CN115125331A - EB virus detection method based on in-situ hybridization method - Google Patents
EB virus detection method based on in-situ hybridization method Download PDFInfo
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Abstract
The invention discloses an EB virus detection method based on an in-situ hybridization method, which adopts digoxin labeled probe designed by a special EBER sequence to be specifically complementary and hybridized with an EBER target sequence in a tissue or a cell to qualitatively analyze EB virus in the cell or tissue sample, and comprises the steps of sample dewaxing treatment, enzyme treatment, dripping EBER probe and matched secondary antibody, DAB color development, counterstaining, dehydration transparency, mounting and the like. According to the detection method provided by the invention, the mouse anti-digoxin antibody, the goat anti-mouse antibody and the enzyme-labeled anti-goat IgG polymer are selected as the secondary antibody system used with the EBER probe, so that the specificity of hybridization and combination of the probe and nucleic acid in cells or tissue slices is good, a hybridization signal can be expanded, and the detection accuracy and sensitivity are improved.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to an EB virus detection method based on an in-situ hybridization method.
Background
The EB virus (Epstein-Barr virus, EBV) belongs to herpes virus, has a genome length of 172kb, is related to various tumors and non-tumor diseases, and is commonly used for treating nasopharyngeal carcinoma, gastric cancer, nasal NK/T cell lymphoma, Hodgkin lymphoma and the like. According to the classification criteria of the international cancer research institute for oncogenic factors, epstein barr virus is listed in the first group of oncogenic factors. The respiratory tract is considered to be the largest site of EBV latency by current research, is mainly transmitted by human saliva and has human-like lymphocyte characteristics. The epithelial cells of the pharyngeal tissues of the human body are infected firstly, and then are spread to the B lymphocytes of the peripheral blood to be in a latent infection state. Most people in the world become infected with EBV in infancy and carry it for life.
EBER is a small RNA encoded by epstein-barr virus, an expression product of epstein-barr virus, is present in high copies in infected nuclei, and is transcribed continuously during any period of epstein-barr virus infection, including the latent and viral replication stages. At present, a plurality of methods are used for detecting EBV, such as serum antigen-antibody reaction, immunohistochemistry, Polymerase Chain Reaction (PCR) and the like, but the EREB in situ hybridization detection technology has accurate positioning, high sensitivity and strong specificity, and becomes a recognized EB virus standard detection method. Positive detection of EBER in situ hybridization has become the gold standard for determining EBV infection of organ tissue specimens. In addition, the EREB in situ hybridization technology has a positioning function when detecting EBV, and can determine the relationship between virus and tissues and cells. The clinical significance of detecting EB virus in-situ infection is as follows: (1) the kit is helpful for diagnosis and differential diagnosis of EB virus related diseases: such as the identification of nasopharyngeal carcinoma from other tumors of the nasopharynx; EBER is positive in nearly 100% of tumor cell nuclei of nasopharyngeal carcinoma, extranodal NK/T cell lymphoma (nasal type) is positive in 100% theoretically, most diffuse large B cell lymphomas related to chronic inflammation are positive, the positive rate of plasmablast lymphoma is 60-75%, and Hodgkin lymphoma is positive; (2) understanding the etiology or factors related to diseases makes understanding of the diseases more profound; (3) understanding prognosis and guiding treatment: when the pathologist reports the presence of epstein barr virus infection in the lesion, the clinician may use some known antiviral therapies such as interferon, ganciclovir, epstein barr virus specific cytotoxic T cells, and chinese traditional medicines.
In Situ Hybridization (In Situ Hybridization) refers to the process of hybridizing specifically labeled nucleic acids of known sequence as probes to nucleic acids In cells or tissue sections to precisely quantify the specific nucleic acid sequence, and may be performed on cell or tissue specimens. At present, the detection quality of the in situ hybridization method is often determined by the following factors: (1) selection of probe, primary antibody and secondary antibody systems: the selection of the probe, the primary antibody and the secondary antibody system should select the probe, the primary antibody and the secondary antibody with strong specificity, high sensitivity and good binding capacity as much as possible, so as to enlarge hybridization signals; (2) quality of tissue fixation dehydration: the tissue fixation is not timely or insufficient, so that the cell nucleic acid is easy to break and the protein is dissolved, so that the tissue cell structure after the gastric enzyme digestion is fuzzy, and the hybridization detection result cannot be interpreted or is negative; the tissue dehydration is poor, so that strong non-specific background staining can be caused, and the interpretation of a detection result is influenced; (3) gastric enzyme digestion quality: the result of gastric enzyme digestion will affect the degree of hybridization binding between the probe and the target nucleic acid, resulting in reduced positive or false negative results; (4) temperature and time of denaturation and hybridization: in the hybridization denaturation process, particularly, the DNA denaturation hybridization temperature must reach a preset temperature, the denaturation temperature is too low, so that a DNA double strand cannot be opened, a probe cannot be combined with target DNA, and a false negative detection result occurs; (5) standardization of operation flow: non-specific background staining in tissues can be caused by dry films, unused hybridization washing solution, insufficient PBS washing, overlong DAB color development time and other factors in the detection process. The specificity of the probe and the selection of the primary antibody and the secondary antibody are particularly critical in the detection process of the in situ hybridization method.
Therefore, there is a need in the art to develop a detection method with high sensitivity, good specificity and large hybridization signal, which can improve detection accuracy, and can also be operated conveniently and improve detection efficiency.
Disclosure of Invention
In order to solve the technical problems, the invention provides an EB virus detection method based on an in-situ hybridization method, which adopts EBER probe to be specifically complementary and hybridized with an EBER target sequence in a sample to carry out qualitative analysis on the EB virus in a cell or tissue sample, and comprises the following steps:
1) preparing an EBER detection kit reagent, wherein the EBER detection kit reagent comprises an EBER probe, a gastric enzyme working solution, a mouse anti-digoxin antibody, a goat anti-mouse antibody, an enzyme-labeled anti-goat IgG polymer, a DAB chromogen concentrated solution and a DAB substrate buffer solution;
2) sample preparation: taking a cell or tissue sample to be detected, fixing, dehydrating and embedding paraffin to prepare a wax block, wherein the thickness of the sample is 3-5 mu m, adhering the sample on a polylysine glass slide, drying in the air, and baking the sheet at 60 ℃ for 1-2 hours;
3) enzyme treatment: dripping 1-3 drops of a gastric enzyme working solution according to the size of the sample, incubating for 10 minutes at 37 ℃, washing with purified water for 2 times, and finally drying the sample by using air;
4) and (3) dropwise adding a probe: according to the size of the sample, dripping an EBER probe, adding a silicified cover glass, sealing edges by using rubber cement, and carrying out hybridization incubation at 37 ℃ for 2-4 hours or overnight;
5) removing rubber cement, immersing the sample in PBS buffer solution for 10 minutes, and washing with PBS buffer solution for 3 times, each time for 2 minutes;
6) dropping a mouse anti-digoxin antibody: adding 1-3 drops of mouse anti-digoxin antibody to the sample to completely cover the sample, incubating for 30 minutes at 37 ℃, and washing 3 times with PBST for 5 minutes each;
7) adding a goat anti-mouse antibody dropwise: adding 1-3 drops of goat anti-mouse antibody to the sample to completely cover the sample, incubating for 10 minutes at room temperature, washing 3 times with PBST, each time for 5 minutes;
8) dropwise adding enzyme-labeled anti-goat IgG polymer: adding 1-3 drops of enzyme-labeled goat IgG-resistant polymer into the sample until the sample is completely covered, incubating the sample at room temperature for 30 minutes, and washing the sample with PBST for 3 times, each time for 5 minutes;
9) DAB color development: preparing DAB working solution, adding the DAB working solution into the sample until the sample is completely covered for dyeing, and washing the sample by using purified water after dyeing is finished;
10) counterdyeing: performing counterstaining by using hematoxylin, and washing by using PBS or tap water to return blue after counterstaining;
11) and (3) dehydrating and transparency: sequentially soaking the sample in 70% -100% alcohol, soaking in pure xylene for transparency treatment for 2 times, 3 minutes each time, and sealing the sample after the end.
Specifically, in the step 1), the EBER probe, the pepsin working solution, the mouse anti-digoxin antibody, the goat anti-mouse antibody and the enzyme-labeled anti-goat IgG polymer are all ready-to-use reagents.
Specifically, in the step 1), the DAB is 3, 3' -diaminobenzidine.
Specifically, in step 2), the dewaxing treatment refers to soaking and dewaxing 3-5 μm paraffin sections in xylene for 2 times and 10 minutes each time, then soaking in absolute ethyl alcohol for 2 times and 3 minutes each time, and finally air drying for 5-10 minutes.
In particular, in steps 3), 6), 7) and 8), the incubation refers to incubation in an in situ hybridization apparatus or a wet cell.
Specifically, in the step 8), the enzyme-labeled anti-sheep IgG polymer is a polymer HRP-conjugated secondary antibody.
Specifically, in the step 9), the DAB working solution is prepared by mixing the DAB chromogen concentrated solution and a DAB substrate buffer solution in proportion, wherein one drop of DAB chromogen concentrated solution is added into every 800 mu L of DAB substrate buffer solution.
Specifically, in the step 10), the blue return refers to that the cell nucleus in the observation sample is blue.
Specifically, in step 11), the concentration gradient is 70%, 85%, 95%, and 100%.
Specifically, in step 11), the mounting refers to mounting the sample by using neutral gum.
Specifically, in step 4), the hybridization incubation time is 2-4 hours or overnight, which means that the hybridization incubation time is 2-4 hours or overnight in an in situ hybridization instrument or a wet box.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the detection method provided by the invention, a mouse-digoxin-resistant antibody, a goat-anti-mouse antibody and an enzyme-labeled goat-anti-IgG polymer are selected as a secondary antibody system used for the matched EBER probe, wherein the enzyme-labeled goat-anti-IgG polymer is preferably a polymer HRP-coupled secondary antibody, so that the specificity of hybridization and combination of the probe and nucleic acid in cells or tissue slices is good, a hybridization signal can be enlarged, and the accuracy and the sensitivity of detection are improved.
2. The detection method provided by the invention optimizes and adjusts the deformation and hybridization temperature and time, simplifies the operation, and simultaneously can ensure that the EBER probe and the nucleic acid in the sample are well hybridized and combined, thereby enhancing the dyeing effect and being beneficial to improving the detection accuracy.
Drawings
FIG. 1 is a graph showing the staining results of EB virus detection of gastric cancer tissues in the first embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example one test method provided by the present invention was used to test 1 sample of gastric cancer tissue infected with EB Virus
In one embodiment, the detection method provided by the invention is used for performing qualitative analysis on the EB virus in the cells or the tissue sample by using a digoxin labeled probe specifically designed by using the specific sequence of the EBER to be complementary and hybridized with the EBER target sequence in the tissues or the cells, and comprises the following steps:
1) preparing an in-situ hybridization reagent, wherein the in-situ hybridization reagent comprises an EBER probe, a pepsin working solution, a mouse anti-digoxin antibody, a goat anti-mouse antibody, an enzyme-labeled anti-goat IgG polymer, a DAB chromogen concentrated solution and a DAB substrate buffer solution, DAB is 3, 3' -diaminobenzidine, and the EBER probe, the pepsin working solution, the mouse anti-digoxin antibody, the goat anti-mouse antibody and the enzyme-labeled anti-goat IgG polymer are ready-to-use reagents which are used directly for detection without dilution or adjustment of use concentration;
2) sample preparation: placing the formalin-fixed paraffin embedded section in a thermostat at 60 ℃ for baking for 1-2 hours, storing at room temperature, then placing the paraffin section with the size of 3-5 micrometers in dimethylbenzene for soaking and dewaxing for 2 times, each time for 10 minutes, then placing the paraffin section in absolute ethyl alcohol for soaking for 2 times, each time for 3 minutes, and finally drying for 5-10 minutes in air to obtain a tissue sample;
3) enzyme treatment: dripping 1-3 drops of gastric enzyme working solution into the tissue sample, incubating in an in-situ hybridization instrument or a wet box for 10 minutes at 37 ℃, washing with purified water for 2 times, and finally drying the gastric cancer tissue sample by using air;
4) and (3) dropwise adding a probe: dripping a proper amount of EBER probe according to the size of the tissue, adding a silicified cover glass, sealing the edges by using rubber cement, and hybridizing and incubating for 2-4 hours or overnight (in an in-situ hybridization instrument or a wet box) at 37 ℃;
5) removing rubber cement, immersing the gastric cancer tissue sample in PBS buffer solution for 10 minutes, washing with the PBS buffer solution for 3 times, each time for 2 minutes, lifting the glass slide for several times during the washing period to promote the cover glass to slide off, and drawing a circle with a water-blocking pen;
6) dropping a mouse anti-digoxin antibody: adding 1-3 drops of mouse anti-digoxin antibody to the tissue sample until the tissue sample is completely covered, incubating in an in situ hybridization instrument or wet box for 30 minutes at 37 ℃, and washing 3 times with PBST for 5 minutes each;
7) dropping a goat anti-mouse antibody: adding 1-3 drops of goat anti-mouse antibody to the tissue sample until the tissue sample is completely covered, incubating in an in situ hybridization instrument or a wet box for 10 minutes at room temperature, and washing with PBST for 3 times, each for 5 minutes;
8) dropwise adding enzyme-labeled anti-goat IgG polymer: the enzyme-labeled goat IgG resisting polymer is a polymer HRP-coupled secondary antibody, 1-3 drops of the polymer HRP-coupled secondary antibody are added into the tissue sample until the tissue sample is completely covered, the mixture is incubated in an in-situ hybridization instrument or a wet box for 30 minutes at room temperature, and PBST is used for washing for 3 times, and each time lasts for 5 minutes;
9) DAB color development: preparing DAB working solution, adding one drop of DAB chromogen concentrated solution into every 800 mu L of DAB substrate buffer solution, preparing a proper amount of DAB working solution according to the proportion, adding the DAB working solution into the tissue sample until the tissue sample is completely covered for dyeing, and washing the tissue sample by using purified water after dyeing is finished;
10) counterdyeing: performing counterstaining by using hematoxylin, and washing by using PBS or tap water to return blue after counterstaining;
11) and (3) dehydrating and transparency: sequentially soaking the tissue sample in 70%, 85%, 95% and 100% ethanol, soaking in pure xylene for 2 times (3 min each time), and sealing with neutral gum.
As shown in figure 1, tumor cells in the EB virus infected gastric cancer tissue section of the example show nuclear positive expression, which shows that EB virus in the human tissue sample is infected in situ, and the detection method provided by the invention has accurate positioning.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (11)
1. An EB virus detection method based on an in situ hybridization method is characterized in that an EBER probe is adopted to be specifically complementary and hybridized with an EBER target sequence in a sample to carry out qualitative analysis on the EB virus in a cell or tissue sample, and comprises the following steps:
1) preparing an EBER detection kit reagent, wherein the EBER detection kit reagent comprises an EBER probe, a pepsin working solution, a mouse anti-digoxin antibody, a goat anti-mouse antibody, an enzyme-labeled anti-goat IgG polymer, a DAB chromogen concentrated solution and a DAB substrate buffer solution;
2) sample preparation: taking a cell or tissue sample to be detected, fixing, dehydrating and embedding paraffin to prepare a wax block, wherein the thickness of the sample is 3-5 mu m, adhering the sample on a polylysine glass slide, drying in the air, and baking the sheet at 60 ℃ for 1-2 hours;
3) enzyme treatment: dripping 1-3 drops of a gastric enzyme working solution according to the size of the sample, incubating for 10 minutes at 37 ℃, washing with purified water for 2 times, and finally drying the sample by using air;
4) and (3) dropwise adding a probe: according to the size of the sample, dripping an EBER probe, adding a silicified cover glass, sealing edges by using rubber cement, and carrying out hybridization incubation at 37 ℃ for 2-4 hours or overnight;
5) removing rubber cement, immersing the sample in PBS buffer solution for 10 minutes, and washing for 3 times with PBS buffer solution for 2 minutes each time;
6) dropping a mouse anti-digoxin antibody: adding 1-3 drops of mouse anti-digoxin antibody to the sample to completely cover the sample, incubating for 30 minutes at 37 ℃, and washing 3 times with PBST for 5 minutes each;
7) dropping a goat anti-mouse antibody: adding 1-3 drops of goat anti-mouse antibody to the sample to completely cover the sample, incubating for 10 minutes at room temperature, washing 3 times with PBST, each time for 5 minutes;
8) dropwise adding enzyme-labeled anti-sheep IgG polymer: adding 1-3 drops of enzyme-labeled goat IgG-resistant polymer into the sample until the sample is completely covered, incubating the sample at room temperature for 30 minutes, and washing the sample with PBST for 3 times, each time for 5 minutes;
9) DAB color development: preparing DAB working solution, adding the DAB working solution into the sample until the sample is completely covered for dyeing, and washing the sample by using purified water after dyeing is finished;
10) counterdyeing: performing counterstaining by using hematoxylin, and washing by using PBS or tap water to return blue after counterstaining;
11) and (3) dehydrating and transparency: sequentially soaking the sample in 70% -100% alcohol, soaking in pure xylene for transparency treatment for 2 times, 3 minutes each time, and sealing the sample after the end.
2. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the step 1), the EBER probe, the pepsin working solution, the mouse anti-digoxin antibody, the goat anti-mouse antibody and the enzyme-labeled anti-goat IgG polymer are all ready-to-use reagents, and the EBER probe is a digoxin labeled probe designed by a unique sequence.
3. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the step 1), the DAB is 3, 3' -diaminobenzidine.
4. The EB virus detection method based on the in situ hybridization method as claimed in claim 1, wherein in the step 2), the dewaxing treatment is to soak and dewax 3-5 μm paraffin sections in xylene for 2 times and 10 minutes each time, then soak in absolute ethyl alcohol for 2 times and 3 minutes each time, and finally air-dry for 5-10 minutes.
5. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the steps 3), 6), 7) and 8), the incubation refers to incubation in an in situ hybridization instrument or a wet box.
6. The EB virus detection method based on the in situ hybridization method of claim 1, wherein in the step 8), the enzyme-labeled anti-sheep IgG polymer is a polymer HRP-conjugated secondary antibody.
7. The EB virus detection method based on the in-situ hybridization method as claimed in claim 1, wherein in the step 9), the DAB working solution is formed by mixing the DAB chromogen concentrated solution and the DAB substrate buffer solution according to a proportion, and the proportion is that one drop of the DAB chromogen concentrated solution is added into every 800 μ L of the DAB substrate buffer solution.
8. The EB virus detection method based on the in situ hybridization method as claimed in claim 1, wherein in the step 10), the bluing refers to observing that cell nuclei in the sample are blue.
9. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the step 11), the concentration gradient is 70%, 85%, 95% and 100%.
10. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the step 11), the mounting refers to mounting the sample by using neutral gum.
11. The EB virus detection method based on the in situ hybridization method according to claim 1, wherein in the step 4), the hybridization incubation is 2-4 hours or overnight, which means that the hybridization incubation is 2-4 hours or overnight in an in situ hybridization instrument or a wet box.
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| CN116590468A (en) * | 2023-02-22 | 2023-08-15 | 卡秋(江苏)生物科技有限公司 | In situ hybridization kit for EB virus detection and use method thereof |
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| WO2013032404A1 (en) * | 2011-08-26 | 2013-03-07 | Temasek Life Sciences Laboratory Limited | Human enterovirus specific antibodies and their uses in diagnostics |
| CN113528513A (en) * | 2021-08-12 | 2021-10-22 | 基因科技（上海）股份有限公司 | In-situ hybridization probe complex, preparation method and application thereof |
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| WO2013032404A1 (en) * | 2011-08-26 | 2013-03-07 | Temasek Life Sciences Laboratory Limited | Human enterovirus specific antibodies and their uses in diagnostics |
| CN113528513A (en) * | 2021-08-12 | 2021-10-22 | 基因科技（上海）股份有限公司 | In-situ hybridization probe complex, preparation method and application thereof |
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