ultrathin section is 70-100nm, and is supported by copper mesh.

10. The method for rapid detection of retrovirus by cell ultrathin section electron microscopy as claimed in claim 1, wherein in step IV, the specific staining method is: dyeing the citric acid lead pieces for 10-15 minutes at normal temperature.

Technical Field

The invention relates to the fields of biology and medical health, in particular to the field of biological product safety detection and rapid detection and judgment of unknown virus morphology, and specifically relates to a rapid method for detecting retrovirus by using a cell ultrathin section electron microscope.

Background

The biological product is prepared by using microorganisms, cells, animal or human tissues, body fluids and the like as raw materials and applying the traditional technology or the modern biotechnology, and is used for preventing, treating and diagnosing human diseases. The biological product for human use includes: bacterial vaccines (including toxoids), viral vaccines, antitoxins and antisera, blood products, cytokines, growth factors, enzymes, in vivo and in vitro diagnostic products, and other bioactive agents such as toxins, antigens, allergens, monoclonal antibodies, antigen-antibody complexes, immunomodulatory and probiotic agents.

Some problems such as safety are encountered during the production and application of biological products. For example, the use of mouse hybridoma in antibody drugs and monoclonal antibodies makes the monoclonal antibody products easily have the possibility of endogenous virus pollution; the polyclonal antibody is mainly prepared by adopting animal serum, and the safety problem of animal serum sources also exists. At present, many gene products relate to the construction of protein expression vectors, and the constructed cell lines are easy to have the hidden danger of virus infection. For example, human lymphoblast cell lines transfected with herpes virus (EBV) are fused with mouse myeloma cells, and the secreted monoclonal antibodies are susceptible to infection by mouse retroviruses. Furthermore, the expression cell line used in the preparation of the recombinant protein itself contains endogenous viruses, for example, the endogenous retrovirus exists in Chinese hamster ovary cells, which also causes the safety hazard of the secreted protein. Since all biological products are likely to be contaminated with microorganisms such as bacteria and viruses, the most important difference between the biological products and conventional drugs in terms of the safety of the drugs is: there is a possibility of endogenous viral contamination of the biological product. Thus, WHO, FDA in the united states, CFDA in china and related european departments have made corresponding regulations and provisions: the biological products with endogenous pollution exist, the production process must have a process for removing/inactivating the endogenous pollution, and the process and the products must be subjected to biological safety detection to eliminate the potential virus pollution of the preparation process and the products of the biological products.

In addition, virus detection is also required in the medical health field. Viral infection can be caused by various viruses, but the clinical manifestations of the infection include aversion to cold, fever, general malaise, weakness, anorexia and other general toxic symptoms and the manifestations of inflammation of invaded tissues and organs. Viral infections of the human body are classified into recessive infections, dominant infections, and lentivirus infections. Infection in most cases is recessive. A few are dominant infections. In the dominant infection, most viral infections are acute infections, the onset of diseases is acute, the course of diseases is short, the viral infections are self-healed within 1-2 weeks, and the majority of the dominant infections are latent infections (such as herpes virus infections) and chronic infections (such as hepatitis B virus infections).

Electron microscopy plays an important role in virology research, and in particular, in the identification of viral particles, morphological research and virus detection, the transmission electron microscopy technique of ultrathin sections is the most direct and effective way to detect the presence of viral particles. Clear and accurate results can be obtained by the ultrathin section electron microscope technology no matter from the aspects of virus morphology and virology. However, the traditional ultrathin section sample preparation method is complex, the period is long, a large amount of manpower and time cost are required to be invested, and the quick detection of the ultrathin section electron microscope technology cannot be realized, so that the ultrathin section electron microscope technology cannot be better applied to the pharmaceutical field with severe competition. Therefore, it is the fundamental idea to solve the current problems to optimize and innovate according to the principles of the conventional method.

Disclosure of Invention

The invention aims to provide a method for rapidly detecting retrovirus by using a cell ultrathin section electron microscope. Compared with the traditional method, the method greatly improves the efficiency of sample preparation, shortens the traditional sample preparation period of 5-7 days to 1 day, and makes an ultrathin section technology for rapidly detecting viruses possible. Compared with the traditional virus detection method, the optimized ultrathin section electron microscope technology for detecting the virus greatly improves the specificity, the accuracy and the detection efficiency of virus detection.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for rapidly detecting retrovirus by a cell ultrathin section electron microscope comprises the following steps:

I. collecting samples, washing and then fixing by glutaraldehyde and osmium acid;

II, uranium acetate lump dyeing;

III, after acetone dehydration, performing gradient permeation on the epoxy resin, and polymerizing the pure resin of the sample;

and IV, ultrathin section, detection by a transmission electron microscope after dyeing.

As a preferred technical solution of the present invention, in step I, the sample collection is: collecting 1-2X 107 cell samples;

as a preferred technical solution of the present invention, in step I, the specific cleaning method is: washing with 1 × PBS for 3-5 times;

as a preferred technical solution of the present invention, in step I, the double immobilization specific method is: fixing with 5% glutaraldehyde (volume percentage) for 8-12 hr, and fixing with 1% osmic acid (mass volume percentage) for 0.5-1 hr.

As a preferable technical scheme of the method, in the step II, the uranium acetate block is dyed and placed in a refrigerator at the temperature of 2-8 ℃ for 2-3 hours, so that the shaking of a sample is reduced;

as a preferred technical solution of the present invention, in step III, the acetone dehydration specifically comprises: dehydrating with 70% acetone, 90% acetone and 100% acetone for 5 min, and shaking on a shaking table during dehydration.

As a preferred technical solution of the present invention, in step III, the specific method for gradient permeation of epoxy resin is: acetone: epoxy =1:1 infiltration 20 minutes later pure resin was infiltrated for 2 hours while shaking on a shaker.

As a preferred technical solution of the present invention, in step III, the pure resin polymerization method specifically comprises: the sample was polymerized in neat resin at 60 ℃ for not less than 10 hours.

As a preferable technical scheme of the invention, in the step IV, the ultrathin section is 70-100nm and is supported by a copper net.

As a preferred technical solution of the present invention, in step IV, the dyeing method specifically comprises: and dyeing the lead citrate slice for 10-15 minutes at normal temperature.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides a more visual, visual and rapid detection method for detecting the virus in the cell bank;

2. the sample processing mode used by the invention greatly shortens the operation time of sample preparation; the sample preparation of the traditional method needs 7 days, the sample preparation of the optimized method only needs 1 day, the time cost and the labor cost are greatly reduced, the flexibility of project arrangement is improved, the output of unit personnel is increased, and the influence of interference factors is reduced;

3. the invention uses uranyl acetate block for dyeing, increases slice contrast and reduces dye liquor pollution;

4. compared with the traditional method, the sample resin block is hard, so that slicing is convenient;

5. the staining before slicing can eliminate pollution;

6. the invention uses the bare network to directly bear, compared with the traditional method, the invention removes the influence of the bearing film and reduces the interference factor;

7. the invention greatly improves the cell contrast, provides clearer display for the cell sub-microstructure, increases the richness of cell slices, enhances the definition of organelles and other particles, eliminates the influence of other interfering substances on observing viruses and increases the discrimination of each structure;

8. the observation of other possible particle inclusions by the present invention provides a more efficient detection route.

Drawings

FIG. 1 is a schematic diagram showing a comparative sample prepared by the conventional method and the method of the present invention using SP 2/0-H-A3-cells in example 1; in FIG. 1, A, B is a photograph of a specimen prepared by a conventional method using SP 2/0-H-A3-cells; a. b is a sample picture of the ultrathin section prepared by using SP 2/0-H-A3-cells by the optimization method; white arrows are indicated as golgi, black arrows as mitochondria, and white bold arrows as endoplasmic reticulum.

FIG. 2 is a schematic comparison of endogenous viral particles in electron micrographs prepared by conventional and inventive methods using SP 2/0-H-A3-cells as described in example 1; in FIG. 2, C is an electron microscope picture prepared by using SP 2/0-H-A3-cells in a conventional method, and C is an electron microscope picture prepared by using SP 2/0-H-A3-cells in an optimized method of the present invention; the white arrows indicate endogenous viral particles.

FIG. 3 is a comparative schematic of secreted viral particles prepared by conventional and inventive methods using SP 2/0-H-A3-cells as shown in example 1 under electron microscopy; in FIG. 3, D is an electron microscope picture prepared by using SP 2/0-H-A3-cells in a conventional method, and D is an electron microscope picture prepared by using SP 2/0-H-A3-cells in an optimized method of the present invention; the white arrows indicate the secretory viral particles.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.