CN116590468A - In situ hybridization kit for EB virus detection and use method thereof - Google Patents
In situ hybridization kit for EB virus detection and use method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a group of in situ hybridization probes for EB virus detection and a use method thereof, wherein the sequence of the in situ hybridization probes for EB virus detection is shown as SEQ ID No:1 and No:4, can be used for detecting EB virus coded small RNA molecules, namely EBER-1 and EBER-2. The combined reagent comprises hybridization solution, blocking solution, digestion solution, HRP-labeled digoxin antibody, DAB concentrated color development solution and DAB substrate buffer solution. The in situ hybridization kit for EB virus detection has the advantage of high detection sensitivity.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a group of in-situ hybridization probes for EB virus detection and a combined reagent thereof.
Background
EB virus (Epstein-Barr virus, EBV) belongs to the family Herpesviridae, is type IV herpesvirus, a human lymphotropic herpesvirus. EBV is a double-stranded DNA virus, the genome of which is about 172kb, is a linear molecule in the viral particle, and when it enters the infected cell, its DNA circularizes and replicates itself. According to clinical statistics, approximately 95% of adults have been infected with EBV or are in a state of latent infection. EBV transmission is mainly through saliva, body fluids, organ transplants, blood transfusion, and the like. After epithelial cell infection (e.g., of the oropharynx), virus particles are produced by intracellular assembly and released into the blood to infect B lymphocytes. The infected B lymphocyte can continuously generate a large amount of EBV particles, and continuously enter the blood circulation, so that systemic infection is caused, infectious diseases such as infectious mononucleosis, chronic active EB virus infection and the like are caused, and even in certain cases, the latent infected EB virus can cause nasopharyngeal carcinoma and gastric cancer which are derived from epithelial cells, and malignant tumors such as Hodgkin's lymphoma and the like.
The current EBV detection methods are mainly classified into 3 types, namely a PCR-fluorescent probe method, an immunohistochemical method and an in situ hybridization probe method. Among them, the PCR-fluorescent probe method has the highest detection sensitivity, but has the most strict requirements on operators, detection equipment and sites, and is not easy to popularize. The immunohistochemical method is characterized in that the detection sensitivity of the method is the lowest because the transmembrane protein expressed by the EB virus is detected, and the condition of missed diagnosis is easy to occur. In the case of the in situ hybridization probe method, the EBV-encoded small RNA molecule EBER1 or EBER2 is detected. The number of copies of EBER1 and EBER2 in each cell was 10 6 ～10 7 Is a marked phenomenon of EBV latent infection. EBER1 and EBER2 are not as easily degraded as other RNA molecules in cells because they have stable secondary structures and are not translated into proteins. Thus, in situ hybridization probe methods are currently the most common EBV detection method.
In situ hybridization probes are classified into RNA probes and DNA probes, the former has better detection sensitivity and specificity, but the synthesis cost is relatively high and the in situ hybridization probes cannot be stored for a long time, so most of the existing EBV in situ hybridization detection kits use DNA probes. To improve the detection sensitivity of DNA probes, patent CN 109337958A adopts a method of modifying two digoxins on DNA probes to increase the signal intensity of the detection of its kit. This approach, while viable, is not critical, as increasing the concentration of the DNA probe can achieve the same result, and even more so current chemical techniques have been able to efficiently and cost-effectively modify 3 Digoxins at the 5 'end, 3' end, and intermediate positions of the DNA probe.
Based on the above, the key point of the invention is to ensure that the DNA probe can be designed to stably combine with the target to be detected. Because EBER-1 and EBER-2 are RNA and have complex secondary structures, the binding effects of different areas of the EBER-1 and the EBER-2 are different, and the detection results are different.
Disclosure of Invention
The invention aims to provide a group of in-situ hybridization probes for EBV detection and a combined reagent thereof, which can detect EBV coded small RNA molecules, namely EBER-1 and EBER-2, can obviously increase the detection rate of EBV and provide valuable reference information for early diagnosis of EBV patients.
The invention designs a plurality of groups of probes which are respectively combined with different areas of the EBER-1 and the EBER-2 to screen out a group of probes with the most stable combination effect. Meanwhile, in order to improve the sensitivity of EBV detection, the invention learns the thought of the PCR-fluorescent probe method and carries out multiple detection on the target to be detected. The detection sensitivity of EBV can be doubled compared with the detection of EBER-1 or EBER-2 alone, and the detection of EBER-1 and EBER-2 simultaneously.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
detecting an in situ hybridization probe of a small RNA molecule-EBER 1 coded by EB virus, wherein the probe sequence is shown as SEQ ID NO:1-NO: 3.
Detecting an in situ hybridization probe of a small RNA molecule-EBER 2 coded by the EB virus, wherein the probe sequence is shown as SEQ ID NO:4-NO: 6.
Preferably, the in situ hybridization probe for detecting EBER-1 is a sequence shown as SEQ ID NO. 1;
the in situ hybridization probe for detecting the human EBER-2 is a sequence shown as SEQ ID NO. 4.
An in situ hybridization kit for EB virus detection, which comprises one or more of DNA probe groups for detecting EBER-1 and EBER-2, wherein the sequences of the DNA probe groups are shown as SEQ ID NO. 1-NO:3 and SEQ ID NO:4-NO: shown at 6.
The probe is diluted by using hybridization solution to a final concentration of 5-10 ng/. Mu.L.
The kit also comprises a sealing solution, a digestive juice, an HRP-labeled digoxin antibody, a DAB concentrated color development solution and a DAB substrate buffer solution.
The blocking solution is a hydrogen peroxide solution of 1-5%, more preferably a hydrogen peroxide solution of 3%, and the digestion solution is a pepsin solution of 100 μg/mL.
The using method of the kit comprises the following steps:
the synthesized probe was dissolved in purified water, and the concentration thereof was diluted to 5 to 10 ng/. Mu.L using hybridization solution (6 XSSC; 5 XDenhardt; 0.5% SDS; 100. Mu.g/ml Salmon sperm DNA);
and respectively adding DAB substrate buffer solution and DAB concentrated color development solution, and uniformly mixing to prepare DAB working solution.
Placing fresh paraffin slice into xylene, dewaxing for 3×10min, removing excessive liquid, placing into absolute ethanol for 3×3 min, and air drying for 5-10min;
closing: dripping 80-100 mu L of 5% hydrogen peroxide blocking solution into the dried slices, incubating for 10min at room temperature in a dark place, washing with pure water, gradient dehydrating for 2min with 75%, 95% and 100% ethanol solution, and air drying;
digestion: dripping 80-100 mu L of 100 mu g/mL pepsin digestion solution into the dried slice, incubating for 5-30min at 37 ℃, washing with pure water, and then carrying out gradient dehydration on 75%, 95% and 100% ethanol solutions for 2min respectively, and air-drying;
hybridization: dripping 5-10 mu L of 1-2 ng/mu L probe hybridization solution into the dried slice, covering a siliconized cover slip, sealing edges by rubber cement, and carrying out hybridization incubation for 2-4 hours or overnight at 37 ℃;
washing: carefully removing rubber cement, putting the slice into PBS buffer solution, removing the cover glass, and then soaking and cleaning for 10min;
combining: dripping 30-50 mu L of HRP-labeled Digoxin antibody on the slice, incubating for 30min at 37 ℃, and then putting into PBS buffer solution for washing for 10min;
color development: dripping 80-100 mu L of newly prepared DAB working solution on the slice, and incubating for 5-10min at room temperature;
counterstaining-dewatering-sealing sheet: washing the redundant DAB working solution with purified water, then dripping 10-30 mu L of hematoxylin staining solution, incubating at room temperature for 10-30s, then carrying out gradient dehydration on 75%, 95% and 100% ethanol solutions for 2min respectively, air-drying, and finally sealing the neutral resin;
reading: the hybridization of the probe was observed under a microscope, and positive staining was localized to the nucleus.
The invention has the beneficial effects that:
1. the group of in-situ hybridization probes for EB virus detection and the combined reagent thereof provided by the invention can detect the EBER-1 and the EBER-2 simultaneously, and compared with the independent detection of the EBER-1 or the EBER-2, the sensitivity of the EBV detection is improved by 1 time. However, multiplex detection may occur with binding between probes, resulting in unnecessary false positive results. Therefore, the invention avoids the complementary pairing of more than 5 bases between probes based on the base complementary pairing principle at the beginning of probe design.
2. The group of in situ hybridization probes for EB virus detection and the combined reagent thereof improve the effect of EBV in situ hybridization detection by optimizing the final concentration of the probes and the concentration and the components of part of the combined reagent.
And 3. The EBER-1 and the EBER-2 are RNA and have complex secondary structures, so that the binding effects of different areas of the EBER-1 and the EBER-2 are different, and further, the detection results are different. In order to overcome the problem, the invention designs a plurality of groups of probes which are respectively combined with different areas of the EBER-1 and the EBER-2, and then a group of probes with the most stable combination effect is screened out through multiple experimental verification.
Drawings
FIG. 1 is a graph showing experimental results observed by an optical microscope after each in situ hybridization probe in example 1 is used for pathological section, wherein A-F are SEQ ID NOs: 1-NO:6, and the result shows SEQ ID NO:1 and NO:4, the binding effect of the corresponding probe with EBER-1 or EBER-2 is better, and SEQ ID NO; 2. NO:3 and NO:6, the probes corresponding to the SEQ ID NO:5, the probes corresponding to the probes are not successfully combined;
FIG. 2 is a graph showing the results of the simultaneous detection of EBER-1 and EBER-2 and the detection of EBER-1 or EBER-2 alone in example 2 of the present invention; a) The method is a using effect graph of multiple detection; b) Is SEQ ID NO:1, a using effect diagram; c) Is SEQ ID NO:4, the result shows that multiple detection detects more positive sites than single detection;
FIG. 3 is a graph showing the results of experiments observed by an optical microscope after the probe hybridization solution of each concentration in example 3 of the present invention is used for pathological sections, A) is a graph showing the effect of using the probe hybridization solution of 1-2 ng/. Mu.L; b) 5-10 ng/. Mu.L of probe hybridization solution; c) The use effect graph of 25-50 ng/mu L of probe hybridization solution shows that 1-2 ng/mu L of probe hybridization solution has fewer positive sites, the background detected by 25-50 ng/mu L of probe hybridization solution is overweight, and the detection effect of only 5-10 ng/mu L of probe hybridization solution is optimal;
FIG. 4 is a graph showing the results of experiments observed by an optical microscope after the blocking solution of each hydrogen peroxide concentration in example 4 of the present invention was used for pathological sections, A) a 1% hydrogen peroxide solution; b) 3% hydrogen peroxide solution; c) The result of 5% hydrogen peroxide solution shows that the blocking effect of 1% concentration is slightly worse and background interference exists; the blocking effect of 5% concentration is too high, and the positive sites are blocked, so that the blocking effect of 3% concentration is optimal;
FIG. 5 is a graph showing the results of experiments observed by an optical microscope after pepsin and proteinase k digests were used in pathological sections in example 5 of the present invention, A) pepsin digests; b) The result of proteinase k digestion solution shows that the pathological section after proteinase k digestion has heavy staining background and has no digestion effect like pepsin.
Detailed Description
For ease of understanding, the present invention is specifically exemplified below. It should be apparent to those skilled in the art that the examples are merely provided to aid in understanding the present invention and should not be construed as limiting the invention in any way.
Example 1 screening of in situ hybridization probes for EBV detection
In order to verify the design quality of in situ hybridization probes and determine the optimal binding region, the invention designs 3 groups of DNA probes for the sequences of EBER-1 and EBER-2 coded by EBV respectively for parallel comparison, and the sequences are shown in the table 1, and can bind to the region in which a secondary structure is not formed in the EBER-1 or EBER-2 sequence.
TABLE 1 in situ hybridization probe sequences
The sequences of EBER-1 and EBER-2 are shown in Table 2.
TABLE 2 sequences of EBER-1 and EBER-2
The in situ hybridization kit for EB virus detection in the embodiment and the using method thereof are as follows:
1. reagent preparation
1.1 entrusting Gene Synthesis company to synthesize the sequences described in Table 1 and modify digoxin at its 5', 3' end or in the middle;
1.2 dissolving the synthesized probe in purified water, and diluting the concentration to 1.about.2 ng/. Mu.L using hybridization solution (6 XSSC; 5 XDenhardt; 0.5% SDS; 100. Mu.g/ml Salmon sperm DNA);
1.3 according to 19:1 respectively adding DAB substrate buffer solution and DAB concentrated color development solution according to the proportion, and uniformly mixing the DAB substrate buffer solution and the DAB concentrated color development solution to prepare DAB working solution.
2. Operational flow
2.1 dewaxing: placing fresh paraffin slice into xylene, dewaxing for 3×10min, removing excessive liquid, placing into absolute ethanol for 3×3 min, and air drying for 5-10min;
2.2 blocking: dripping 80-100 mu L of 5% hydrogen peroxide blocking solution into the dried slices, incubating for 10min at room temperature in a dark place, washing with pure water, gradient dehydrating for 2min with 75%, 95% and 100% ethanol solution, and air drying;
2.3 digestion: dripping 80-100 mu L of 100 mu g/mL pepsin digestion solution into the dried slice, incubating for 5-30min at 37 ℃, washing with pure water, and then carrying out gradient dehydration on 75%, 95% and 100% ethanol solutions for 2min respectively, and air-drying;
2.4 hybridization: dripping 5-10 mu L of 1-2 ng/mu L probe hybridization solution into the dried slice, covering a siliconized cover slip, sealing edges by rubber cement, and carrying out hybridization incubation for 2-4 hours or overnight at 37 ℃;
2.5 washing: carefully removing rubber cement, putting the slice into PBS buffer solution, removing the cover glass, and then soaking and cleaning for 10min;
2.6 binding: dripping 30-50 mu L of HRP-labeled Digoxin antibody on the slice, incubating for 30min at 37 ℃, and then putting into PBS buffer solution for washing for 10min;
2.7 developing: dripping 80-100 mu L of newly prepared DAB working solution on the slice, and incubating for 5-10min at room temperature;
2.8 counterstaining-dewatering-sealing sheet: washing the redundant DAB working solution with purified water, then dripping 10-30 mu L of hematoxylin staining solution, incubating at room temperature for 10-30s, then carrying out gradient dehydration on 75%, 95% and 100% ethanol solutions for 2min respectively, air-drying, and finally sealing the neutral resin;
2.9 reading: the hybridization of the probe was observed under a microscope, and positive staining was localized to the nucleus.
SEQ ID NO:1-NO:6 is shown in fig. 1, wherein the probe for EBER-1 is shown in SEQ ID NO:1 is superior to the probe corresponding to SEQ ID NO in detection effect: 2 and NO:3, a step of; in the probe against EBER-2, SEQ ID NO:4 is superior to the probe corresponding to SEQ ID NO:5 and NO:6.
example 2 comparison of multiplex and singleplex assays
To increase the sensitivity of the in situ hybridization detection of EBV, the invention uses the sequence of SEQ ID NO:1 and NO:4 to be hybridized, diluted to 5-10 ng/. Mu.L with hybridization solution, and the procedure of example 1 was applied, and the hybridization reaction was carried out under the same conditions as in SEQ ID NO:1 or NO:4, and comparing the probes corresponding to the probes when used alone. As a result of comparison, as shown in FIG. 2, the mixed probe was able to detect EBER-1 and EBER-2 simultaneously, and the detection signal intensity was 1-fold higher than that of the probe alone.
Example 3 optimization of the concentration of Probe hybridization solution
In order to optimize the concentration of the probe hybridization solution, the invention sets three groups of probe hybridization solutions of 1-2 ng/mu L, 5-10 ng/mu L and 25-50 ng/mu L, and the operation flow in the example 1 is applied to carry out parallel detection comparison on pathological sections.
As shown in FIG. 3, the results of the comparison are best when the probe hybridization solution of 5-10 ng/. Mu.L is used in the in situ hybridization of EB virus.
EXAMPLE 4 Hydrogen peroxide blocking fluid concentration optimization
In order to optimize the concentration of the hydrogen peroxide sealing liquid, the invention sets three groups of hydrogen peroxide sealing liquids of 1%, 3% and 5%, and the operation procedure in the example 1 is applied to carry out parallel detection comparison on pathological sections.
As shown in FIG. 4, the blocking solution consisting of 3% hydrogen peroxide solution was most effective in the in situ hybridization of EB virus.
Example 5 comparison of the digestion Effect of pepsin and proteinase k
To determine the digestion effect of pepsin and proteinase k, the procedure of example 1 was applied to two proteases at the same concentration, and a parallel assay comparison was performed on pathological sections.
The comparison results show in FIG. 5 that there is no significant difference in digestion effect between pepsin and proteinase k, but proteinase k is more expensive in terms of raw material cost. Thus, the present invention finally selects pepsin as the main component of the digestive juice.
In summary, the invention discloses a group of in situ hybridization probes for EB virus detection and a reagent used in combination, which can detect EBV-encoded small RNA molecules, namely EBER-1 and EBER-2. Compared with the independent detection of the EBER-1 or the EBER-2, the detection rate of the EBV is improved by 1 time, and valuable reference information is provided for early diagnosis of EBV patients.
The applicant therefore states that the detailed detection reagents and detection methods of the present invention are described by the above examples, but the embodiments of the present invention are not limited by the above examples, and any other substitution pattern such as addition, modification, substitution, combination, simplification, etc. without departing from the spirit and principles of the present invention should fall within the scope of the present invention.
Claims (10)
1. The in situ hybridization probe for detecting the small RNA molecule-EBER 1 coded by the EB virus is characterized in that the probe sequence is shown as SEQ ID NO. 1-NO: 3.
2. The in situ hybridization probe for detecting EB virus-encoded small RNA molecule-EBER 1 according to claim 1, wherein the probe sequence is shown in SEQ ID NO. 1.
3. The in situ hybridization probe for detecting the small RNA molecule-EBER 2 coded by the EB virus is characterized in that the probe sequence is shown as SEQ ID NO. 4-NO: 6.
4. The in situ hybridization probe for detecting EB virus-encoded small RNA molecule-EBER 2 according to claim 3, wherein the probe sequence is shown as SEQ ID NO. 4.
5. An in situ hybridization kit for EB virus detection, wherein the kit comprises the probe according to claim 1 and/or 2.
6. The kit for in situ hybridization according to claim 5, wherein the kit comprises a probe sequence shown in SEQ ID NO. 1 and a probe sequence shown in SEQ ID NO. 4.
7. The kit for in situ hybridization for EB virus detection according to claim 5 or 6, wherein said probe is diluted with hybridization solution to a final concentration of 5 to 10 ng/. Mu.L.
8. The kit for in situ hybridization for EB virus detection according to claim 7, wherein the kit further comprises a blocking solution, a digestive solution, an HRP-labeled digoxin antibody, a DAB concentrated color-developing solution and a DAB substrate buffer solution.
9. The kit for in situ hybridization for EB virus detection according to claim 7, wherein the blocking solution is a hydrogen peroxide solution with a concentration of 3%, and the digestion solution is a pepsin solution with a concentration of 100. Mu.g/mL.
10. The application method of the in situ hybridization kit for EB virus detection is characterized by comprising the following steps:
dissolving the synthesized probe with purified water, and diluting the concentration of the probe to 5-10 ng/mL by using hybridization solution;
respectively adding DAB substrate buffer solution and DAB concentrated color development solution, and uniformly mixing to prepare DAB working solution;
dewaxing: sequentially dewaxing fresh paraffin slices in dimethylbenzene, removing redundant liquid, putting the paraffin slices in absolute ethyl alcohol, and then air-drying for 5-10min;
closing: dripping 80-100 mu L of hydrogen peroxide blocking solution into the dried slices, incubating for 10min at room temperature in a dark place, washing with pure water, gradient dehydrating with 75%, 95% and 100% ethanol solution for 2min, and air drying;
digestion: dripping 80-100 mu L of 100 mu g/mL pepsin digestive juice into the dried slice, incubating for min at 37 ℃, washing with pure water, and then carrying out gradient dehydration on 75%, 95% and 100% ethanol solutions for 2min respectively, and air-drying;
hybridization: dropwise adding 5-10 mu L of probe hybridization solution into the dried slice, covering a siliconized cover slip, sealing edges by rubber cement, and carrying out hybridization incubation for 2-4 hours or overnight at 37 ℃;
washing: carefully removing rubber cement, putting the slice into PBS buffer solution, removing the cover glass, and then soaking and cleaning for 10min;
combining: dripping 30-50 mu L of HRP-labeled Digoxin antibody on the slice, incubating for 30min at 37 ℃, and then putting into PBS buffer solution for washing for 10min;
color development: dripping 80-100 mu L of newly prepared DAB working solution on the slice, and incubating for 5-10min at room temperature;
counterstaining-dewatering-sealing sheet: washing the excessive DAB working solution with purified water, then dripping 10-30 mu L of hematoxylin staining solution, incubating at room temperature for 10-30s, gradient dehydrating with 75%, 95% and 100% ethanol solution for 2min, air drying, and sealing with neutral resin;
reading: the hybridization of the probe was observed under a microscope, and positive staining was localized to the nucleus.
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