CN117051114A - In situ hybridization probe set for Kappa and Lambda light chain mRNA detection - Google Patents
In situ hybridization probe set for Kappa and Lambda light chain mRNA detection Download PDFInfo
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Abstract
The invention discloses an in situ hybridization probe set for detecting Kappa and Lambda light chain mRNA, which comprises a probe I for detecting Kappa light chain mRNA and a probe II for detecting Lambda light chain mRNA, wherein the sequence of the probe I is shown as SEQ ID NO:1-SEQ ID NO:3, the sequence of the probe II is SEQ ID NO:4-SEQ ID NO: shown at 6. The in situ hybridization probe can detect human immunoglobulin Kappa and Lambda light chain mRNA simultaneously on the same tissue section, shortens detection time and reduces the dosage of reagents and samples.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to an in-situ hybridization probe set for detecting Kappa and Lambda light chain mRNA.
Background
Human-produced immunoglobulins (Ig) in which the light chain has Kappa and Lambda types; the expression of Kappa type plasma cells/Lambda type plasma cells in normal lymph nodes was about 2/1; expressing Kappa type plasma cells/Lambda type plasma cells in abnormal lymph nodes more than 4/1 or less than 1/2; kappa/Lambda probes can label B lymphocytes with light chains, plasma cells and immunoblasts, as compared to classical ones: multiple Myeloma (MM).
Multiple myeloma is a malignant plasma cytopathy that produces monoclonal immunoglobulins that invade and destroy adjacent bone tissue, the tumor cells of which originate from plasma cells in the bone marrow, which are cells that develop into the final functional stage of B lymphocytes. Multiple myeloma can therefore be assigned to the range of B-lymphocytic lymphomas, and Kappa/Lambda assays are used for diagnosis and typing of multiple myeloma, and patients can be monitored for later disease by increasing or decreasing the light chain of both sera. In pathological diagnosis, the two Ig light chain immunohistochemical staining markers are used for staining and positioning, but because Ig is a secreted protein, the immunohistochemical staining, especially Kappa, often has heavy background staining, and the in situ hybridization is used for replacing the immunohistochemical staining, so that the result is clearer and easy to interpret. In situ hybridization probes are classified into RNA probes and DNA probes, the former has better detection sensitivity and specificity, so that most of the existing Kappa/Lambda in situ hybridization detection reagents are RNA probes.
Currently, in the detection of plasma cell lymphoma diseases, two in situ hybridization probes are required to be used for dyeing to detect Kappa and Lambda light chain mRNA respectively, the operation is complex, the detection time is long, and the reagent and sample consumption is large.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an in-situ hybridization probe set for detecting Kappa and Lambda light chain mRNA, which can detect human immunoglobulin Kappa and Lambda light chain mRNA simultaneously.
In order to achieve the above object, the present invention adopts the following technical scheme:
an in situ hybridization probe set for detecting Kappa and Lambda light chain mRNA, wherein the probe set comprises a probe I for detecting Kappa light chain mRNA and a probe II for detecting Lambda light chain mRNA, and the sequence of the probe I is shown as SEQ ID NO:1-SEQ ID NO:3, the sequence of the probe II is SEQ ID NO:4-SEQ ID NO: shown at 6.
Preferably, digoxin is modified at the 5' carboxy terminus of each of the first and second probes.
The preparation method of the in situ hybridization probe set comprises the following steps:
s1, preparing a probe I: diluting the probe I of the target Kappa light chain mRNA with the hybridization solution to obtain a probe I working solution;
s2, preparing a probe II: diluting a probe II of target Lambda light chain mRNA with the hybridization solution to form a probe II working solution;
s3, preparing an in situ hybridization probe set: and (3) the prepared probe I working solution and probe II working solution are mixed according to the volume ratio of 1:1, mixing.
Preferably, in the foregoing steps S1 and S2, the hybridization solution includes the following components: sodium citrate buffer salts, non-specific blockers, sodium dodecyl sulfate, and salmon sperm deoxyribonucleic acid.
The detection method of Kappa and Lambda light chain mRNA by the in situ hybridization probe set comprises the following steps:
(1) Preparing a color development working solution;
(2) Detecting in situ hybridization of Kappa light chain mRNA probes;
(3) In situ hybridization of Lambda light chain mRNA probes was detected.
Use of an in situ hybridization probe set for simultaneously detecting mRNA levels of human immunoglobulin Kappa and Lambda using an in situ hybridization method.
An in situ hybridization detection kit for detecting Kappa and Lambda light chain mRNAs, comprising the in situ hybridization probe set.
The invention has the advantages that: the in situ hybridization probe set provided by the invention can detect human immunoglobulin Kappa and Lambda light chain mRNA simultaneously, compared with single detection, the expression condition of human immunoglobulin Kappa and Lambda light chain mRNA can be displayed on the same pathological slice, the detection time is shortened, the dosage of reagents and samples is reduced, and valuable auxiliary diagnosis information is provided for pathologists.
Drawings
FIG. 1 is a graph of experimental results observed in an optical microscope after each in situ hybridization probe in example 1 of the present invention is used for pathological sections (A is a graph of the use effect of SEQ ID NO: 1+SEQ ID NO:4, B is a graph of the use effect of SEQ ID NO: 2+SEQ ID NO:5, and C is a graph of the use effect of SEQ ID NO: 3+SEQ ID NO: 6);
FIG. 2 is a graph showing the comparison between the simultaneous detection of human immunoglobulin Kappa and Lambda light chain mRNAs and the separate detection of human immunoglobulin Kappa and Lambda light chain mRNAs in example 2 of the present invention (A is a graph showing the effect of using SEQ ID NO:1 for Kappa, B is a graph showing the effect of using SEQ ID NO:4 for Lambda, C is a graph showing the effect of using a mixture of SEQ ID NO:1 for Kappa and SEQ ID NO:4 for Lambda);
FIG. 3 is a graph showing the results of experiments observed by an optical microscope after the probe hybridization solutions of various concentrations in example 3 were used for pathological sections (A is a graph showing the effect of using 1-2 ng/. Mu.L probe hybridization solution, B is a graph showing the effect of using 5-10 ng/. Mu.L probe hybridization solution, and C is a graph showing the effect of using 25-50 ng/. Mu.L probe hybridization solution).
Description of the embodiments
The invention is described in detail below with reference to the drawings and the specific embodiments.
The invention relates to an in situ hybridization probe set for detecting Kappa and Lambda light chain mRNA, which comprises a probe I for detecting Kappa light chain mRNA and a probe II for detecting Lambda light chain mRNA, wherein the sequence of the probe I is shown in SEQ ID NO:1-SEQ ID NO:3, the sequence of the probe II is SEQ ID NO:4-SEQ ID NO:6, the sequences of KAPPA and LAMBDA are shown in Table 1 and the sequences of KAPPA and LAMBDA are shown in Table 2.
TABLE 1 in situ hybridization probe sequences
TABLE 2 sequences of KAPPA and LAMBDA
Example 1
The preparation method of the in situ hybridization probe set comprises the following steps:
s1, preparing a probe I: diluting the probe I of the target Kappa light chain mRNA and the hybridization solution into 5-10 ng/mu L of probe I working solution;
s2, preparing a probe II: diluting a probe II of target Lambda light chain mRNA and the hybridization solution into 5-10 ng/mu L of probe II working solution;
s3, preparing an in situ hybridization probe set: and (3) the prepared probe I working solution and probe II working solution are mixed according to the volume ratio of 1:1, mixing.
The detection method of Kappa and Lambda light chain mRNA by the in situ hybridization probe set comprises the following steps:
1. preparing a color development working solution:
1.1 modifying digoxin at the 5' carboxyl end of the probe I and the probe II;
1.2 Dissolving the synthesized probe with purified water, and diluting the concentration of the probe to 5-10 ng/. Mu.L by using hybridization solution; the hybridization solution comprises the following components: 6 XSSC (sodium citrate buffer salt), 5 XDenhardt (non-specific blocker), 0.5% SDS (sodium dodecyl sulfate) and 100. Mu.g/ml Salmon sperm DNA (salmon sperm deoxyribonucleic acid);
1.3 The following steps are 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;
1.4 And (3) respectively adding a BCIP solution, an NBT solution and an alkaline phosphatase chromogenic buffer solution according to the proportion of 300:150:1, and uniformly mixing to prepare the alkaline phosphatase chromogenic working solution.
2. Detection of Kappa light chain mRNA probe in situ hybridization:
2.1 Dewaxing: sequentially dewaxing fresh paraffin slice with xylene for 3×10min, removing excessive liquid, adding into absolute ethanol for 3×3 min, and air drying for 5-10 min;
2.2 Repairing: placing the slice in pure water, repairing at 95deg.C for 3-10min, taking out, soaking in pure water, and allowing the glass slide to return to room temperature;
2.3 Digestion: dripping 50-100 mu L pepsin digestive juice into the slices, incubating for 10-20min at 37 ℃, washing with pure water, then dehydrating for 2min in a gradient way by 75%, 95% and 100% ethanol solution, and airing;
2.4 Hybridization of probe I: dripping 5-10 mu L of 1-2 ng/muL of probe I 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 AP enzyme binding: dropwise adding 30-50 mu L of AP-labeled Biotin antibody to the slice, incubating at 37 ℃ for 30min, and then putting into PBS buffer solution for cleaning for 10min;
2.7 Color development: and (3) dropwise adding 50-100 mu L of newly prepared alkaline phosphatase chromogenic working solution to the slices, incubating for 5-20min at room temperature, and then washing out the redundant alkaline phosphatase chromogenic working solution by using purified water.
3. Detection of Lambda light chain mRNA probe in situ hybridization:
3.1 Blocking: dripping 50-100 mu L of 3-5% hydrogen peroxide blocker, incubating for 3-5min at 37 ℃, and then placing into PBS buffer solution for cleaning for 10min;
3.2 Repairing: placing the slice in pure water, repairing at 95deg.C for 3-10min, taking out, soaking in pure water, and allowing the glass slide to return to room temperature;
3.3 Digestion: dripping 50-100 mu L pepsin digestive juice into the slices, incubating for 10-20min at 37 ℃, washing with pure water, then dehydrating for 2min in a gradient way by 75%, 95% and 100% ethanol solution, and airing;
3.4 Probe II hybridization: dripping 5-10 mu L of 1-2 ng/muL of probe II hybridization solution into the dried slice, covering a siliconized cover slip, sealing edges by rubber cement, and carrying out hybridization incubation at 37 ℃ for 2-4 hours or overnight;
3.5 Washing: carefully removing rubber cement, putting the slice into PBS buffer solution, removing the cover glass, and then soaking and cleaning for 10min;
3.6 HRP enzyme 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;
3.7 Color development: dripping 80-100 mu L of newly prepared DAB chromogenic working solution on the slice, incubating for 5-10min at room temperature, and then cleaning the redundant DAB chromogenic working solution by purified water;
3.8 Lining dyeing: dripping 80-100 mu L of nuclear solid red color development liquid on the slice, and cleaning the excessive nuclear solid red color development liquid by purified water after 8-12 min;
3.9 Reading: the hybridization of the probe was observed under a microscope, positive staining was localized to the cytoplasm, kappa was blue and Lambda was brown.
The probe hybridization is shown in FIG. 1, where in situ hybridization probes against human immunoglobulin Kappa and Lambda light chain mRNAs, the results show SEQ ID NOs: 1 and SEQ ID NO:4, the binding effect of the corresponding probe and human immunoglobulin Kappa and Lambda light chain mRNA is better; SEQ ID NO;2 and NO:5, though the probes corresponding to the probes are combined, the positive expression is too shallow to be easily identified; SEQ ID NO:3 and NO:6, and the corresponding probe is not successfully combined.
Example 2
To increase the sensitivity and specificity of in situ hybridization probe detection of human immunoglobulin Kappa and Lambda light chain mRNAs, the invention uses the sequence of SEQ ID NO:1 and SEQ ID 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 and SEQ id no: the probes corresponding to 4 were compared in case of use alone, and the results are shown in FIG. 2.
As can be seen from FIG. 2, the mixed probe is capable of detecting human immunoglobulin Kappa and Lambda light chain mRNA simultaneously, and there is no significant difference compared to the two probes used alone, and the detection of human immunoglobulin Kappa and Lambda light chain mRNA simultaneously has no significant effect on probe localization.
Example 3
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 procedure in the example 1 is applied to parallel detection and comparison of pathological sections, and the results are shown in FIG. 3.
As can be seen from FIG. 3, 1-2 ng/. Mu.L of probe hybridization solution has fewer positive sites, 25-50 ng/. Mu.L of probe hybridization solution has an excessively heavy background, and 5-10 ng/. Mu.L of probe hybridization solution has the best detection effect.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.
Claims (7)
1. An in situ hybridization probe set for Kappa and Lambda light chain mRNA detection, characterized in that the probe set comprises a probe i for detecting Kappa light chain mRNA and a probe ii for detecting Lambda light chain mRNA, the sequences of the probes i are as shown in SEQ ID NO:1-SEQ ID NO:3, the sequence of the probe II is SEQ ID NO:4-SEQ ID NO: shown at 6.
2. The set of in situ hybridization probes for Kappa and Lambda light chain mRNA detection according to claim 1, wherein digoxin is modified at the 5' carboxy terminus of both probe I and probe II.
3. The method for preparing an in situ hybridization probe set according to claim 1, comprising the steps of:
s1, preparing a probe I: diluting the probe I of the target Kappa light chain mRNA with the hybridization solution to obtain a probe I working solution;
s2, preparing a probe II: diluting a probe II of target Lambda light chain mRNA with the hybridization solution to form a probe II working solution;
s3, preparing an in situ hybridization probe set: and (3) the prepared probe I working solution and probe II working solution are mixed according to the volume ratio of 1:1, mixing.
4. The method for preparing an in situ hybridization probe set according to claim 3, wherein in the steps S1 and S2, the hybridization solution comprises the following components: sodium citrate buffer salts, non-specific blockers, sodium dodecyl sulfate, and salmon sperm deoxyribonucleic acid.
5. The method for detecting Kappa and Lambda light chain mRNA by the in situ hybridization probe set according to claim 1, comprising the steps of:
(1) Preparing a color development working solution;
(2) Detecting in situ hybridization of Kappa light chain mRNA probes;
(3) In situ hybridization of Lambda light chain mRNA probes was detected.
6. Use of the in situ hybridization probe set of claim 1 for simultaneous detection of mRNA levels of human immunoglobulin Kappa and Lambda using an in situ hybridization method.
7. An in situ hybridization assay kit for detecting Kappa and Lambda light chain mRNAs, comprising the in situ hybridization probe set according to claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311325908.5A CN117051114A (en) | 2023-10-13 | 2023-10-13 | In situ hybridization probe set for Kappa and Lambda light chain mRNA detection |
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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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| WO2002083941A2 (en) * | 2001-04-11 | 2002-10-24 | Histaggen Gmbh & Co. Kg | Highly specific detection system for detecting nucleotide sequences coding for lambda and kappa light chains |
| CN110257483A (en) * | 2019-07-10 | 2019-09-20 | 厦门通灵生物医药科技有限公司 | Hybridization solution, preparation method and detection kit in situ hybridization |
| 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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