CN117305458B - Use of TKTmRNA detection reagent in preparation of multiple myeloma screening kit and kit - Google Patents
Use of TKTmRNA detection reagent in preparation of multiple myeloma screening kit and kit Download PDFInfo
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- CN117305458B CN117305458B CN202311366926.8A CN202311366926A CN117305458B CN 117305458 B CN117305458 B CN 117305458B CN 202311366926 A CN202311366926 A CN 202311366926A CN 117305458 B CN117305458 B CN 117305458B
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- 206010035226 Plasma cell myeloma Diseases 0.000 title claims abstract description 57
- 208000034578 Multiple myelomas Diseases 0.000 title claims abstract description 47
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 44
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 238000012216 screening Methods 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 20
- 210000004180 plasmocyte Anatomy 0.000 claims abstract description 15
- 238000011529 RT qPCR Methods 0.000 claims description 9
- 201000010099 disease Diseases 0.000 abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 11
- 102000018358 immunoglobulin Human genes 0.000 abstract description 11
- 108020004999 messenger RNA Proteins 0.000 description 13
- 201000000050 myeloid neoplasm Diseases 0.000 description 10
- 238000003745 diagnosis Methods 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 101150024271 TKT gene Proteins 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
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- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
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- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
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- 150000007523 nucleic acids Chemical class 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 206010006002 Bone pain Diseases 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 208000010190 Monoclonal Gammopathy of Undetermined Significance Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to the field of bioscience, and particularly relates to application of TKTmRNA detection reagent in preparation of a multiple myeloma screening kit and the kit. The present invention for the first time found that TKTmRNA levels in bone marrow plasma cells of patients with multiple myeloma were significantly higher than those of patients with monoclonal immunoglobulin diseases (MGUS) of unknown significance and healthy controls. The reagent for detecting TKTmRNA levels is used for preparing the multiple myeloma screening kit, so that the multiple myeloma can be effectively screened.
Description
Technical Field
The invention belongs to the field of medical science, and particularly relates to application of TKTmRNA detection reagents in preparation of a multiple myeloma screening kit and the kit.
Background
Multiple Myeloma (MM) is a malignant plasma cytopathy characterized by abnormal proliferation of plasma cells in the bone marrow. The proliferated plasma cells produce monoclonal immunoglobulin, which causes damage to target organs, and causes bad conditions such as anemia, bone pain, bone destruction, and kidney function impairment, thus bringing heavy burden to the family and society of patients. The incidence rate of MM in China is about one ten thousandth, and the MM accounts for about 1% of all cancers.
Multiple myeloma is not clearly distinguished clinically from the unknown monoclonal immunoglobulin disease (MGUS), especially in the early stages of the disease. Meanwhile, due to the high heterogeneity of myeloma, diagnosis also becomes quite complex, bringing a great economic burden to patients and their families. Therefore, the development of a novel and effective myeloma diagnosis method has extremely important significance for early diagnosis of diseases, improvement of treatment effects and improvement of life quality of patients.
The specific function of the TKT gene, TKTmRNA, which is messenger RNA (mRNA) of the TKT gene transcribed from the TKT gene and carrying information encoding the TKT protein, is not completely understood. At present, the relevant report about the diagnosis and treatment of the parkinsonism by TKT is provided.
Currently, TKTmRNA is not known in the art relating to multiple myeloma.
Disclosure of Invention
The invention aims to provide an application of TKTmRNA detection reagent in preparing a multiple myeloma screening kit and the kit.
The invention provides an application of a reagent for detecting TKTmRNA levels in preparation of a multiple myeloma screening kit.
Further, the reagents for detecting TKTmRNA levels include real-time quantitative PCR detection reagents, northern Blot detection reagents, high throughput sequencing reagents, gene chip reagents, or nucleic acid Blot detection reagents.
Further, the reagent for detecting TKTmRNA level is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Further, the reagent for detecting TKTmRNA level is a Northern Blot detection reagent.
Further, the reagent for detecting TKTmRNA level is a reagent for detecting TKT mRNA in plasma cells in human bone marrow.
The invention also provides a multiple myeloma screening kit comprising reagents for detecting TKTmRNA.
Further, the reagents for detecting TKTmRNA levels include real-time quantitative PCR detection reagents, northern Blot detection reagents, high throughput sequencing reagents, gene chip reagents, or nucleic acid Blot detection reagents.
Further, the reagent for detecting TKTmRNA level is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Further, the reagent for detecting TKTmRNA level is a Northern Blot detection reagent.
Further, the reagent for detecting TKTmRNA level is a reagent for detecting TKTmRNA level in plasma cells in human bone marrow.
The invention has the beneficial effects that: the kit can screen the risk degree of multiple myeloma of a to-be-detected population by detecting the level of TKTmRNA in bone marrow: if TKTmRNA levels are low, the risk of multiple myeloma is low, and if TKTmRNA levels are high, the risk of multiple myeloma is high. The reagent for detecting TKTmRNA is used for preparing the multiple myeloma screening kit, and the kit has high specificity and high sensitivity and can realize the effective screening of multiple myeloma. The invention provides effective basis for patients to take relevant therapeutic measures or decisions, and has good clinical application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a graph showing results of TKTmRNA expression levels in patients with multiple myeloma and in unknown monoclonal immunoglobulin diseases (MGUS).
FIG. 2 is a graph of the expression levels of TKTmRNA between healthy controls (NC), unidentified monoclonal immunoglobulin diseases (MGUS) and Multiple Myeloma (MM) in the GSE6477 database.
Fig. 3 ROC profile for healthy controls (NC) in gse6477 database as control.
Figure 4 ROC profile of unknown monoclonal immunoglobulin disease (MGUS) in gse6477 database as control.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
MGUS refers to an unknown monoclonal immunoglobulin disease (Monoclonal Gammopathy of Undetermined Significance). This is a benign plasmacytoma characterized by low levels of M protein in the blood, but without causing significant signs or symptoms. MGUS may develop multiple myeloma.
Example 1 real-time quantitative PCR (qPCR) detection of TKTmRNA levels in bone marrow plasma cells in relation to multiple myeloma
1. Experimental method
Sample collection and processing
1. Sample collection
The study subjects were bone marrow samples collected from 7 patients with unknown monoclonal immunoglobulin disease (MGUS) and 12 patients with multiple myeloma. Diagnosis of MGUS and multiple myeloma, both recruited from the people hospitals in the province of Sichuan, were diagnosed and confirmed according to the diagnostic criteria of the International Myeloma Working Group (IMWG) guidelines (2014 edition) and the Chinese multiple myeloma diagnosis guidelines (2022 revision). Basic information of the subjects is shown in table 1:
Table 1 basic information of subjects
| Basic information | MGUS | Multiple myeloma |
| Number of people (n) | 7 | 12 |
| Average age (Y) | 43 | 67 |
| Male proportion (%) | 57.1 | 75 |
2. Experimental treatment
2.1 Bone marrow sample collection
1-2Ml of patient bone marrow is collected by bone marrow puncture under the conventional sterile condition and is placed in an EDTA anticoagulated purple head blood collection tube, and the mixture is inverted and uniformly mixed.
2.2 Bone marrow plasma cell separation
To the EDTA anticoagulated bone marrow samples, 20. Mu.L of CD138+ magnetic bead reagent (Meitianfu Biotech Co.) was added to label the plasma cells, and the mixture was placed in a refrigerator at 4℃for 15min.
The magnetic bead sorting column is arranged on a sorter, 3ml of PBS buffer solution is added to the magnetic bead sorting column, and after PBS completely passes through the sorting column, incubated samples are added for sorting. The addition of 3ml of PBS buffer was repeated twice.
Taking down the separation column, adding 3ml PBS buffer solution, rapidly pushing the separation column plunger core rod for eluting, repeating the eluting for 3 times, and loading the eluent into a 10ml centrifuge tube for centrifugation (3000 r/min,10 min). After centrifugation 200. Mu.l of supernatant was kept with pellet, mixed and loaded into a 1.5ml EP tube, and the resulting sample was plasma cells isolated from bone marrow.
(II) mRNA nucleic acid extraction and amplification
MRNA of bone marrow plasma cells isolated in the above step was extracted using TRIzol, and mRNA concentration and purity were determined.
MRNA was synthesized into cDNA using a reverse transcription kit for mRNA whose concentration and purity were satisfactory.
Quantitative amplification analysis was performed on the synthesized cDNA using qPCR kit.
The relative expression level was calculated using the method 2 -ΔΔCq.
The primer sequences for TKT are as follows:
TKT forward 5'-3' (SEQ ID No. 1): ACTTCGACAAGGCCAGCTAC;
TKT reverse 5'-3' (SEQ ID No. 2): GCTGTGTCCATCCACGATGA.
2. Experimental results
The results of TKTmRNA level experiments on patients with multiple myeloma and unidentified monoclonal immunoglobulin diseases (MGUS) are shown in FIG. 1. It can be seen that the levels of isolated plasma cell (CD138+) detection TKTmRNA in multiple myeloma patients are significantly higher than those of TKTmRNA in monoclonal immunoglobulin disease (MGUS) patients of unknown significance.
Experimental results show that the invention detects the level of TKTmRNA in plasma cells isolated from patients through experiments, and proves that TKTmRNA can be used as a marker of multiple myeloma and can provide effective basis for patients to take relevant therapeutic measures or decisions.
Examples 2, TKTmRNA expression in GSE6477 (database)
1. Experimental method
By selecting myeloma study data in the database, TKTmRNA levels in myeloma patients were analyzed. Data GSE6477 from the GEO (Gene Expression Omnibus) database of NCBI. The study analyzed the differences in expression of TKTmRNA between healthy controls (NC), unidentified monoclonal immunoglobulin diseases (MGUS) and Multiple Myeloma (MM).
2. Experimental results
The results of analysis of TKT mRNA levels using GSE6477 database are shown in figure 2. Healthy control group (NC) 12 persons, MGUS group total 22 persons, multiple myeloma group 49 persons in GSE6477 database.
The average level of TKT mRNA in bone marrow plasma cells of patients with multiple myeloma was 11.15, and the average level of TKT mRNA in healthy control (NC) bone marrow was 9.75. The multiple myeloma group was statistically significant compared to the healthy control group (p < 0.0001). The ROC assay results for the multiple myeloma group and the healthy control group showed a specificity of 91.67% and a sensitivity of 97.96%, indicating that TKT mRNA can specifically distinguish multiple myeloma from healthy control, as shown in fig. 3.
The average level of TKTmRNA in bone marrow of MGUS patients was 9.40. The multiple myeloma group was statistically significant compared to the MGUS group (p < 0.0001). ROC analysis of the multiple myeloma group versus healthy control group resulted in a specificity of 95.83% and a sensitivity of 97.96%, indicating TKTmRNA can distinguish multiple myeloma from MGUS specifically, as shown in fig. 4.
Experimental results show that TKTmRNA can be used for auxiliary diagnosis of clinical multiple myeloma. The invention combines experiments and database analysis, and proves that TKTmRNA level in bone marrow plasma cells of patients with multiple myeloma is obviously higher than that of normal people and MGUS patients, TKTmRNA can be used as a marker of multiple myeloma, and provides effective basis for patients to take relevant therapeutic measures or decision.
In summary, the kit of the invention can screen the risk degree of multiple myeloma of the population to be tested by detecting the level of TKTmRNA in bone marrow: if TKTmRNA levels are low, the risk of multiple myeloma is low, and if TKT mRNA levels are high, the risk of multiple myeloma is high. The reagent for detecting TKTmRNA is used for preparing the multiple myeloma screening kit, and the kit has high specificity and high sensitivity and can realize the effective screening of multiple myeloma. The invention provides effective basis for patients to take relevant therapeutic measures or decisions, and has good clinical application prospect.
Claims (2)
1. Use of a reagent for detecting TKTmRNA levels in the preparation of a multiple myeloma screening kit, said reagent for detecting TKTmRNA levels comprising real-time quantitative PCR detection reagents, said reagent for detecting TKTmRNA levels being reagents for detecting TKTmRNA in plasma cells in human bone marrow.
2. The use according to claim 1, wherein the reagent for detecting TKTmRNA levels is a real-time quantitative PCR detection reagent comprising a primer sequence as shown in SEQ ID No.1 or SEQ ID No. 2.
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| AU2003286515A1 (en) * | 2002-10-21 | 2004-05-13 | Regents Of The University Of Minnesota | Transgenic non-human animals with expanded mature b cell and plasma cell populations |
| CN101999002A (en) * | 2008-02-04 | 2011-03-30 | 彼帕科学公司 | Methods of diagnosing and treating PARP-mediated diseases |
| CN103562404A (en) * | 2010-09-16 | 2014-02-05 | Cbs生物科学有限公司 | Composition or kit for making a prognosis of liver cancer, and method for making a prognosis of liver cancer |
| EP2652505A2 (en) * | 2010-12-13 | 2013-10-23 | Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. | Methods of diagnosing and treating pancreatic cancer |
| WO2014044848A1 (en) * | 2012-09-21 | 2014-03-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting multiple myeloma treatment response |
| WO2014124280A1 (en) * | 2013-02-08 | 2014-08-14 | Institute For Myeloma & Bone Cancer Research | Improved diagnostic, prognostic, and monitoring methods for multiple myeloma, chronic lymphocytic leukemia, and b-cell non-hodgkin lymphoma |
| WO2019171110A1 (en) * | 2018-03-04 | 2019-09-12 | Mazumdar Shaw Medical Foundation | Salivary protein biomarkers for the diagnosis and prognosis of head and neck cancers, and precancers |
| WO2020252303A1 (en) * | 2019-06-12 | 2020-12-17 | The Regents Of The University Of California | Engineered off-the-shelf immune cells and methods of use thereof |
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