CN117737308A - Kit for EBER detection and application thereof - Google Patents
Kit for EBER detection and application thereof Download PDFInfo
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- CN117737308A CN117737308A CN202311795451.4A CN202311795451A CN117737308A CN 117737308 A CN117737308 A CN 117737308A CN 202311795451 A CN202311795451 A CN 202311795451A CN 117737308 A CN117737308 A CN 117737308A
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- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 4
- 206010015108 Epstein-Barr virus infection Diseases 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
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- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 4
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 4
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for EBER detection and application thereof, wherein the kit comprises an EBER1 probe with nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 8 and an EBER2 probe with nucleotide sequences shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 15. The probe set and the kit for EBER detection use probes which are respectively combined with the EBER1 and the EBER2 in a specific way, can detect two small RNAs coded by the EB virus simultaneously, have shorter length, increase the sensitivity of probe detection, ensure the specificity simultaneously, and can realize the detection efficiency of more than 99 percent in clinical experiments.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a kit for EBER detection and application thereof.
Background
The detection gold standard of the EB virus on the tissue is EBER in situ hybridization, and EBER is small RNA encoded by the EB virus and comprises EBER1 and EBER2. The detection probes in the current clinical application are designed aiming at EBER1 and are single probes with longer sequences, and have the defects of lower sensitivity, incomplete combination of small RNA which is subjected to partial degradation, easy elution and finally lower color development or false negative result.
In order to solve the above technical problems, the applicant's prior invention patent application CN115948608A discloses a probe set and a kit for EBER detection, which use probes specifically bound to EBER1 and EBER2 respectively, so that two small RNAs encoded by epstein barr virus can be detected simultaneously, the length of the probe design is shorter, the sensitivity of probe detection is increased, and the specificity is ensured simultaneously. Although the sensitivity and the specificity of the prior invention can reach 100% in vitro experiments, the effectiveness verification result in clinical experiments is 98.18%, and a certain room for improvement still exists.
In view of the above, the inventors of the present application have further optimized a primer set in a kit, and have completed the present invention accordingly.
Disclosure of Invention
Based on the above reasons, the invention provides a kit for EBER detection and application thereof. Specifically, in order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:
one aspect of the invention relates to a kit for EBER detection, which comprises an EBER1 probe with nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 8 and an EBER2 probe with nucleotide sequences shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 15.
In a preferred embodiment of the present invention, the 3' -end of the EBER1 probe and the EBER2 probe are labeled with digoxin.
In a preferred embodiment of the invention, the kit further comprises a primer having a nucleotide sequence shown in SEQ ID NO. 14.
In a preferred embodiment of the invention, it is characterized in that: an EBER1 probe with nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 8 and an EBER2 probe with nucleotide sequences shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 15 are selected in a molar ratio of 8:12:5:15:15:5.
In a preferred embodiment of the invention, it is characterized in that: the kit also comprises TE buffer solution, probe hybridization buffer solution, gastric enzyme working solution, PBS buffer solution, anti-digoxin antibody, DAB working solution and deionized water.
In a preferred embodiment of the invention, it is characterized in that: the pH of the TE buffer was 8.
In a preferred embodiment of the invention, it is characterized in that: the anti-digoxin antibody was HRP-labeled.
In another aspect, the invention also relates to the use of the kit in the preparation of a diagnostic reagent for diagnosing epstein barr virus infection.
In a preferred embodiment of the present invention, the epstein barr virus infection refers to gastric cancer EBER positive, lymphoma EBER positive and/or nasopharyngeal cancer EBER positive.
Advantageous effects
The probe set and the kit for EBER detection use probes which are respectively combined with the EBER1 and the EBER2 in a specific way, can detect two small RNAs coded by the EB virus simultaneously, have shorter length, increase the sensitivity of probe detection, ensure the specificity simultaneously, and can realize the detection efficiency of more than 99 percent in clinical experiments.
Drawings
FIGS. 1-3 show the detection results of the probe set 6 of the present invention for tumor samples.
FIGS. 4-6 show the results of detection of the same tumor sample by a commercial probe combination.
Detailed Description
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1:
the preparation method comprises the following steps:
synthesizing each sequence into a single-stranded DNA probe, and marking the 3' -end by digoxin; the TE buffer with pH of 8.0 is used for dissolving the dry powder of every 2OD fully; and taking 1 volume of each dissolved probe, adding 6 volumes of mixed probes, and diluting the mixed probes by using 100 volumes of hybridization buffer solution to obtain the combined probe.
The application is as follows:
the method is used for in situ hybridization detection of the EB virus.
The effect is as follows:
can effectively detect the small RNA coded by the EB virus in tissues and clear the virus infection.
Principle of:
the probe sequence is complementary with the base sequence of EBER in the tissue, and at proper temperature and time, the probe passes through the cell nucleus hole to enter the cell nucleus, and is complementary pairing-combined with the base of EBER, the 3' -labeled digoxin of the probe is combined with the HRP-labeled anti-digoxin antibody, and finally the HRP is catalyzed by DAB to generate a tan precipitate, which can be observed under an optical microscope.
Dosage form:
a solution.
The detection dosage is as follows:
manually detecting the dosage of each sample to be 10ul; full-automatic immunohistochemical apparatus (Ultra 30/Ultra 60) was used in an amount of 150ul per case.
Test for assay effectiveness:
experimental materials
1. Designing a probe: the total number of probes designed according to the EBER1 sequence is 8, and the total number of probes designed according to the EBER2 sequence is 7, and the total number of probes is 15.
2. Sample: the number of EB virus infected samples is 1000, and the number of other virus infected samples is 200.
Design of test for validity of probe
1. The probe design information is as follows:
2. 1ml of TE buffer of PH8.0 was used to dissolve 2OD probes per tube as probe stock; the probe stock solution was diluted 1:100 with probe hybridization buffer as working solution.
3. Experimental method
3.1 dewaxing
Slice samples were dewaxed with fresh xylene (10 min. Times.3). The dewaxed sections were placed in fresh 100% ethanol for 3 minutes, 3 cylinders. And (5) performing air drying for 5-10 minutes to perform enzyme treatment.
3.2 enzymatic treatment
The dried sections were then incubated with 80-100ul of gastric enzyme working solution for 30 minutes at 37℃depending on the tissue size. After discarding the gastric enzyme working fluid, alcohol was dehydrated stepwise (75%, 95% and 100%), and each gradient was left for 2 minutes. Air-drying and hybridization.
3.3 hybridization of probes
According to the tissue size, 10ul of designed probes are respectively dripped into each slice, and the slices are covered with a cover slip and sealed by rubber cement. Incubate in wet box at 37℃for 3 hours. After hybridization, the sections were immersed in PBS buffer for 10 minutes until the coverslip spontaneously dropped. The water-blocking pen is circled. The sections were washed 2 min x 3 times with PBS buffer, removed and excess buffer was removed.
3.4 detection and visualization procedure
Appropriate amount of HRP-labeled anti-digoxin antibody was added to the sections, incubated at 37 ℃ for 30 minutes, excess liquid was removed, and the slides were rinsed 3 times with PBS buffer for 2 minutes each.
Wiping water at the edge of the slice, and adding a proper amount of freshly prepared DAB working solution. Color development was performed for 5 minutes, and the color development was observed under a microscope. And washing the slices with deionized water, and then performing counterstaining and sealing.
3.5 counterstaining procedure
Hematoxylin counterstain, staining for 40 seconds. Washing with ionized water for a short time, dehydrating conventionally, transparency, and sealing.
4 experimental results
Experimental results prove that the hybridization sensitivity of the long probe is obviously reduced, and the hybridization specificity is easily reduced due to the too short probe length. The sensitivity and the specificity of the protection probe sequences of the patent application are obviously higher.
Note that: sensitivity: the positive cells in the EBER positive sample are all detected by the designed probes; specificity: the designed probe only specifically detects EBER positive signals, and no nonspecific staining exists.
After screening out the designed sequences with insufficient sensitivity and specificity, a probe combination experiment is performed.
Test for verifying sensitivity and specificity of probe combination
1. Probe combination mode
2. 1ml of TE buffer of PH8.0 was used to dissolve 2OD probes per tube as probe stock; different probe combinations were performed as working solutions according to the above table using probe hybridization buffers.
3. Experimental method
3.1 dewaxing
Slice samples were dewaxed with fresh xylene (10 min. Times.3). The dewaxed sections were placed in fresh 100% ethanol for 3 minutes, 3 cylinders. And (5) performing air drying for 5-10 minutes to perform enzyme treatment.
3.2 enzymatic treatment
The dried sections were then incubated with 80-100ul of gastric enzyme working solution for 30 minutes at 37℃depending on the tissue size. After discarding the gastric enzyme working fluid, alcohol was dehydrated stepwise (75%, 95% and 100%), and each gradient was left for 2 minutes. Air-drying and hybridization.
3.3 hybridization of probes
According to the tissue size, 10ul of designed probes are respectively dripped into each slice, and the slices are covered with a cover slip and sealed by rubber cement. Incubate in wet box at 37℃for 3 hours. After hybridization, the sections were immersed in PBS buffer for 10 minutes until the coverslip spontaneously dropped. The water-blocking pen is circled. The sections were washed 2 min x 3 times with PBS buffer, removed and excess buffer was removed.
3.4 detection and visualization procedure
Appropriate amount of HRP-labeled anti-digoxin antibody was added to the sections, incubated at 37 ℃ for 30 minutes, excess liquid was removed, and the slides were rinsed 3 times with PBS buffer for 2 minutes each.
Wiping water at the edge of the slice, and adding a proper amount of freshly prepared DAB working solution. Color development was performed for 5 minutes, and the color development was observed under a microscope. And washing the slices with deionized water, and then performing counterstaining and sealing.
3.5 counterstaining procedure
Hematoxylin counterstain, staining for 40 seconds. Washing with ionized water for a short time, dehydrating conventionally, transparency, and sealing.
4. Experimental results
The experimental results show that different probe combination modes have certain difference in sensitivity and specificity of hybridization, wherein the combination of which the sensitivity and the specificity reach 100% is combination 6 and combination 8, and the combination 6 is the probe combination mode of patent protection.
Note that: sensitivity: the positive cells in the EBER positive sample are all detected by the designed probes; specificity: the designed probe only specifically detects EBER positive signals, and no nonspecific staining exists.
Enlarged sample validity verification experiment of probe combination
1. Expanding sample selection modes: the other EB virus detection methods are selected to confirm that the EB virus infected sample tissues are 1000 (including 400 samples of gastric cancer EBER positive samples, 350 samples of lymphoma EBER positive samples and 250 samples of nasopharyngeal carcinoma EBER positive samples).
2. And detecting clinical samples by using the combination 6 and the combination 8, designing a probe validity verification experiment together, and detecting by using a full-automatic immunohistochemical instrument (Ultra 30/Ultra 60) standard program. The standard procedure is as follows:
3. validity verification result
| Total number of cases | Invalidation data | Valid data |
| 1000 | 6 | 994 |
Note that: invalid data rejection basis: falling slices and no tumor tissue.
The results of the detection of combination 6 and commercially available probes on the same sample are shown in FIGS. 1-6. Combination 6 valid data analysis:
the sample verification result proves that the detection effective rate of the probe combination 6 is 99.7%, and compared with the prior application, the effective rate of the probe combination 6 is remarkably improved.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (9)
1. A kit for EBER detection comprises an EBER1 probe with nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 8 and an EBER2 probe with nucleotide sequences shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 15.
2. The kit of claim 1, wherein the 3' ends of the EBER1 probe and EBER2 probe are labeled with digoxin.
3. The kit of claim 1, further comprising a primer having a nucleotide sequence set forth in SEQ ID NO. 14.
4. The kit of claim 1, wherein: an EBER1 probe with nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 6 and SEQ ID NO. 8 and an EBER2 probe with nucleotide sequences shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 15 are selected in a molar ratio of 8:12:5:15:15:5.
5. The kit of claim 1, wherein: the kit also comprises TE buffer solution, probe hybridization buffer solution, gastric enzyme working solution, PBS buffer solution, anti-digoxin antibody, DAB working solution and deionized water.
6. The kit of claim 1, wherein: the pH of the TE buffer was 8.
7. The kit of claim 1, wherein: the anti-digoxin antibody was HRP-labeled.
8. Use of the kit of any one of claims 1-7 for the preparation of a diagnostic reagent for diagnosing epstein barr virus infection.
9. The use according to claim 8, wherein the epstein barr virus infection is gastric cancer EBER positive, lymphoma EBER positive and/or nasopharyngeal carcinoma EBER positive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311795451.4A CN117737308A (en) | 2023-12-25 | 2023-12-25 | Kit for EBER detection and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311795451.4A CN117737308A (en) | 2023-12-25 | 2023-12-25 | Kit for EBER detection and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117737308A true CN117737308A (en) | 2024-03-22 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311795451.4A Pending CN117737308A (en) | 2023-12-25 | 2023-12-25 | Kit for EBER detection and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117737308A (en) |
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2023
- 2023-12-25 CN CN202311795451.4A patent/CN117737308A/en active Pending
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