CN114459851A - A method for detection of virus in UPB using ultrathin section electron microscopy - Google Patents

Abstract

The invention discloses a method for detecting virus in UPB by using ultrathin section electron microscope technology, comprising the following steps: 1) centrifugation and precipitation collection of UPB samples; 2) double fixation of glutaraldehyde and osmic acid; 3) uranyl acetate block staining 4) Acetone dehydration and gradient resin infiltration; 5) Sample polymerization treatment; 6) Sectioning and staining; 7) Transmission electron microscope detection; The present invention expands the ultra-thin section electron microscope according to the characteristics of UPB samples and the principle of ultra-thin section transmission electron microscope technology The ability of the technology to detect viruses makes it possible to detect viruses in UPB using ultrathin section electron microscopy. Compared with the traditional method of detecting viruses in cells, ultra-thin section electron microscopy technology can be widely used in the detection of biological products or in the field of medical and health, as well as the biological safety detection of biological products.

Description

A method for detection of virus in UPB using ultrathin section electron microscopy

technical field

The invention relates to a virus detection method, in particular to a virus detection method using ultra-thin section electron microscope technology.

Background technique

Biological products are mainly made from microorganisms, cells, animal or human tissue and body fluids, etc., using traditional or modern biotechnology, and are used for the prevention, treatment or diagnosis of human diseases. Biological products for human use include: bacterial vaccines (including toxoids), viral vaccines, antitoxins and antiserum, blood products, cytokines, growth factors, enzymes, in vivo and in vitro diagnostic products, and other biologically active preparations such as toxins , antigens, allergens, monoclonal antibodies, antigen-antibody complexes, immune regulation and probiotics, etc. Due to the characteristics that most biomedical products are manufactured by cells, bacteria and other organisms, there may be residual contamination of some microorganisms, viruses or proteins in the production process, which may pose certain challenges to the safety of the products. . In order to ensure the consistency and safety of the product, it is generally necessary to establish a cell bank and test the cells in the bank. The detection items generally include identification, microbial contamination detection, detection of internal and external viral factors, retrovirus detection and specific virus detection. detection, etc. Although the banked cells were tested for exogenous and exogenous viral factors, some viruses were not detected in the cell bank or accidental virus contamination was introduced in the production process, so the virus in the cell harvest solution (UPB) was detected and then Control appears to be very important.

In addition, the medical field also needs virus testing. Viral infection can be caused by a variety of viruses, but the clinical manifestations include chills, fever, general fatigue, loss of appetite and other symptoms of systemic poisoning and inflammation of the affected tissues and organs. Human viral infections are divided into recessive infections, dominant infections, and lentiviral infections. In most cases the infection is latent infection. A few are overt infections. Among the dominant infections, most viral infections are acute infections, with rapid onset and short course of disease, and most of them heal within 1 to 2 weeks. virus infection, etc.). Laboratory tests are required to make the relevant diagnosis. The application of electron microscopy, etc., can directly check the virus particles and virus antigens in the specimen, which is often used for rapid and early diagnosis.

Electron microscopy plays an important role in virological research, especially in the identification of virus particles, morphological studies and virus detection. Ultra-thin section transmission electron microscopy is the most direct and effective way to detect virus particles. exist. Both in terms of virus morphology and virion, clear and accurate results can be obtained by ultrathin section electron microscopy. However, the traditional ultra-thin section electron microscopy technology can only be detected in cell samples, which makes the ultra-thin section electron microscopy technology unable to be better applied in the highly competitive pharmaceutical field. The invention can expand the application range of the ultra-thin section electron microscope technology to different sample types, and enrich the detection method of virus particles in UPB.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above-mentioned deficiencies of the prior art, and to provide a method for detecting viruses in UPB samples using ultra-thin section electron microscopy, so as to achieve accurate and highly specific detection of viruses in UPB samples.

To achieve the above object, the present invention adopts the following technical solutions:

A method for detecting viruses in UPB using ultrathin section electron microscopy, comprising the following steps:

1) Centrifugation and precipitation collection of UPB samples;

2) Double fixation of glutaraldehyde and osmic acid;

3) Uranium acetate block staining;

4) Acetone dehydration and gradient resin infiltration;

5) Sample polymerization treatment;

6) Sectioning and staining;

7) Transmission electron microscope detection.

Further, the method includes the following specific steps:

Step 1: Take the UPB sample, centrifuge, discard the supernatant to collect the precipitate;

Step 2: The precipitation was fixed with glutaraldehyde overnight, then the glutaraldehyde fixative was removed, agar was added for pre-embedding, and PBS was washed; then post-fixed with osmic acid;

Step 3: After washing with water, stain with uranyl acetate, and then wash with water;

Step 4: Dehydration and overtreatment with 70%, 95%, 100% acetone, respectively;

Step 5: Infiltrate with acetone: epoxy resin = 1:1 (volume ratio), then infiltrate with pure resin;

Step 6: After embedding the sample in pure resin, polymerize at 60°C overnight;

Step 7: Ultra-thin section 70-100nm, copper mesh to collect and carry the section;

Step 8: 3% (mass concentration) lead citrate staining;

Step 9: Dry and collect the slices, observe 10 copper mesh holes in a transmission electron microscope, count the number of virus particles in them, and then calculate the virus concentration in the original UPB.

Further, the sample centrifugation conditions in step 1 are: 100000g, 2h.

Further, in step 2, the volume concentration of glutaraldehyde is 5%, the mass concentration of agar is 3%, and the mass concentration of osmic acid is 1%.

Further, the volume concentration of uranyl acetate in step 3 is 3%.

Further, in step 5, the pure resin is infiltrated for 2h.

Further, in step 6, the polymerization is carried out at 60° C. for at least 10 h.

The advantages of the present invention are:

1. The present invention can apply the ultrathin section electron microscope technology to the detection of virus in UPB, enrich the technology of UPB virus detection, and can clearly observe the morphological structure of virus particles.

2. The present invention provides an intuitive and visual detection method for virus detection in the cell harvest liquid (UPB); and provides an effective detection method for the observation of particles that may exist in other UPBs.

3. According to the characteristics of UPB samples, the present invention expands the ability of ultra-thin section electron microscopy to detect viruses, and makes it possible to use ultra-thin section electron microscopy to detect viruses in UPB. Compared with the traditional method of detecting viruses in cells, the ultra-thin section electron microscopy technique can be widely used in the detection of biological products or in the medical and health fields, as well as the biological safety detection of biological products.

Description of drawings

Figure 1 is an example of virus particles in UPB under an electron microscope at 10,000 times magnification;

Figure 2 is an example of viral particles in UPB observed under an electron microscope at 30,000x magnification.

Detailed ways

Embodiment 1: The method for detecting virus in this embodiment specifically includes the following steps:

Step 1: Take 4mL UPB sample (CHO cell culture supernatant), centrifuge (ultra-high speed 100000g, centrifugation for 2h), discard the supernatant to collect the precipitate;

Step 2: The precipitate was fixed with 5% glutaraldehyde overnight, then the glutaraldehyde fixative was removed, 50 μL of 3% agar was added for pre-embedding, and 1×PBS was washed 3 times for 10 minutes; then 1% osmic acid was post-fixed for 0.5 -1 hour;

Step 3: Wash 3 times with MilliQ for 10 minutes, stain with 3% uranyl acetate for 1 hour, and wash with MilliQ 3 times for 10 minutes;

Step 4: 70%, 95%, 100% acetone dehydration and overtreatment for 5 minutes each;

Step 5: Acetone:Epoxy = 1:1 infiltration for 15 minutes, then pure resin infiltration for 2 hours;

Step 6: After embedding the sample in pure resin, polymerize at 60°C overnight;

Step 7: Ultra-thin section 70-100nm, copper mesh to collect and carry the section;

Step 8: 3% lead citrate staining for 10-15 minutes;

Step 9: Dry and collect the slices, observe 10 copper mesh holes in a transmission electron microscope, count the number of virus particles in them, and then calculate the virus concentration in the original UPB.

Embodiment 2: The method for detecting virus in this embodiment specifically includes the following steps:

Step 1: Take 4mL UPB sample (Vero cell culture supernatant), centrifuge (ultra-high speed 100000g, centrifugation for 2h), discard the supernatant to collect the precipitate;

Step 2: The precipitate was fixed with 5% glutaraldehyde overnight, then the glutaraldehyde fixative was removed, 50 μL of 3% agar was added for pre-embedding, and 1×PBS was washed 3 times for 10 minutes; then 1% osmic acid was post-fixed for 0.5 -1 hour;

Step 3: Wash 3 times with MilliQ for 10 minutes, stain with 3% uranyl acetate for 1 hour, and wash with MilliQ 3 times for 10 minutes;

Step 4: 70%, 95%, 100% acetone dehydration and overtreatment for 5 minutes each;

Step 5: Acetone:Epoxy = 1:1 infiltration for 15 minutes, then pure resin infiltration for 2 hours;

Step 6: After embedding the sample in pure resin, polymerize at 60°C overnight;

Step 7: Ultra-thin section 70-100nm, copper mesh to collect and carry the section;

Step 8: 3% lead citrate staining for 10-15 minutes;

Step 9: Dry and collect the slices, observe 10 copper mesh holes in a transmission electron microscope, count the number of virus particles in them, and then calculate the virus concentration in the original UPB.

Embodiment 3: The method for detecting virus in this embodiment specifically includes the following steps:

Step 1: Take 4 mL of UPB sample with virus added, centrifuge (ultra-high speed 100000g, centrifugation for 2h), discard the supernatant to collect the precipitate;

Step 2: The precipitate was fixed with 5% glutaraldehyde overnight, then the glutaraldehyde fixative was removed, 50 μL of 3% agar was added for pre-embedding, and 1×PBS was washed 3 times for 10 minutes; then 1% osmic acid was post-fixed for 0.5 -1 hour;

Step 3: Wash 3 times with MilliQ for 10 minutes, stain with 3% uranyl acetate for 1 hour, and wash with MilliQ 3 times for 10 minutes;

Step 4: 70%, 95%, 100% acetone dehydration and overtreatment for 5 minutes each;

Step 5: Acetone:Epoxy = 1:1 infiltration for 15 minutes, then pure resin infiltration for 2 hours;

Step 6: After embedding the sample in pure resin, polymerize at 60°C overnight;

Step 7: Ultra-thin section 70-100nm, copper mesh to collect and carry the section;

Step 8: 3% lead citrate staining for 10-15 minutes;

Step 9: Bake dry and collect the slices, observe 10 copper mesh holes in a transmission electron microscope, count the number of virus particles and record it as x, and then calculate the virus concentration c in the original UPB. The calculation process is as follows:

c=x÷5780μm ³ ×5×10 ¹⁰ μm ³ ÷V

Among them, the total volume of 10 copper mesh holes is: 5780 μm ³ ; the total volume of the precipitation plus agar-embedded block is 5×10 ¹⁰ μm ³ ; the volume of the original UPB is V.

The effect of electron microscopy is shown in Figure 1 and Figure 2. It can be seen from the figures that the structure and morphology of virus particles in UPB observed by ultra-thin section electron microscopy are clear, the contrast is obvious, and it is easy to distinguish from non-viral substances, indicating that the use of ultra-thin section electron microscopy technology can be used. Viral particles were successfully identified and detected in UPB.

It can be seen from the above description that this method is a convenient, clear and accurate detection method for detecting viruses in UPB, which is intuitive, clear, and simple to calculate.

Claims (7)

1. a method for detecting virus in UPB using ultrathin section electron microscope technology, is characterized in that, comprises the following steps: 1) Centrifugation and precipitation collection of UPB samples; 2) double fixation with glutaraldehyde and osmic acid; 3) uranyl acetate staining; 4) acetone dehydration and gradient resin infiltration; 5) sample polymerization treatment; 6) sectioning and staining; 7) Transmission electron microscope detection. 2. a kind of method that uses ultrathin section electron microscope technology to detect virus in UPB as claimed in claim 1, is characterized in that, comprises following concrete steps: 1). Take the UPB sample, centrifuge, discard the supernatant to collect the precipitate; 2). The precipitation was fixed with glutaraldehyde overnight, then the glutaraldehyde fixative was removed, agar was added for pre-embedding, and PBS was washed; then post-fixed with osmic acid; 3). After washing with water, stain with uranyl acetate, and then wash with water; 4). Use 70%, 95%, 100% acetone for dehydration and overtreatment respectively; 5). Use the volume ratio of acetone: epoxy resin = 1:1 to infiltrate, and then infiltrate with pure resin; 6). After the sample is embedded in pure resin, polymerize at 60℃ overnight; 7).Ultra-thin section 70-100nm, copper mesh to collect and carry the section; 8). Use the mass concentration of 3% lead citrate for staining; 9). Bake dry and collect the sections, observe 10 copper mesh holes in a transmission electron microscope, count the number of virus particles, and then calculate the virus concentration in the original UPB. 3. a kind of method using ultrathin section electron microscope technology to detect virus in UPB as claimed in claim 1 or 2, is characterized in that, in 1), the sample centrifugation condition is: 100000g, 2h. 4. a kind of method that uses ultrathin section electron microscope technique to detect virus in UPB as claimed in claim 1 or 2, it is characterized in that, 2) in glutaraldehyde volume concentration is 5%, agar mass concentration is 3%, osmium The acid mass concentration is 1%. 5. a kind of method using ultrathin section electron microscope technology to detect virus in UPB as claimed in claim 1 or 2, is characterized in that, the volume concentration of uranyl acetate in 3) is 3%. 6. a kind of method using ultra-thin section electron microscope technology to detect virus in UPB as claimed in claim 1 or 2, is characterized in that, in 5), pure resin penetrates 2h. 7. A method for detecting virus in UPB using ultra-thin section electron microscope technology as claimed in claim 1 or 2, characterized in that, in 6), polymerization is carried out at 60° C. for at least 10 h.

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