CN110714081B

CN110714081B - Complete set of reagent for quantitatively detecting OC-STAMP gene expression level and application thereof

Info

Publication number: CN110714081B
Application number: CN201911111997.7A
Authority: CN
Inventors: 阮国瑞; 黄晓军; 王子龙; 周亚兰; 吴利新
Original assignee: Peking University People's Hospital (peking University Second Clinical Medical College)
Current assignee: Beijing Jinyu Medical Examination Laboratory Co ltd
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2022-09-27
Anticipated expiration: 2039-11-14
Also published as: CN110714081A

Abstract

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 the monitoring of the disease progression and/or recurrence and remission depth, and plays an important role in the field of medical detection.

Description

Complete set of reagent for quantitatively detecting OC-STAMP gene expression level and application thereof

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. Infiltration of bone marrow can lead to suppression of normal hematopoiesis and the appearance of anemia, infection, and bleeding. Infiltration in bone can stimulate osteoclasts to enhance their osteolytic effect, 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. Currently, the treatment of MM is dominated by 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 (Dendrocyte Expressed given vector 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 anchoring receptor under induction of RANKL (receptor activator of nuclear factor-. kappa.b ligand) and interacts with DC-STAMP during monocyte fusion to form osteoclasts.

Disclosure of Invention

It is an object of the present invention 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 segment 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 multiple myeloma patients;

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 the prediction of progression and/or relapse 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 diagnosing multiple myeloma;

K4) detecting the OC-STAMP gene;

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

K7) predicting or aiding in predicting progression and/or recurrence 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 carry out RQ-PCR amplification on the A and the probe A;

x6) detecting the relative expression level of the reference internal reference gene of the OC-STAMP 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 are 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 a 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, wherein 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, the cell to be detected is or is selected as 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) 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 is a RQ-PCR fluorescence standard curve of the 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 the change in the expression level of the OC-STAMP gene and the disease status 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 from Invitrogen; RNAsin is a product of huamei biotechnology corporation; dNTP is a product 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 New England 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 Bio-Tech Co., Ltd; 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 a product of banna 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 Miguel J, 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 response criterion 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.

Example 1 design and Synthesis of specific primer pairs and probes

Design of specific primer pair and probe

A specific primer pair A (consisting of an upstream primer OC-FP and a downstream primer OC-RP, the size of an amplification product is 101bp, and the nucleotide sequence of the amplification product is shown as a sequence 7) designed aiming at an OC-STAMP gene (NCBI gene bank serial number: NM-080721.3) and a probe A (probe OC-T-probe):

an upstream primer OC-FP: 5'-TGTGGACTGGGCTCAGAAGTT-3' (SEQ ID NO: 1 of the sequence Listing);

the downstream primer OC-RP: 5'-TTCATCCCTTTCCTCTTCAACC-3' (SEQ ID NO: 2 of the sequence Listing);

probe OC-T-probe: FAM-CACGGTCAAGTATGATGTGGCATACACTGTC-BHQ (the nucleotide sequence is the sequence 3 in the sequence table).

A specific primer pair B (consisting of an upstream primer ABL1-F and a downstream primer ABL1-R, wherein the size of an amplification product is 124bp, and the nucleotide sequence of the amplification product is shown as a sequence 8) and a probe B (a probe ABL1-T-probe) which are designed aiming at an ABL1 gene (an internal reference gene; an NCBI gene bank serial number: NM-005157):

an upstream primer ABL 1-F: 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' (SEQ ID NO: 4 of the sequence Listing);

the downstream primer ABL 1-R: 5'-GATGTAGTTGCTTGGGACCCA-3' (SEQ ID NO: 5 of the sequence Listing);

probe ABL 1-T-probe: FAM-CCATTTTTGGTTTGGGCTTCACACCATT-BHQ (the nucleotide sequence is the sequence 6 in the sequence table).

Second, synthesis of specific primer pairs and probes

And (3) respectively synthesizing the specific primer pair A, the probe A, the specific primer pair B and the probe B which are designed in the step one. Specific primer pairs B and Probe B can be found in the references "Gabert J, Beillard E, van der Velden VH, et al.Standard differentiation and quality control primers of 'real-time' quantitative reverse transcription polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe age Cancer promoter. Leukemia.2003, 17: 2318-57".

Example 2 detection of sensitivity of specific primer pairs and probes to Positive control cells

First, preparation of relevant Positive control/plasmid

1. Preparation of Positive control cDNA (cDNA containing OC-STAMP Gene fragment)

cDNA of bone marrow mononuclear cells of multiple myeloma patients with positive OC-STAMP gene expression is used as a positive control.

2. Preparation of internal reference control plasmid (plasmid containing ABL1 Gene fragment)

Taking cDNA of bone marrow mononuclear cells of normal people (volunteers) as a template, adopting an upstream primer: 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' and the downstream primer: 5'-GATGTAGTTGCTTGGGACCCA-3' PCR amplification, the nucleotide sequence of the amplification product is shown as sequence 8 in the sequence list (124bp), the amplification product is cloned into pMD18-T plasmid after purification, the recombinant plasmid is transformed into Escherichia coli DH5 alpha competence, the positive clone is screened to extract plasmid and purified for sequencing, the plasmid with correct sequencing is recorded as internal reference control plasmid.

The internal reference plasmid is a vector obtained by inserting a DNA molecule shown in a sequence 8 into ECOR V enzyme cutting sites of the pMD18-T plasmid.

3. Negative control plasmid

pMD18-T plasmid.

Second, positive control cDNA sensitivity detection

The positive control cDNA obtained in step one was diluted with sterile double-distilled water for injection in 10-fold gradient to obtain each dilution (10 per microliter each) ⁴ 、10 ³ 、10 ² 、10 ¹ 、10 ⁰ One copy of the OC-STAMP gene fragment); the reference control plasmid obtained in example 2 was diluted with sterile double-distilled water for injection in 10-fold gradient to give each dilution (10 per microliter) ⁶ 、10 ⁵ 、10 ⁴ 、10 ³ 、10 ² 、10 ¹ 、10 ⁰ Single copy ABL1 gene fragment); the copy number of the OC-STAMP gene fragment or the ABL1 gene fragment was calculated by measuring the absorbance value.

Each positive control cDNA dilution was subjected to RQ-PCR using the specific primer set A obtained in example 1 and the probe A on a fluorescent real-time quantitative PCR instrument (7500-FAST model ABI, USA). The internal control plasmids were subjected to RQ-PCR using the specific primer set B and probe B obtained in example 1 on a fluorescent real-time quantitative PCR apparatus (7500-FAST model ABI, USA).

PCR reaction (10. mu.l): upstream primer 0.9. mu.M, downstream primer 0.9. mu.M, probe 0.25. mu.M, 2 × TaqMan Universal PCR public System 5. mu.l (ABI, USA), plasmid 1. mu.l; the balance of deionized water.

And (3) PCR reaction conditions: 2min at 50 ℃ for 1 cycle; 10min at 95 ℃ for 1 cycle; 95 ℃ for 15s, 60 ℃ for 1min, 40 cycles.

Internal reference control plasmid RQ-PCR fluorescence standard curve as shown in FIG. 1 (threshold 0.082), function log ₁₀ ABL1 is (Ct-38.57)/-3.3, the correlation coefficients are all above 0.99, and the sensitivity for detecting the internal reference gene is 10 copies. RQ-PCR fluorescence standard curve of positive control cDNA is shown in FIG. 2 (threshold 0.082) and function log ₁₀ The OC-STAMP is (Ct-41.37)/-3.6, the correlation coefficients are all over 0.99, and the sensitivity for detecting OC-STAMP gene fragments can reach 10 copies.

Example 3 detection of cell lines and multiple myeloma patients

First, assembly of kit

The kit consists of the following components: the specific primer pair A and the probe A aiming at the OC-STAMP gene, the specific primer pair B and the probe B aiming at the ABL1 gene, which are prepared in the example 1; positive control cDNA and internal control plasmid prepared in example 2.

Secondly, applying the kit of the first step to detect the cell line

The expression level of the OC-STAMP gene was examined in each cell line separately. Each cell line sample was tested in duplicate 3 times. The method comprises the following specific steps:

1. total RNA from each cell was extracted and reverse transcribed to cDNA.

2. Using cDNA as a template, and using specific primers to carry out RQ-PCR on a first probe and a first probe on a fluorescent real-time quantitative PCR instrument (7500-FAST type of ABI company in America); RQ-PCR was performed on a fluorescent real-time quantitative PCR instrument (7500-FAST model, ABI, USA) using the cDNA as a template and the specific primer set B and the probe B. The threshold was fixed at 0.082 for each RQ-PCR batch.

The PCR reaction system and the PCR reaction conditions were the same as those in example 2.

A standard curve was prepared using the positive control cDNA and the internal control plasmid, respectively. The copy numbers of the OC-STAMP gene and the ABL1 gene in each cell line were obtained by comparison with the standard curve. The relative expression level (%) of the OC-STAMP gene was expressed as the ratio of the copy number of the OC-STAMP gene to the copy number of the ABL1 gene.

The results are shown in Table 1. The results show that: OC-STAMP is highly expressed in multiple myeloma cell lines (U266 and RPMI-8226), is expressed in lower amounts in other blood tumor cell lines, and is negative in relative expression levels in normal human bone marrow cells.

TABLE 1 relative expression levels of the OC-STAMP Gene in hematological tumor cell lines

Thirdly, the kit of the first step is applied to detect the multiple myeloma patient

Several patients with multiple myeloma and healthy volunteers were tested separately. The method comprises the following specific steps:

1. RNA from each bone marrow specimen (or RNA from a peripheral blood specimen) was extracted under sterile conditions using a TRIzol kit (Invitrogen, USA) with reference to the kit instructions, and reverse-transcribed into cDNA.

A standard curve was prepared using the positive control cDNA and the internal control plasmid, respectively. The copy numbers of the OC-STAMP gene and the ABL1 gene in each patient were obtained by comparison with a standard curve. The relative expression level (%) of the OC-STAMP gene was expressed as the ratio of the copy number of the OC-STAMP gene to the copy number of the ABL1 gene.

A total of 288 bone marrow specimens from 157 multiple myeloma patients were examined, of which 157 specimens were initially diagnosed, 99 specimens were obtained for remission (complete remission, very good partial remission and partial remission) after treatment, and 32 specimens were obtained for disease progression or recurrence. In addition, 42 bone marrow specimens of healthy volunteers were examined.

The method for detecting the relative expression levels of the respective genes was the same as in example 2. The results are shown in FIG. 3. The expression level of the OC-STAMP gene was significantly higher in the primary diagnosis specimens (median 0.50%, range 0-120.73%) and in the disease progression/recurrence specimens (median 0.25%, range 0-47.22%) than in the remission specimens (median 0.02%, range 0-4.69%, P <0.001) and healthy volunteer specimens (median 0.02%, range 0-0.10%, P < 0.001).

Example 4 correlation of expression level of OC-STAMP Gene with patient disease status

The expression level of the OC-STAMP gene was analyzed in several multiple myeloma patients as follows in step three of example 3: 99 sets of initial diagnosis-remission follow-on bone marrow specimens and 29 sets of initial diagnosis-remission-disease progression/relapse continuous follow-on bone marrow specimens (where remission specimens were from the remission specimens described above). The remission specimens were classified into 3 groups of complete remission (CR, N ═ 27), very good partial remission (VGPR, N ═ 33), and partial remission (PR, N ═ 39), depending on the depth of remission.

The results are shown in FIG. 4. The results show that: in the CR group, the expression level of OC-STAMP decreased in 26 patients when remission was obtained; in the VGPR group, the expression level of OC-STAMP decreased in 28 patients when remission was obtained; in the PR group, the expression level of OC-STAMP decreased in 32 patients when remission was obtained; the expression level of OC-STAMP increased in 27 patients at the time of disease progression/recurrence.

The above results indicate that the trend of the OC-STAMP gene expression level in the bone marrow follow-up sample of an individual patient is consistent with the disease state of the patient. The OC-STAMP gene expression level is reduced in the initial patients with Complete Remission (CR), Very Good Partial Remission (VGPR) and Partial Remission (PR) following therapeutic intervention. And the OC-STAMP gene expression level is increased in patients with disease progression/relapse.

Example 5 correlation between expression level of OC-STAMP Gene and depth of remission

99 remission samples from example 4 were analyzed as in step three of example 4. The remission specimens were divided into 3 groups of complete remission (CR, N ═ 27), very good partial remission (VGPR, N ═ 33), and partial remission (PR, N ═ 39), depending on the depth of remission.

The results are shown in FIG. 5. The results show that: the expression level of the OC-STAMP gene was lowest in the Complete Remission (CR) group (median 0.01%; range 0-0.07%) and significantly lower than that of the Very Good Partial Remission (VGPR) group (median 0.04%; range 0-4.69%; P ═ 0.0012) and Partial Remission (PR) group (median 0.06%; range 0-1.48%; P ═ 0.0002). However, no significant difference was observed in the expression level of OC-STAMP gene between the VGPR group and the PR group (P ═ 0.60).

The above results indicate that the expression level of the OC-STAMP gene decreases with the increase of remission depth of multiple myeloma.

Sequence listing

<110> Beijing university Hospital (second clinical medical college of Beijing university)

<120> reagent set for quantitatively detecting OC-STAMP gene expression level and application thereof

<160>9

<170>PatentIn version 3.5

<210>1

<211>21

<212>DNA

<213>Artificial Sequence

<400>1

tgtggactgg gctcagaagt t 21

<210>2

<211>22

<212>DNA

<213>Artificial Sequence

<400>2

ttcatccctt tcctcttcaa cc 22

<210>3

<211>31

<212>DNA

<213>Artificial Sequence

<400>3

cacggtcaag tatgatgtgg catacactgt c 31

<210>4

<211>31

<212>DNA

<213>Artificial Sequence

<400>4

tggagataac actctaagca taactaaagg t 31

<210>5

<211>21

<212>DNA

<213>Artificial Sequence

<400>5

gatgtagttg cttgggaccc a 21

<210>6

<211>28

<212>DNA

<213>Artificial Sequence

<400>6

ccatttttgg tttgggcttc acaccatt 28

<210>7

<211>101

<212>DNA

<213>Artificial Sequence

<400>7

tgtggactgg gctcagaagt tgccaactgt gcccatcacg ctcacggtca agtatgatgt 60

ggcatacact gtcctgggct tcatcccttt cctcttcaac c 101

<210>8

<211>124

<212>DNA

<213>Artificial Sequence

<400>8

tggagataac actctaagca taactaaagg tgaaaagctc cgggtcttag gctataatca 60

caatggggaa tggtgtgaag cccaaaccaa aaatggccaa ggctgggtcc caagcaacta 120

catc 124

<210>9

<211>2123

<212>DNA

<213>Artificial Sequence

<400>9

agggattccc aagtcccagc gattccccca cttcctctcc acctgccaca gctgccagcg 60

accgcccgcc tgaaaccact gccatttgga cagcatgcca ggccacccag gagcagctga 120

gcaacttgtc aagaccgggt ggaggtcctg gcacttgggg ttctggaagg cccttgcccc 180

actgcaggct gcctgggacg ccttctccca gcctgttcca gccagctgtg gccagctgct 240

gacccagctc ctcctgtgtg cctccctggc tgctgctgct gcaggtctgg tttatcactg 300

gctggcatcc ttgctgcttt atcctcctgg accttcagcc atggttgcca ctgtctgtgg 360

cctcctggtc ttcctgagcc tgggcctggt acccccagtc cgctgcctgt ttgcactcag 420

cgtgcccacc ctgggtatgg agcagggccg ccggctgctc ctgtcctaca gcactgccac 480

cctggccatt gctgtggtgc ccaacgtcct ggccaacgtg ggtgcggccg ggcaggtgct 540

gaggtgtgtc accgagggct ccctggagag tctcctcaat accactcacc agctgcatgc 600

agcatccagg gctctgggcc ccacaggcca ggcaggcagc cggggcctga catttgaggc 660

ccaggacaat ggctctgcct tctaccttca catgctcagg gtcactcagc aggtcctgga 720

ggatttctct ggcctggagt ccctggcccg ggcagcagcg ctagggaccc agcgagtggt 780

cacagggctg tttatgttgg gcctcctggt ggagtcggca tggtacctcc attgctacct 840

gacagacctg cggtttgaca atatctacgc cactcaacag ctgacccagc ggttggcaca 900

ggcccaggct acacacctcc tggcccctcc acccacctgg ctgctccagg cggctcagct 960

gaggctgtca caggaggagc tgttgagttg tcttctaagg ctggggctgc ttgccctgct 1020

cctcgtggcc acggctgtgg cggtggccac agaccatgta gccttcctcc tggcacaggc 1080

tactgtggac tgggctcaga agttgccaac tgtgcccatc acgctcacgg tcaagtatga 1140

tgtggcatac actgtcctgg gcttcatccc tttcctcttc aaccagctgg ctccggagag 1200

ccccttcctc tccgtccaca gctcctacca atgggagctc cgcctcacct ccgcccgctg 1260

cccactgcta cccgcccggc gtccccgcgc agctgccccg ctggccgcgg gggccctgca 1320

gctcctggcg ggctccacgg tgctcctgga ggcctacgcc cgccgcctgc ggcatgccat 1380

cgccgcttcc ttcttcacag cccaggaggc gaggagggtc cgccacctgc acgcccggct 1440

ccagcgaaga cacgacaggc accaaggcca gcagctgccc ctaggggatc cttcttgcgt 1500

ccccacaccc agacctgcct gcaagcctcc ggcatggata gactacaggc tggatgcctt 1560

aagaaccgag agcagtgagg gagaagggaa agagctttgg agttgcagag acctgagttg 1620

taaccttggt cctgtgccgc ctccctgtgt gaccttgggt aagtcacttc acctctctga 1680

gcctcggttt ctacatctgc ataacgacag catatttacc attgatgtga cctacttccc 1740

acgcagggat gtggtcagga tggaaggaaa tactgggcat gataggcctg gataaccggt 1800

aaagaaccat gcaaaggcga agacaaggag tgcagagaga gctcatggtt cctccaggct 1860

ggttggcgat caggctcatc tcatctgcac caactgctct acttgttaga tggagacctt 1920

gcatcatgaa tttctcgaaa tgctcctgga acttatttat atgcctcaaa atcctctaaa 1980

ctcatttata gtaacccata gttttaattt tataaataaa cgtatttatt aaatcttaga 2040

ttacgttatt cctctgcctc aaattccttc agggcctttc cattgccagg attagatttt 2100

gccaaattaa aatataggac act 2123

Claims

1. The use of a kit of reagents in the manufacture of a product; the function of the product is at least one of the following K1) -K6):

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

K4) predicting or aiding in predicting the efficacy of treatment for multiple myeloma;

K5) predicting or aiding in predicting progression and/or recurrence of multiple myeloma;

K6) predicting or aiding in predicting the depth of remission of multiple myeloma;

the kit comprises a primer pair A and a probe A; the primer pair A consists of a primer OC-FP and a primer OC-RP;

2. Use according to claim 1, characterized in that:

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

3. Use according to claim 1 or 2, characterized in that: 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;

4. Use according to claim 3, characterized in that:

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

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

5. Use according to claim 1, characterized in that: 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.OC-STAMPThe application of the gene as a marker in the preparation of a reagent for diagnosing or assisting in diagnosing multiple myeloma.