阅读说明：本技术 定量检测oc-stamp基因表达水平的成套试剂及其应用 (Complete set of reagent for quantitatively detecting OC-STAMP gene expression level and application thereof ) 是由 阮国瑞 黄晓军 王子龙 周亚兰 吴利新 于 2019-11-14 设计创作，主要内容包括：本发明公开了定量检测OC-STAMP基因表达水平的成套试剂及其应用。所述成套试剂包括由引物OC-FP和引物OC-RP组成的引物对甲、探针甲、由引物ABL1-F和引物ABL1-R组成的引物对乙、探针乙、阳性对照和内参对照质粒。所述引物OC-FP如序列1所示,所述引物OC-RP如序列2所示,所述探针甲如序列3所示,所述引物ABL1-F如序列4所示,所述引物ABL1-R如序列5所示,所述探针乙如序列6所示。本发明提供的成套试剂可定量检测OC-STAMP基因的表达水平,用于多发性骨髓瘤患者辅助诊断、病程进展和/或复发及缓解深度的监测,将在医学检测领域发挥重要的作用。(The invention discloses a complete set of reagent for quantitatively detecting OC-STAMP gene expression level and application thereof. The kit comprises a primer pair A consisting of a primer OC-FP and a primer OC-RP, a probe A, a primer pair B consisting of a primer ABL1-F and a primer ABL1-R, a probe B, a positive control and an internal reference control plasmid. The primer OC-FP is shown as a sequence 1, the primer OC-RP is shown as a sequence 2, the probe A is shown as a sequence 3, the primer ABL1-F is shown as a sequence 4, the primer ABL1-R is shown as a sequence 5, and the probe B is shown as a sequence 6. The kit provided by the invention can quantitatively detect the expression level of the OC-STAMP gene, is used for the auxiliary diagnosis of multiple myeloma patients, and monitoring the disease progression and/or recurrence and remission depth, and plays an important role in the field of medical detection.)

1. A kit comprising a primer pair A and a probe A; the primer pair A consists of a primer OC-FP and a primer OC-RP;

the target sequence of the primer pair A contains a specific DNA fragment A; the specific DNA fragment A is a DNA molecule shown as a sequence 7 in a sequence table;

the probe A is a single-stranded DNA molecule consisting of 20-35 nucleotides, and is the same as or complementary with a partial segment in the specific DNA fragment A.

2. The kit of claim 1, wherein:

the primer OC-FP is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

the primer OC-RP is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the probe A is a single-stranded DNA molecule shown in a sequence 3 in a sequence table.

3. The kit of claim 1 or 2, wherein: the kit also comprises a primer pair B and a probe B; the primer pair B consists of a primer ABL1-F and a primer ABL 1-R;

the target sequence of the primer pair B contains a specific DNA fragment B; the specific DNA fragment B is a DNA molecule shown as a sequence 8 in a sequence table;

the probe B is a single-stranded DNA molecule consisting of 20-35 nucleotides, and is the same as or complementary with a partial section in the specific DNA fragment B.

4. The kit of claim 3, wherein:

the primer ABL1-F is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

the primer ABL1-R is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;

the probe B is a single-stranded DNA molecule shown as a sequence 6 in the sequence table.

5. The kit of any one of claims 1 to 4, wherein: the kit further comprises a positive control and/or an internal reference control plasmid;

the positive control is cDNA of bone marrow mononuclear cells of patients with multiple myeloma;

the internal reference plasmid is a recombinant plasmid obtained by inserting a DNA molecule shown as a sequence 8 in a sequence table into a cloning vector or an expression vector.

6. Use of a kit according to any one of claims 1 to 5 in the manufacture of a product; the function of the product is at least one of the following K1) -K8):

K1) diagnosing or aiding in the diagnosis of multiple myeloma;

K2) diagnosing or aiding in diagnosing whether the subject is a multiple myeloma patient;

K3) identifying or assisting in identifying whether the test cell is a multiple myeloma cell;

K4) detecting the OC-STAMP gene;

K5) detecting the expression level of the OC-STAMP gene;

K6) predicting or aiding in predicting the efficacy of treatment for multiple myeloma;

K7) predicting or aiding in predicting progression and/or recurrence of multiple myeloma;

K8) predicting or aiding in predicting the depth of remission of multiple myeloma.

7. A product comprising a kit of parts according to any one of claims 1 to 5.

8. The product of claim 7, wherein: the function of the product is at least one of the following K1) -K8):

K1) diagnosing or aiding in the diagnosis of multiple myeloma;

K2) diagnosing or aiding in diagnosing whether the subject is a multiple myeloma patient;

K3) identifying or assisting in identifying whether the test cell is a multiple myeloma cell;

K4) detecting the OC-STAMP gene;

K5) detecting the expression level of the OC-STAMP gene;

K6) predicting or aiding in predicting the efficacy of treatment for multiple myeloma;

K7) predicting or aiding in predicting progression and/or recurrence of multiple myeloma;

K8) predicting or aiding in predicting the depth of remission of multiple myeloma.

Application of OC-STAMP gene as a marker in preparation of a reagent for diagnosing or assisting in diagnosing multiple myeloma.

10. The specific DNA fragment A of any one of claims 1 to 5.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a complete set of reagents for quantitatively detecting OC-STAMP gene expression level and application thereof, in particular to a primer pair and a probe for quantitatively detecting OC-STAMP gene expression level and application thereof in multiple myeloma diagnosis.

Background

Multiple Myeloma (MM) is the second most common hematological malignancy that originates from plasma cells, accounting for 10-15% of all hematological tumors. Myeloma cells proliferate maliciously in the bone marrow and secrete monoclonal immunoglobulins, which can cause organ and tissue damage. Bone marrow infiltration can lead to suppression of normal hematopoiesis and thus to symptoms of anemia, infection, and hemorrhage. Infiltration in bone can stimulate osteoclasts to enhance their osteolytic action, which can lead to bone pain, bone defects, hypercalcemia, and pathological fractures. The disease is well developed in the elderly, the average age of the disease is about 70 years old, and the proportion of male and female is (1.6-3): 1. The current treatment of MM is predominantly drug therapy. In terms of prognosis stratification and curative effect judgment, cytogenetic indexes and biochemical indexes are often relied on, and specific molecular biological markers are lacked.

OC-STAMP (osteo plastic proteins-membrane protein) is a novel gene found in osteoclasts, whose expression is up-regulated during differentiation of monocytes into osteoclasts. The gene expression product of OC-STAMP has high similarity with DC-STAMP (cell Expressed derived Transmembrane protein) protein family, is conserved at the carboxyl terminal, and has a main function as a key molecule in the process of fusing and differentiating osteoclasts. Recent studies have shown that the OC-STAMP gene encodes a cell membrane-anchored receptor under induction of RANKL (receptor activator of nuclear factor- κ B ligand) and interacts with DC-STAMP during monocyte fusion to form osteoclasts.

Disclosure of Invention

One object of the present invention is to provide a kit for the aided diagnosis of multiple myeloma.

The kit for the auxiliary diagnosis of the multiple myeloma comprises a primer pair A and a probe A; the primer pair A consists of a primer OC-FP and a primer OC-RP;

the target sequence of the primer pair A contains a specific DNA fragment A; the specific DNA fragment A is a DNA molecule shown as a sequence 7 in a sequence table;

the probe A is a single-stranded DNA molecule consisting of 20-35 nucleotides, and is the same as or complementary with a partial segment in the specific DNA fragment A.

In the reagent set, the primer OC-FP is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

the primer OC-RP is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the probe A is a single-stranded DNA molecule shown in a sequence 3 in a sequence table.

The kit also comprises a primer pair B and a probe B; the primer pair B consists of a primer ABL1-F and a primer ABL 1-R;

the target sequence of the primer pair B contains a specific DNA fragment B; the specific DNA fragment B is a DNA molecule shown as a sequence 8 in a sequence table;

the probe B is a single-stranded DNA molecule consisting of 20-35 nucleotides, and is the same as or complementary with a partial section in the specific DNA fragment B.

In the kit, the primer ABL1-F is a single-stranded DNA molecule shown as a sequence 4 in a sequence table;

the primer ABL1-R is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;

the probe B is a single-stranded DNA molecule shown as a sequence 6 in the sequence table.

The kit also comprises a positive control plasmid and/or an internal reference control plasmid;

the positive control is cDNA of bone marrow mononuclear cells of patients with multiple myeloma;

the internal reference plasmid is a recombinant plasmid obtained by inserting a DNA molecule shown as a sequence 8 in a sequence table into a cloning vector or an expression vector. The cloning vector may specifically be the pMD18-T vector.

In the kit, the end of the probe A is provided with a fluorescent label; further, the 5 'end and the 3' end of the probe A are provided with fluorescent labels; furthermore, the 5 'end of the probe A is provided with a FAM fluorescent label, and the 3' end of the probe A is provided with a BHQ fluorescent label;

the tail end of the probe B is provided with a fluorescent label; further, the 5 'end and the 3' end of the probe B are provided with fluorescent labels; furthermore, the 5 'end of the probe B is provided with a FAM fluorescent label, and the 3' end of the probe B is provided with a BHQ fluorescent label.

In the above-mentioned kit of parts,

the molar ratio of the primer OC-FP, the primer OC-RP and the probe A can be 90:90: 25;

the molar ratio of the primer ABL1-F, the primer ABL1-R and the probe B can be 90:90: 25.

The preparation method of any one of the above-mentioned kits also belongs to the protection scope of the invention.

Any one of the kits described above can be prepared by packaging said primer OC-FP and/or said primer OC-RP and/or said probe A and/or said primer ABL1-F and/or said primer ABL1-R and/or said probe B and/or said positive control and/or said internal reference control plasmid separately.

The application of the kit in the preparation of products also belongs to the protection scope of the invention; the function of the product is at least one of the following K1) -K8):

K1) diagnosing or aiding in the diagnosis of multiple myeloma;

K2) diagnosing or aiding in diagnosing whether the subject is a multiple myeloma patient;

K3) identifying or assisting in identifying whether the test cell is a multiple myeloma cell;

K4) detecting the OC-STAMP gene;

K5) detecting the expression level of the OC-STAMP gene;

K6) predicting or aiding in predicting the efficacy of treatment for multiple myeloma;

K7) predicting or aiding in predicting progression and/or recurrence of multiple myeloma;

K8) predicting or aiding in predicting the depth of remission of multiple myeloma.

It is another object of the invention to provide a product.

The invention provides a product comprising the kit as described above.

The function of the product is at least one of the following K1) -K8):

K1) diagnosing or aiding in the diagnosis of multiple myeloma;

K2) diagnosing or aiding in diagnosing whether the subject is a multiple myeloma patient;

K3) identifying or assisting in identifying whether the test cell is a multiple myeloma cell;

K4) detecting the OC-STAMP gene;

K5) detecting the expression level of the OC-STAMP gene;

K6) predicting or aiding in predicting the efficacy of treatment for multiple myeloma;

K7) predicting or aiding in predicting progression and/or recurrence of multiple myeloma;

K8) predicting or aiding in predicting the depth of remission of multiple myeloma.

The product also comprises a device with the following data processing and conclusion display functions of X1), X2), X3), X4), X5), X6) or X7):

x1) or to aid in diagnosing whether the subject is a multiple myeloma patient: detecting the expression level of the OC-STAMP gene in the cDNA of the person to be detected and the cDNA of the normal person, and if the expression level of the OC-STAMP gene in the cDNA of the person to be detected is higher than that of the cDNA of the normal person, determining that the person to be detected is or is a candidate for a patient with multiple myeloma; if the expression level of the OC-STAMP gene in the cDNA of the testee is lower than that of the cDNA of the normal person, the testee is not or is not candidate to be a multiple myeloma patient;

x2) or to aid in diagnosing whether the subject is a multiple myeloma patient: detecting the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the person to be detected and the cDNA of the normal person, if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the person to be detected is higher than that of the cDNA of the normal person, the person to be detected is or is selected as a multiple myeloma patient; if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the testee is lower than that of the cDNA of the normal person, the testee is not or is not a candidate for the multiple myeloma patient;

x3) or to assist in identifying or identifying whether the test cell is a multiple myeloma cell: detecting the expression level of the OC-STAMP gene in the cDNA of the cell to be detected and the cDNA of the normal cell, and if the expression level of the OC-STAMP gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, determining that the cell to be detected is or is candidate to be a multiple myeloma cell; if the expression level of the OC-STAMP gene in the cDNA of the cell to be detected is lower than that of the cDNA of the normal cell, the cell to be detected is not or is not candidate to be the multiple myeloma cell;

x4) or to assist in identifying or identifying whether the test cell is a multiple myeloma cell: detecting the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected and the cDNA of the normal cell, and if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, determining that the cell to be detected is or is candidate to be a multiple myeloma cell; if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected is lower than that of the cDNA of the normal cell, the cell to be detected is not the multiple myeloma cell or the candidate cell is not the multiple myeloma cell;

x5) detecting the expression level of the OC-STAMP gene: comprises the steps of taking cDNA of a sample to be detected as a template, and adopting the primers in the reagent set to perform RQ-PCR amplification on the probe A and the primer A;

x6) detecting the relative expression level of the OC-STAMP gene reference internal reference gene: comprises the steps of using cDNA of a sample to be detected as a template, adopting a primer pair A and a probe A in the reagent set to carry out RQ-PCR amplification, and adopting a primer pair B and a probe B in the reagent set to carry out RQ-PCR amplification;

x7) predicting or aiding in predicting the depth of remission in patients with multiple myeloma: obtaining the expression level of the OC-STAMP gene in cDNA of a patient with multiple myeloma or the relative expression level of a reference gene of the OC-STAMP gene according to the method of X5) or X6): the lower the expression level of the OC-STAMP gene in the cDNA of the multiple myeloma patient or the relative expression level of the reference internal reference gene of the OC-STAMP gene, the deeper the remission depth of the multiple myeloma patient. The multiple myeloma patients are multiple myeloma patients who are initially diagnosed with multiple myeloma and relieved after treatment.

The application of the OC-STAMP gene as a marker in the preparation of a reagent for diagnosing or assisting in diagnosing multiple myeloma also belongs to the protection scope of the invention.

The following Y1) or Y2) or Y3) or Y4) or Y5) or Y6) or Y7) also belong to the scope of protection of the invention:

y1) method of diagnosing or aiding in the diagnosis of whether a subject is a multiple myeloma patient: detecting the expression level of the OC-STAMP gene in the cDNA of the person to be detected and the cDNA of the normal person, and if the expression level of the OC-STAMP gene in the cDNA of the person to be detected is higher than that of the cDNA of the normal person, determining that the person to be detected is or is a candidate for a patient with multiple myeloma; if the expression level of the OC-STAMP gene in the cDNA of the testee is lower than that of the cDNA of the normal person, the testee is not or is not candidate to be a multiple myeloma patient;

y2) method of diagnosing or aiding in the diagnosis of whether a subject is a multiple myeloma patient: detecting the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the person to be detected and the cDNA of the normal person, if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the person to be detected is higher than that of the cDNA of the normal person, the person to be detected is or is selected as a multiple myeloma patient; if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the testee is lower than that of the cDNA of the normal person, the testee is not or is not a candidate for the multiple myeloma patient;

y3) or a method for aiding in the identification or the identification of whether a test cell is a multiple myeloma cell: detecting the expression level of the OC-STAMP gene in the cDNA of the cell to be detected and the cDNA of the normal cell, and if the expression level of the OC-STAMP gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, determining that the cell to be detected is or is candidate to be a multiple myeloma cell; if the expression level of the OC-STAMP gene in the cDNA of the cell to be detected is lower than that of the cDNA of the normal cell, the cell to be detected is not or is not candidate to be the multiple myeloma cell;

y4) or a method for aiding in the identification or the identification of whether a test cell is a multiple myeloma cell: detecting the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected and the cDNA of the normal cell, and if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected is higher than that of the cDNA of the normal cell, determining that the cell to be detected is or is candidate to be a multiple myeloma cell; if the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected is lower than that of the cDNA of the normal cell, the cell to be detected is not the multiple myeloma cell or the candidate cell is not the multiple myeloma cell;

y5), the method comprises the steps of using cDNA of a sample to be detected as a template and adopting the primers in the reagent set to perform RQ-PCR amplification on the probe A and the A;

y6), the method comprises the steps of using cDNA of a sample to be detected as a template, adopting the primer in the reagent set to perform RQ-PCR amplification on the probe A and the primer in the reagent set, and adopting the primer in the reagent set to perform RQ-PCR amplification on the probe B and the primer in the reagent set;

y7) a method for predicting or aiding in predicting the depth of remission in a patient with multiple myeloma comprising the steps of: obtaining the expression level of the OC-STAMP gene in cDNA of a patient with multiple myeloma or the relative expression level of a reference gene of the OC-STAMP gene according to the method of X5) or X6): the lower the expression level of the OC-STAMP gene in the cDNA of the multiple myeloma patient or the relative expression level of the OC-STAMP gene reference internal reference gene is, the deeper the remission depth is. The multiple myeloma patients are multiple myeloma patients who are initially diagnosed with multiple myeloma and relieved after treatment.

The specific DNA fragment A also belongs to the protection scope of the invention.

The expression level of the OC-STAMP gene in the cDNA of any one of the testees is higher than that of the cDNA of a normal person, and specifically, the expression level of the OC-STAMP gene in the cDNA of the testee is obviously higher than that of the cDNA of the normal person.

The relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of any one of the testees is higher than that of the cDNA of a normal person, and specifically, the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the testee is obviously higher than that of the cDNA of the normal person.

The expression level of the OC-STAMP gene in the cDNA of any one of the cells to be detected is higher than that of the cDNA of a normal cell, and specifically, the expression level of the OC-STAMP gene in the cDNA of the cell to be detected is obviously higher than that of the cDNA of the normal cell.

The relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of any one of the cells to be detected is higher than that of the cDNA of a normal cell, and particularly, the relative expression level of the OC-STAMP gene reference internal reference gene in the cDNA of the cell to be detected is obviously higher than that of the cDNA of the normal cell.

The relative expression level of any one of the OC-STAMP gene reference internal reference genes can be specifically the ratio of the expression level of the OC-STAMP to the expression level of the internal reference gene.

The expression level of any one of the OC-STAMP genes and the expression level of any one of the reference genes can be copy numbers obtained according to a standard curve and a CT value.

The reference gene can be human ABL1 gene and the like.

The OC-STAMP gene sequence is shown as a sequence 9 in a sequence table.

The applicant designs a primer pair and a probe for quantitatively detecting the OC-STAMP gene, verifies the result in a large number of MM patients, and shows that the expression level of the OC-STAMP gene can be used for diagnosis/auxiliary diagnosis, disease progression/recurrence and remission depth monitoring of multiple myeloma patients, thereby playing an important role in the field of medical detection.

Drawings

FIG. 1 shows the standard curve of fluorescence of internal reference plasmid RQ-PCR.

FIG. 2 shows RQ-PCR fluorescence standard curve of positive control cDNA.

FIG. 3 shows the expression of the OC-STAMP gene in MM disease status specimens and healthy control specimens.

FIG. 4 is a graph of the relationship between changes in the expression level of the OC-STAMP gene and the disease state of a patient.

FIG. 5 is a graph showing the relationship between the expression level of the OC-STAMP gene and the depth of remission.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The biomaterials, reagents and their sources in the following examples are as follows:

kits are products of Invitrogen corporation; RNAsin is a product of huamei biotechnology corporation; dNTPs are products of Pharmecia; Mo-MLV reverse transcriptase and 5 × Standard buffer are both products of Promega corporation; the DNA ligase is a product of Takara corporation; universal PCR Master mix is a product of Tiangen Biotechnology Ltd; phusion High-Fidelity PCR Kit is a product of NewEngland Biolabs; RPMI-8226 cells are products of American ATCC (American Standard Biometrics Collection), catalog number CCL-155; u266 cells are the product of ATCC in the United states, Cat No. TIB-196; kasumi cells and HEL cells are both products of Shanghai Bye Biotech, Inc.; the K562 cells are products of Kunming cell banks of Chinese academy of sciences; 6T-CEM cells are the product of ATCC in the United states with product catalog number CCL-119; SUP-B15 is the American ATCC product, catalog number CRL-1929^TM(ii) a BALL-1 cells are the product of Baina organisms, Cat # BNCC 339548; nalm-6 cells are a product of Baina organisms, with the cargo number BNCC 338256; ramos cells are American ATCC products having catalog number CRL-1596^TM(ii) a Raji cells are a product of Beijing Xin Tang Biotech, Inc., catalog number CL 009.

The following examples refer to multiple myeloma diagnosis and staging criteria: greipp PR, San MiguelJ, Durie BG, et al. International stabilizing System for Multiple Myeloma. journal of clinical Oncology,2005,15: 3412-. Evaluation criteria of efficacy (including grouping criteria of depth of remission) refer to literature: the method of Durie BG, Harousseau JL, Miguel JS, et al, International unifonm responsecriteria for multiple mylomas, Leukemia.2006,20: 1467-.

Data results in the following examples were statistically analyzed using SPSS22.0, Graphpad Prism 7. And (3) comparing the differences of the two groups of data, wherein chi-square test is adopted for classified variable data, t test is adopted for continuous variable data, and the difference is less than 0.05, so that the statistical significance is achieved.