CN102242209B - Quantitative detection kit based on multiple genes for assisting diagnosis of patients with multiple myeloma - Google Patents

Abstract

The invention discloses a quantitative detection kit based on multiple genes for assisting diagnosis of patients with multiple myeloma. The invention provides a kit comprising a primer probe composition A (a primer pair A composed of nucleotide sequences as shown in sequences 1 and 2 and a probe A as shown in a sequence 3), a primer probe composition B (a primer pair B composed of nucleotide sequences as shown in sequences 4 and 5 and a probe B as shown in a sequence 6), a primer probe composition C (a primer pair C composed of nucleotide sequences as shown in sequences 7 and 8 and a probe C as shown in a sequence 9) and a primer probe composition D (a primer pair D composed of nucleotide sequences as shown in sequences 10 and 11 and a probe D as shown in a sequence 12). The invention provides a quick, reliable and accurate new approach for the diagnosis of multiple myeloma, provides a base for curative effect observation and dynamic observation of minimal residual diseases, and can play an important role in the field of medical detection.

Description

Immue quantitative detection reagent box based on a plurality of gene assisting to diagnose multiple myeloma patients

Technical field

The present invention relates to a kind of immue quantitative detection reagent box based on a plurality of gene assisting to diagnose multiple myeloma patients.

Background technology

Multiple myeloma (multiple myeloma, MM) is life-threatening plasmocyte malignant tumour, and in hematologic malignancies, sickness rate is positioned at second.Although the treatment of stem cell transplantation and newtype drug can make the state of an illness be effectively controlled, seldom there is the patient can obtain fully and alleviate even and cure, recurrence and dead final result are still inevitable.

The cognition that is people that molecular biological development makes increasing tumor-related gene, these important molecular markers have been brought into play great effect in the diagnosis of Malignancy and treatment, although found to relate to immunoglobulin gene rearrangement, C-MYC, RAS oncogene mutation and human telomerase reverse reversed transcriptive enzyme mRNA overexpression etc. in MM, lacked the molecular target of specific diagnosis, minimal residual disease detection and the prognosis evaluation of generally acknowledged Tumor-assaciated.

Cancer testis antigen (Cancer testis antigen, CT antigen) gene is the gene family relevant to malignant tumour, the coding immunogenic protein, identified in the past more than 40 family between 15 years, expression characteristic is restricted be expressed in healthy tissues testis, ovary and embryo trophocyte, other healthy tissuess are expressed hardly, and the expression of different frequency is arranged in the kinds of tumors tissue, and can induce the antibody-mediated and cell-mediated immune response of T.Expression characteristic based on it in tumour patient and healthy tissues and immunogenicity, CT antigen has been considered to have the target of the human malignancies immunotherapy of application prospect, and likely becomes important diagnosis and prognosis sign.

Summary of the invention

The purpose of this invention is to provide a kind of immue quantitative detection reagent box based on a plurality of gene assisting to diagnose multiple myeloma patients.

The invention provides a kind of assisting to diagnose multiple myeloma patient's test kit, comprise primer probe compositions first, primer probe compositions second and primer probe compositions third; Described primer probe compositions first is comprised of first and probe first Auele Specific Primer; Described primer probe compositions second is comprised of second and probe second Auele Specific Primer; Described primer probe compositions third by Auele Specific Primer to third and probe third form; The primer pair that DNA shown in the sequence 2 of DNA and sequence table shown in the sequence 1 that described Auele Specific Primer is sequence table to first forms; The nucleotide sequence of described probe first is as shown in the sequence 3 of sequence table; The primer pair that DNA shown in the sequence 5 of DNA and sequence table shown in the sequence 4 that described Auele Specific Primer is sequence table to second forms; The nucleotide sequence of described probe second is as shown in the sequence 6 of sequence table; The primer pair that described Auele Specific Primer forms for DNA shown in the sequence 8 of DNA shown in the sequence 7 of sequence table and sequence table third; The nucleotide sequence of described probe third is as shown in the sequence 9 of sequence table.

Described test kit specifically can be comprised of described primer probe compositions first, described primer probe compositions second and described primer probe compositions third.

Described test kit also can comprise primer probe compositions fourth; Described primer probe compositions fourth is comprised of fourth and probe fourth Auele Specific Primer; The primer pair that DNA shown in the sequence 11 of DNA and sequence table shown in the sequence 10 that described Auele Specific Primer is sequence table to fourth forms; The nucleotide sequence of described probe fourth is as shown in the sequence 12 of sequence table.

Described test kit specifically can be comprised of described primer probe compositions first, described primer probe compositions second, described primer probe compositions third and described primer probe compositions fourth.

Described test kit also can comprise positive plasmid first, positive plasmid second and positive plasmid third; Described positive plasmid first is for containing MAGE-C1/CT7 gene (NCBI gene pool sequence number: NM_005462) or the recombinant plasmid of its fragment; Described positive plasmid second is for containing MAGE-A3 gene (NCBI gene pool sequence number: NM_005362) or the recombinant plasmid of its fragment; Described positive plasmid third is for containing MAGE-C2/CT10 gene (NCBI gene pool sequence number: NM_016249) or the recombinant plasmid of its fragment.

Described test kit also cocoa comprises the positive plasmid fourth; Described positive plasmid fourth is for containing SSX-2 gene (NCBI gene pool sequence number: NM_175698/003147) or the recombinant plasmid of its fragment.

Described positive plasmid first can be the recombinant plasmid containing the MAGE-C1/CT7 gene fragment shown in the sequence 13 of ordered list.Described positive plasmid second can be the recombinant plasmid containing the MAGE-A3 gene fragment shown in the sequence 14 of ordered list.Described positive plasmid third can be the recombinant plasmid containing the MAGE-C2/CT10 gene fragment shown in the sequence 15 of ordered list.Described positive plasmid fourth can be the recombinant plasmid containing the SSX-2 gene fragment shown in the sequence 16 of ordered list.

Described positive plasmid first specifically can be the MAGE-C1/CT7 gene fragment shown in the sequence 13 of pMD18-T carrier and sequence table is connected to the recombinant plasmid obtained.Described positive plasmid second specifically can be the MAGE-A3 gene fragment shown in the sequence 14 of pMD18-T carrier and sequence table is connected to the recombinant plasmid obtained.Described positive plasmid third specifically can be the MAGE-C2/CT10 gene fragment shown in the sequence 15 of pMD18-T carrier and sequence table is connected to the recombinant plasmid obtained.Described positive plasmid fourth specifically can be the SSX-2 gene fragment shown in the sequence 16 of pMD18-T carrier and sequence table is connected to the recombinant plasmid obtained.

Described test kit also can comprise for the internal reference primer pair of reference gene and internal reference probe; Described reference gene is abl gene (NCBI gene pool sequence number: NM_005157).

Described internal reference primer pair specifically can be the primer pair that shown in the sequence 18 of DNA shown in the sequence 17 of sequence table and sequence table, DNA forms; The nucleotide sequence of described internal reference probe specifically can be as shown in the sequence 19 of sequence table.

Described test kit also can comprise the internal reference plasmid; Described internal reference plasmid is the recombinant plasmid that contains abl gene or its fragment.Described internal reference plasmid can be the recombinant plasmid containing the abl gene fragment shown in the sequence 20 of ordered list.Described internal reference plasmid specifically can be the abl gene fragment shown in the sequence 20 of pMD18-T carrier and sequence table is connected to the recombinant plasmid obtained.

Described primer probe compositions first, described primer probe compositions second and described primer probe compositions third can be used for preparing the test kit of assisting to diagnose multiple myeloma.

Described primer probe compositions first, described primer probe compositions second, described primer probe compositions third and described primer probe compositions fourth can be used for preparing the test kit of assisting to diagnose multiple myeloma.

42 routine MM patients being followed the tracks of to the detection of sample finds, its MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 gene 100% continuous expression all in expressing a certain CT antigen gene and the patient that fails to respond to any medical treatment, still have at least one of these three kinds of genes of 50% continuous expression in the effective patient for the treatment of, the expression of SSX-2 gene is unstable.The positive group factor of positive rate, expression level and the coexpression of MAGE-C1/CT7, MAGE-A3 and MAGE-C2/CT10 gene is all relevant to MM Bone Marrow of Patients plasmocyte digital display work, the expression variation of gene is consistent with the patient clinical course of disease, and wherein the variation of MAGE-C1/CT7 gene has statistical significance.The positive group factor of MAGE-C1/CT7, MAGE-A3 and MAGE-C2/CT10 gene expression dose and coexpression and the multinomial clinical indices significant correlation that prognosis meaning is arranged, and the high level expression of gene and survival of patients time shorten, overall survival rate descends relevant.Although the SSX-2 gene does not have direct correlation with multinomial clinical indices, can be used as supplementing of above three kinds of genes, increase the susceptibility that detects patient MM.

The expression level of MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 gene is the significant clinical indices of potential MM auxiliary diagnosis, prognosis evaluation and curative effect monitoring.The present invention, for the pathogenesis of understanding MM in depth, is familiar with its regularity of occurrence and development, improves China MM treatment level and has important value, also for seeking new MM targeted therapy strategy, lays the foundation.Test kit of the present invention, not only can be applied to qualitative assisting to diagnose multiple myeloma, can also pass through reference gene, measures the expression level of each gene of patient.The diagnosis that the present invention is multiple myeloma provide one fast, reliably, new way accurately, for the dynamic observation of observation of curative effect, minimal residual disease provides foundation.The present invention will play a significant role at the medical science detection field.

The accompanying drawing explanation

Fig. 1 is internal reference control plasmid RQ-PCR fluorescence standard curve.

The RQ-PCR fluorescence standard curve of the positive control plasmid first of Fig. 2.

The RQ-PCR fluorescence standard curve of the positive control plasmid second of Fig. 3.

The RQ-PCR fluorescence standard curve of the positive control plasmid third of Fig. 4.

The RQ-PCR fluorescence standard curve of the cDNA of the multiple myeloma patients that Fig. 5 is high expression level MAGE-C1/CT7 gene (volunteer).

The correlation results that Fig. 6 is each gene expression amount and plasmocyte number and CD38+/CD138+ plasmocyte number.

The correlation results of the expression amount that Fig. 7 is each gene and survival of patients time.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.

kits: purchased from Invitrogen company.RNAsin: purchased from magnificent biotech company.DNTP: purchased from Pharmecia company.Mo-MLV reversed transcriptive enzyme, 5 * standard buffer solution: purchased from Promega company.DNA ligase: purchased from TakaRa company.Universal PCR Master mix: purchased from sky root Bioisystech Co., Ltd.Phusion High-fidelity PCR Kit: purchased from New England Biolabs company.

Dami cell: purchased from Shanghai female willing biochemical industry company limited, Chinese cell bank classical collection cell centre cell catalogue: QK10058.K562 cell: purchased from Shanghai female willing biochemical industry company limited, Chinese cell bank classical collection cell centre cell catalogue: QK10269.MEG-01 cell: purchased from Shanghai female willing biochemical industry company limited, Chinese cell bank classical collection cell centre cell catalogue: QK10748.The RPMI8226 cell: purchased from U.S. ATCC (the biological product of USS collecting center), catalog number is CCL-155.The U266 cell: purchased from U.S. ATCC, catalog number is TIB-196.KM3 cell: purchased from Shanghai, visit Lik-Sang thing Science and Technology Ltd..The KG-1 cell: purchased from Chinese Academy of Sciences's Kunming cell bank, catalog number is KCB 200552YJ.The HL60 cell: purchased from Chinese Academy of Sciences's cell bank, catalog number is TCHu 23.Nalm-6 cell: purchased from Peking University's disease gene research centre.Cem cell: purchased from U.S. ATCC, catalog number is CCL-119.

Diagnosis of Multiple Myeloma reaches standard reference document by stages: Greipp PR, San Miguel J, Durie BG, et al.International Staging System for Multiple Myeloma.Journal of Clinical Oncology, 2005,15:3412-3420..The judgement criteria reference literature of curative effect: Durie BG, Harousseau JL, Miguel JS, et al.International uniform response criteria for multiple myeloma.Leukemia.2006,20:1467-1473..Statistical study application SPSS software v.13.0.Measurement data adopts variance analysis, and enumeration data adopts chi square test.Dependency between clinical data and CT antigen gene expression is analyzed with straight line correlation.P<0.05 is considered to that statistical significance is arranged.By the dependency of single factor and multiplicity factor of evaluation and existence, adopt the Log-rank check to identify the existence correlative factor, the variable of p<0.05 is included the Cox regression analysis in, and p<0.2 is defined as the factor relevant to the prognosis of surviving.

PCR parameter in embodiment 3 to 9 is all as follows:

PCR reaction system (10 μ l): upstream primer 0.9 μ M, downstream primer 0.9 μ M, probe 0.25 μ M, 2 * TaqMan universal PC R public system, 5 μ l (ABI company, the U.S.), plasmid 1 μ l; All the other are deionized water.

PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 1min, 50 circulations.

Embodiment 1, Auele Specific Primer are to the design with probe

MAGE-C1/CT7 gene (NCBI gene pool sequence number: NM 005462) is positioned at human chromosome Xq26, for MAGE-C1/CT7 gene design Auele Specific Primer to first (be comprised of upstream primer MAG-FP1 and downstream primer MAG-RP1, amplified production is 79bp) and probe first (probe MAG-T-probe1):

Upstream primer MAG-FP1 (sequence 1 of sequence table): 5 '-TTGTCTTCTGGGAACCTTGACTC-3 ';

Downstream primer MAG-RP1 (sequence 2 of sequence table): 5 '-TGAGGGACACATACATCCTAAAAGC-3 ';

Probe MAG-T-probe1 (5 ' → 3 '):

FAM-ACTGCCTGGGCCTCCTCTGCTGT-BHQ (nucleotides sequence is classified the sequence 3 of sequence table as).

MAGE-A3 gene (NCBI gene pool sequence number: NM 005362) is positioned at human chromosome Xq28, for the Auele Specific Primer of MAGE-A3 gene to second (be comprised of upstream primer MAG-FP2 and downstream primer MAG-RP2, amplified production is 172bp) and probe second (probe MAG-T-probe2):

Upstream primer MAG-FP2 (sequence 4 of sequence table): 5 '-GGTGAGGAGGCAAGGTTCTGA-3 ';

Downstream primer MAG-RP2 (sequence 5 of sequence table): 5 '-GTGCTGACTCCTCTGCTCAAGAG-3 ';

Probe MAG-T-probe2 (5 ' → 3 '):

FAM-AGATCTGCCAGTGGGTCTCCATTGCC-BHQ (nucleotides sequence is classified the sequence 6 of sequence table as).

MAGE-C2/CT10 gene (NCBI gene pool sequence number: NM 016249) is positioned at human chromosome Xq27, for MAGE-C2/CT10 gene design Auele Specific Primer to the third (be comprised of upstream primer MAG-FP3 and downstream primer MAG-RP3, amplified production is 137bp) and probe third (MAG-T-probe3):

Upstream primer MAG-FP3 (sequence 7 of sequence table): 5 '-GTGTGAGGCACACAGCCTAAAG-3 ';

Downstream primer MAG-RP3 (sequence 8 of sequence table): 5 '-GGAGGCATGACGACTTCTTCA-3 ';

Probe MAG-T-probe3 (5 ' → 3 '):

FAM-AGGAGTCAAGGCCTGTTGGATCTCATCA-BHQ (nucleotides sequence is classified the sequence 9 of sequence table as).

SSX-2 gene (NCBI gene pool sequence number: NM_175698/003147) be positioned at human chromosome Xp11.22, for SSX-2 gene design Auele Specific Primer to fourth (be comprised of upstream primer SSX-FP and downstream primer SSX-RP, amplified production is 110bp) and probe fourth (probe SSX-T-probe):

Upstream primer SSX-FP (sequence 10 of sequence table): 5 '-TAACCGTGGGAATCAGGTTGA-3 '

Downstream primer SSX-RP (sequence 11 of sequence table): 5 '-CCTCCGAATCATTTCCTTCCT-3 '

Probe SSX-T-probe (5 ' → 3 '):

FAM-CCGAAGATCATGCCCAAGAAGCCAG-BHQ (nucleotides sequence is classified the sequence 12 of sequence table as).

For ABL1 gene (reference gene; NCBI gene pool sequence number: NM 005157) design internal reference primer pair (be comprised of upstream primer ABL1-F and downstream primer ABL1-R, amplified production is 124bp) and internal reference probe (probe ABL1-T-probe):

Upstream primer ABL1-F (sequence 17 of sequence table): 5 '-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ';

Downstream primer ABL1-R (sequence 18 of sequence table): 5 '-GATGTAGTTGCTTGGGACCCA-3 ';

Probe ABL1-T-probe (5 ' → 3 '):

FAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA (nucleotides sequence is classified the sequence 19 of sequence table as).

Synthetic Auele Specific Primer is to first and probe first respectively, and Auele Specific Primer is to second and probe second, and Auele Specific Primer is to third and probe the third, and Auele Specific Primer is to fourth and probe fourth, internal reference primer pair and internal reference probe.(but reference is synthetic: Gabert J, Beillard E, van der Velden VH, et al.Standardization and quality control studies of ' real-time ' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program.Leukemia.2003,17:2318-57.).

The preparation of embodiment 2, relevant plasmid

One, the preparation of positive control plasmid first (plasmid that contains the MAGE-C1/CT7 gene fragment)

The cDNA of BMNC of multiple myeloma patients (volunteer) of the MAGE-C1/CT7 genetic expression positive of take is template, carry out pcr amplification, the nucleotide sequence of amplified production is as shown in the sequence 13 (632bp) of sequence table, after amplified production is purified, be cloned into pMD18-T plasmid (pMD18-T carrier system, Shanghai Hao Jia company, catalog number: D101A), by recombinant plasmid transformed bacillus coli DH 5 alpha (day root biochemical corp, catalog number: CD101-02) competence, screening positive clone is checked order after extracting plasmid purifying, result shows to have obtained positive control plasmid first, and (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 13 of ECOR V site).

Pcr amplification primer pair used is as follows:

CT7FP：5’-ACATCCTCACCCTCAGGAGGG-3’；

CT7RP：5’-GACGAGGATCGTCTCAGGTCAGC-3’。

Two, the preparation of positive control plasmid second (plasmid that contains the MAGE-A3 gene fragment)

The cDNA of BMNC of multiple myeloma patients (volunteer) of the MAGE-A3 genetic expression positive of take is template, carry out pcr amplification, the nucleotide sequence of amplified production is as shown in the sequence 14 of sequence table (423bp), after amplified production is purified, be cloned into the pMD18-T plasmid, by recombinant plasmid transformed bacillus coli DH 5 alpha competence, screening positive clone is checked order after extracting plasmid purifying, result shows to have obtained positive control plasmid second (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 14 of ECOR V site).

Pcr amplification primer pair used is as follows:

Upstream primer: 5 '-GAAGCCGGCCCAGGCTCG-3 ';

Downstream primer: 5 '-GGAGTCCTCATAGGATTGGCT-3 '.

Three, the preparation of positive control plasmid third (plasmid that contains the MAGE-C2/CT10 gene fragment)

The cDNA of BMNC of multiple myeloma patients (volunteer) of the MAGE-C2/CT10 genetic expression positive of take is template, carry out pcr amplification, the nucleotide sequence of amplified production is as shown in the sequence 15 of sequence table (884bp), after amplified production is purified, be cloned into the pMD18-T plasmid, by recombinant plasmid transformed bacillus coli DH 5 alpha competence, screening positive clone is checked order after extracting plasmid purifying, result shows to have obtained positive control plasmid third (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 15 of ECOR V site).

Pcr amplification primer pair used is as follows:

Upstream primer: 5 '-CGGATCGGA GGCATTTGTGAG-3 ';

Downstream primer: 5 '-ATGAACTCACGGGCTCTCTTGAG-3 '.

Four, the preparation of positive control plasmid fourth (plasmid that contains the SSX-2 gene fragment)

The cDNA of BMNC of multiple myeloma patients (volunteer) of the SSX-2 genetic expression positive of take is template, carry out pcr amplification, the nucleotide sequence of amplified production is as shown in the sequence 16 of sequence table (435bp), after amplified production is purified, be cloned into the pMD18-T plasmid, by recombinant plasmid transformed bacillus coli DH 5 alpha competence, screening positive clone is checked order after extracting plasmid purifying, result shows to have obtained positive control plasmid fourth (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 16 of ECOR V site).

Pcr amplification primer pair used is as follows:

Upstream primer: 5 '-GTGCTCAAATACCAGAGAAGATC-3 ';

Downstream primer: 5 '-TTTTGGGTCCAGATCTCTCGTG-3 '.

Five, the preparation of internal reference control plasmid (plasmid that contains the abl gene fragment)

Normal people's (volunteer) the cDNA of BMNC of take is template, carry out pcr amplification, the nucleotide sequence of amplified production is as shown in the sequence 8 of sequence table (124bp), after amplified production is purified, be cloned into the pMD18-T plasmid, by recombinant plasmid transformed bacillus coli DH 5 alpha competence, screening positive clone is checked order after extracting plasmid purifying, result shows to have obtained internal reference control plasmid (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 20 of ECOR V site).

Pcr amplification primer pair used is as follows:

Upstream primer: 5 '-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ';

Downstream primer: 5 '-GATGTAGTTGCTTGGGACCCA-3 '.

Six, negative control plasmid

The pMD18-T plasmid.

The sensitivity of embodiment 3, detection positive control plasmid detects

The positive control plasmid first that embodiment 2 is obtained is carried out gradient dilution and is obtained each diluent (concentration range of diluent is: every microlitre contains 10 with the two water for injection that steams of sterilizing ⁰to 10 ⁵the MAGE-C1/CT7 gene fragment of individual copy); The positive control plasmid second that embodiment 2 is obtained is carried out gradient dilution and is obtained each diluent (concentration range of diluent is: every microlitre contains 10 with the two water for injection that steams of sterilizing ⁰to 10 ⁷the MAGE-A3 gene fragment of individual copy); Positive control plasmid the third use sterilizing that embodiment 2 is obtained is two steams water for injection and carries out gradient dilution and obtain each diluent (concentration range of diluent is: every microlitre contains 10 ⁰to 10 ⁷the MAGE-C2/CT10 gene fragment of individual copy); The internal reference control plasmid that embodiment 2 is obtained carries out gradient dilution and obtains each diluent (concentration range of diluent is: every microlitre contains 10 with the two water for injection that steams of sterilizing ⁰to 10 ⁶the abl gene fragment of individual copy); Calculate the copy number of MAGE-C1/CT7 gene fragment, MAGE-A3 gene fragment, MAGE-C2/CT10 gene fragment, SSX-2 gene fragment or abl gene fragment by measuring absorbance).

The Auele Specific Primer that adopts embodiment 1 to obtain each positive control plasmid first diluent carries out RQ-PCR to first and probe first on the fluorescence real-time quantitative PCR instrument.The Auele Specific Primer that adopts embodiment 1 to obtain with diluent each positive control plasmid carries out RQ-PCR to second and probe second on the fluorescence real-time quantitative PCR instrument.The Auele Specific Primer that adopts embodiment 1 to obtain each positive control plasmid third diluent to third and probe third carry out RQ-PCR on the fluorescence real-time quantitative PCR instrument.The Auele Specific Primer that adopts embodiment 1 to obtain each positive control plasmid fourth diluent carries out RQ-PCR to fourth and probe fourth on the fluorescence real-time quantitative PCR instrument.The internal reference primer pair and the internal reference probe that adopt embodiment 1 to obtain each internal reference control plasmid carry out RQ-PCR on the fluorescence real-time quantitative PCR instrument.All adopt the American AB I 7500-FAST of company type fluorescence real-time quantitative PCR instrument.

The threshold value that the threshold value of ABL1 gene fragment, MAGE-A3 gene fragment, MAGE-C2/CT10 gene fragment and SSX-2 gene fragment amplification curve is decided to be to 0.082, MAGE-C1/CT7 amplification curve is decided to be 0.2.

Internal reference control plasmid RQ-PCR fluorescence standard curve is shown in Fig. 1 (threshold value is 0.082), and function is log ₁₀aBL1=(Ct-38.46)/-3.22, relation conefficient all reaches more than 0.99, and the sensitivity that detects reference gene reaches 10 copies.The RQ-PCR fluorescence standard curve of positive control plasmid first is shown in Fig. 2 (threshold value is 0.2), and function is log ₁₀mAGEC1/CT7=(Ct-31.232)/-3.281, relation conefficient all reaches more than 0.99, and the sensitivity that detects the MAGE-C1/CT7 gene fragment can reach 1 copy.The RQ-PCR fluorescence standard curve of positive control plasmid second is shown in Fig. 3 (threshold value is 0.082), and function is log ₁₀mAGE-A3=(Ct-41.83)/-3.57, relation conefficient all reaches more than 0.99, and the sensitivity that detects the MAGE-A3 gene fragment can reach 10 copies.The RQ-PCR fluorescence standard curve of positive control plasmid third is shown in Fig. 4 (threshold value is 0.082), and function is log ₁₀mAGE-C2/CT10=(Ct-40.41)/-3.43, relation conefficient all reaches more than 0.99, and the sensitivity that detects the MAGE-C2/CT10 gene fragment can reach 10 copies.

CDNA by a routine multiple myeloma patients (volunteer), with the two water for injection 1:10 gradient dilutions that steam of sterilizing, the Auele Specific Primer that adopts embodiment 1 to obtain carries out RQ-PCR (7500-FAST of American AB I company type) to fourth and probe fourth on the fluorescence real-time quantitative PCR instrument.Amplification curve shows and logarithm that Ct value and SSX-2 open beginning template amount to have linear dependence (relation conefficient is greater than 0.99, and detection sensitivity reaches 10 ^-4, threshold value is 0.082; See Fig. 5), its amplification efficiency and abl gene are close, for reducing experimental error, all with the ABL typical curve, calculate gene copy number, and the sensitivity that detects the SSX-2 gene fragment can reach 10 copies.

By the multiple myeloma patients cDNA of 7 MAGEC1/CT7 gene masculines, with sterilizing, two water for injection 1:10 that steam dilute, and are prepared into the dilute sample (10 of 6 series ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6diluted sample), the Auele Specific Primer that adopts embodiment 1 to obtain carries out RQ-PCR to first and probe first on the fluorescence real-time quantitative PCR instrument.The results are shown in Table 1.Result shows, applies Auele Specific Primer provided by the invention first and probe first are detected to the multiple myeloma patients of 7 routine MAGEC1/CT7 gene masculines by RQ-PCR, and sensitivity can reach 10 ^-4or 10 ^-5extent of dilution.

The amplification curve feature of 10 times of serial dilutions of table 1 multiple myeloma patients cDNA sample

Result shows, the real-time quantitative PCR that the present invention sets up detects the method for four kinds of gene expression doses, the plasmid sensitivity detected containing said gene cDNA fragment can reach 1～10 copy, the sensitivity detected in patient's sample can reach 10-4～10-5, more responsive, easy method, for further clinical application is laid a good foundation.

Embodiment 4, detection clone and multiple myeloma patients

One, the assembling of test kit

The test kit first is comprised of following assembly: embodiment 1 prepares Auele Specific Primer to first and probe first, internal reference primer pair and internal reference probe; Positive control plasmid first and the internal reference control plasmid of embodiment 2 preparations.

Test kit second is comprised of following assembly: embodiment 1 prepares Auele Specific Primer to second and probe second, internal reference primer pair and internal reference probe; Positive control plasmid second and the internal reference control plasmid of embodiment 2 preparations.

Test kit third is comprised of following assembly: embodiment 1 prepares Auele Specific Primer to third and probe third, internal reference primer pair and internal reference probe; Positive control plasmid third and the internal reference control plasmid of embodiment 2 preparations.

The test kit fourth is comprised of following assembly: embodiment 1 prepares Auele Specific Primer to fourth and probe fourth, internal reference primer pair and internal reference probe; Positive control plasmid fourth and the internal reference control plasmid of embodiment 2 preparations.

Two, the kit detection cell of applying step one system

Detect respectively the expression level of each gene in each clone.Each clone sample is duplicate detection 3 times all.Step is as follows:

1, extract total RNA of each cell, reverse transcription is cDNA.

2, take cDNA as template, with Auele Specific Primer, to first and probe first, (or Auele Specific Primer is to second and probe second, or Auele Specific Primer is to third and probe the third, or Auele Specific Primer is to fourth and probe fourth) carry out RQ-PCR on fluorescence real-time quantitative PCR instrument (7500-FAST of American AB I company type); Take cDNA as template, with internal reference primer pair and internal reference probe, on fluorescence real-time quantitative PCR instrument (7500-FAST of American AB I company type), carry out RQ-PCR.Analyze MAGE-C1/CT7 gene RQ-PCR as a result the time threshold value be (threshold value is 0.2); The RQ-PCR that analyzes MAGE-A3 gene, MAGE-C2/CT10 gene and SSX-2 gene as a result the time threshold value all to be fixed as threshold value be 0.082.

PCR reaction system and PCR reaction conditions are with embodiment 3.

Use respectively positive control plasmid and internal reference control plasmid production standard curve.The reference standard curve obtains the copy number of each gene and ABL1 gene in each clone.Relative expression's level (%) with copy number with each gene of ratio value representation of the copy number of ABL1 gene of each gene.Annotate: if the relative expression quantity detected result is greater than 0, positive result; Otherwise negative result, be designated as "-".

The results are shown in Table 2, table 3, table 4 and table 5.

Relative expression's level of table 2 MAGE-C1/CT7 gene in blood tumor cell system

Clone	Source	Relative expression's level of MAGE-C1/CT7 gene
			U266	Multiple myeloma patients	929.08
KG-1	Patients with acute myeloid leukemia
			HL60	Patients with acute myeloid leukemia
Nalm-6	B is Patients With Acute Lymphoblastic Leukemia	-

Relative expression's level of table 3 MAGE-A3 gene in blood tumor cell system

Clone	Source	Relative expression's level of MAGE-A3 gene
			RPMI8226	Multiple myeloma patients	438.70
U266	Multiple myeloma patients	7673.33
			KG-1	Patients with acute myeloid leukemia	-
HL60	Patients with acute myeloid leukemia
			Nalm-6	B is Patients With Acute Lymphoblastic Leukemia	-

Relative expression's level of table 4 MAGE-C2/CT10 gene in blood tumor cell system

Clone	Source	Relative expression's level of MAGE-C2/CT10 gene
			RPMI8226	Multiple myeloma patients	13.52
U266	Multiple myeloma patients	16.37
			KM3	Multiple myeloma patients	2.97
KG-1	Patients with acute myeloid leukemia	-
			HL60	Patients with acute myeloid leukemia	-
Dami	Patients with acute myeloid leukemia
			CEM	T cell Acute Lymphoblastic Leukemia patient
Nalm-6	B is Patients With Acute Lymphoblastic Leukemia

Relative expression's level of table 5 SSX-2 gene in blood tumor cell system

Clone	Source	Relative expression's level of SSX-2 gene
			RPMI8226	Multiple myeloma patients	0.04
U266	Multiple myeloma patients	22.63

KM3	Multiple myeloma patients	0.14
			K562	Chronic myelogenous leukemia	-
MEG-01	Chronic myelogenous leukemia	-
			HL60	Patients with acute myeloid leukemia

Three, the test kit of applying step one detects multiple myeloma patients

Respectively some multiple myeloma patients (volunteer) and other volunteers are detected.Step is as follows:

1, extract the RNA (or the RNA of peripheral blood sample also can) of each volunteer's bone marrow prepare under aseptic condition with TRIzol test kit (purchased from American I nvitrogen company) reference reagent box specification sheets, reverse transcription is cDNA.

2, take cDNA as template, with Auele Specific Primer, to first and probe first, (Auele Specific Primer is to second and probe second, Auele Specific Primer is to third and probe the third, or Auele Specific Primer is to fourth and probe fourth) carry out RQ-PCR on fluorescence real-time quantitative PCR instrument (7500-FAST of American AB I company type); Take cDNA as template, with internal reference primer pair and internal reference probe, on fluorescence real-time quantitative PCR instrument (7500-FAST of American AB I company type), carry out RQ-PCR.Analyze MAGE-C1/CT7 gene RQ-PCR as a result the time threshold value be (threshold value is 0.2); The RQ-PCR that analyzes MAGE-A3 gene, MAGE-C2/CT10 gene and SSX-2 gene as a result the time threshold value all to be fixed as threshold value be 0.082.

PCR reaction system and PCR reaction conditions are with embodiment 3.

Use respectively positive control plasmid and internal reference control plasmid production standard curve.The reference standard curve obtains the copy number of each gene and ABL1 gene in each patient.Relative expression's level (%) with copy number with each gene of ratio value representation of the copy number of ABL1 gene of each gene.The results are shown in Table 6.

Relative expression's level of table 6 each gene in patients with hematological tumor and Normal Human Bone Marrow

Annotate: if the relative expression quantity detected result is greater than 0, positive result; Otherwise negative result, be designated as "-".

In multiple myeloma patients marrow, the positive rate of MAGE-C1/CT7 gene, MAGE-A3 gene and MAGE-C2/CT10 gene and expression level are all higher than CML patient, AML patient and B-ALL patient, all negative in the detected result of 22 routine health donors.

Detect multiple myeloma cell line and other clone by Real-time quantitative PCR, just control the expression of each gene in multiple myeloma patients and other leukaemic, result shows multiple myeloma cell line and Bone Marrow Cells of Multiple Myeloma Patients high frequency expression said gene, in other clone and the extremely low rear not expression of patient's expression level, in the health donors medullary cell, express all negative, the prompting said gene is the oncogene relevant to multiple myeloma, is potential multiple myeloma molecular marker.

The dependency of embodiment 5, each gene and curative effect

One, the positive rate of each gene in different curative effect patients

Detect altogether 138 parts of sample of bone marrow (being numbered 1 to 138) of 138 routine multiple myeloma patients (volunteer).In 138 routine multiple myeloma patients, just control patient's 60 examples, patient's 78 examples after treatment.After treatment, in the patient, recurrent intractable patient 10 examples, treat effective patient (partial rcsponse and alleviation fully) 17 examples.

Relative expression's quantity measuring method of each gene is with the step 3 of embodiment 4.If the relative expression quantity detected result is greater than 0, positive result, be designated as+; Otherwise negative result, be designated as-.The results are shown in Table 7.

The result of each each gene test of sample of table 7

Patient's numbering	MAGE-C1/CT7	MAGE-A3	MAGE-C2/CT10	SSX-2
					1	25.64163	14.98714	0.44849	Negative
2	0.16736	Negative	Negative	Negative
					3	0.00136	Negative	0.00516	Negative
4	132.25307	36.23309	0.03843	1.21973
					5	11.24911	0.91551	0.08506	Negative
6	70.24984	0.05975	0.54303	Negative
					7	4512.81203	0.00066	0.00207	Negative
8	0.03037	0.00929	0.00284	Negative
					9	39.28703	45.51421	0.11249	0.35182
10	0.84757	0.00033	Negative	Negative
					11	4.97148	0.00779	0.07235	Negative
12	0.92908	1.31284	1.84152	Negative
					13	0.99267	9.15506	0.28008	Negative
14	0.02382	0.01661	Negative	Negative
					15	11.16666	0.56338	2.27944	Negative
16	1118.35418	5.19583	3.59675	0.26214
					17	211.48738	1.81462	9.15506	0.02821
18	94.28463	3.92870	46.87341	Negative
					19	Negative	Negative	Negative	Negative
20	38.39010	0.02279	0.23648	0.01939
					21	4.10094	0.02133	Negative	Negative
22	121.51115	0.00047	0.92908	Negative
					23	39.57711	14.44589	2.19712	0.04653
24	99.26705	0.22297	0.76171	0.01150
					25	137.20824	0.13323	0.30819	0.00455
26	143.40012	0.34925	1.44459	0.00572
					27	2.64074	0.06291	2.21334	Negative
28	228.94633	0.00005	0.06199	Negative
					29	5.29991	0.26602	0.28008	Negative

30	2.82149	0.01229	0.00462	0.00349
					31	6.45337	0.01543	0.00045	0.00244
32	1458.98811	0.05391	7.23477	Negative
					33	4.31421	Negative	0.00060	Negative
34	9.83261	0.09782	0.00808	Negative
					35	1515.48646	1.34213	16.98369	Negative
36	Negative	Negative	Negative	Negative
					37	Negative	Negative	Negative	Negative
38	291.48707	Negative	0.04452	Negative
					39	647.07866	1.22873	0.26407	Negative
40	31.32088	0.01532	0.08890	0.01828
					41	16.85920	8.07883	4.41944	0.03128
42	0.06819	0.00396	0.00160	0.00032
					43	281.05653	8.69555	37.86821	Negative
44	3502.77403	32.66311	10.60618	0.01053
					45	23.95407	0.00702	0.01015	Negative
46	533.34340	0.21810	0.43230	Negative
					47	Negative	0.00077	Negative	Negative
48	1426.29420	0.00349	0.69735	Negative
					49	21.64934	0.01061	0.00157	Negative
50	Negative	0.00370	Negative	Negative
					51	6017.20980	3.95771	6.33748	Negative
52	218.51581	0.00723	0.91551	0.00001
					53	Negative	Negative	0.00244	Negative
54	1018.40133	0.00037	0.00347	Negative
					55	18.41517	6.15372	0.05045	0.00010
56	47.77631	0.79608	4.72195	0.08824
					57	46.18881	5.71727	3.31716	1.56635
58	1.20109	0.84434	0.00324	0.01601
					59	8.15898	Negative	0.02508	Negative
60	403.06303	3.81478	0.18688	Negative
					61	2729.69013	23.99892	164.91205	0.82591
62	6961.09808	0.05273	0.85686	0.01566
					63	240.48650	0.50082	0.03623	Negative
64	3.83787	0.00066	0.52728	Negative
					65	2814.67782	52.34194	Negative	0.81385
66	27.39679	0.00964	0.07902	0.00054
					67	73.96216	44.19442	109.22918	0.00572
68	8.86751	Negative	0.00611	Negative
					69	358.89643	28.63314	0.73962	7.61709
70	3.15067	0.01256	0.00092	Negative
					71	0.00002	0.00002	Negative	0.00479
72	0.20715	0.01488	0.00411	Negative

73	4.55142	0.23132	0.23648	Negative
					74	Negative	Negative	2.47156	Negative
75	43.88365	Negative	0.00038	Negative
					76	0.09782	0.01068	Negative	Negative
77	3.90117	0.00125	Negative	0.00278
					78	0.10299	Negative	0.00116	Negative
79	1.96497	0.00187	0.04792	Negative
					80	2985.91262	0.16859	0.94285	Negative
81	0.00365	0.00035	Negative	Negative
					82	Negative	Negative	Negative	Negative
83	Negative	Negative	Negative	Negative
					84	Negative	Negative	Negative	Negative
85	Negative	Negative	Negative	Negative
					86	Negative	Negative	Negative	Negative
87	0.00002	Negative	Negative	Negative
					88	34.92785	0.00055	0.00723	Negative
89	4.32298	0.00838	0.35968	Negative
					90	5.10652	0.00131	0.02660	Negative
91	0.00839	Negative	Negative	Negative
					92	221.68875	0.00178	0.00023	Negative
93	0.00713	0.00242	0.00408	0.00032
					94	0.79926	0.00286	Negative	Negative
95	2.14916	0.05312	0.04863	Negative
					96	Negative	Negative	Negative	Negative
97	4.88480	0.03787	0.01092	Negative
					98	0.001382213	Negative	Negative	0.0002418
99	0.20715	0.45514	0.00329	Negative
					100	0.02724	Negative	0.00039	Negative
101	5.37453	Negative	Negative	Negative
					102	0.35442	Negative	Negative	Negative
103	0.00155	Negative	0.00019	Negative
					104	0.05120	0.00175	Negative	Negative
105	0.34925	Negative	Negative	Negative
					106	0.83201	0.01362	0.00354	Negative
107	104.51276	5.67536	2.47156	Negative
					108	2.67988	3.36633	0.99267	0.06245
109	30.25120	0.00220	0.01925	Negative
					110	35.18243	1.21973	0.12654	Negative
111	66.80336	0.01968	0.08696	Negative
					112	Negative	Negative	Negative	Negative
113	602.54637	0.02801	0.08020	Negative
					114	16.43746	0.00629	0.29488	0.00286
115	90.18490	3.49246	0.40759	0.01403

116	559.08268	0.04485	0.07288	0.05759
					117	2620.85047	22.46145	0.63375	0.00713
118	144.36035	Negative	0.00832	Negative
					119	9175.81844	2.05636	17.49087	0.12654
120	3313.15762	0.01466	0.00051	Negative
					121	0.17081	Negative	0.00264	Negative
122	0.00017	Negative	Negative	Negative
					123	0.01566	Negative	Negative	Negative
124	77.39907	0.00606	0.00247	Negative
					125	5.93082	0.00038	0.04387	Negative
126	128.89461	0.10763	Negative	Negative
					127	597.35160	41.66854	14.34001	0.03704
128	219.67207	0.03416	1.41306	Negative
					129	91.21475	0.52342	Negative	Negative
130	2.10658	0.01543	0.00217	Negative
					131	1.44925	0.01294	0.00313	Negative
132	0.03544	Negative	0.00066	Negative
					133	0.00006	0.00009	Negative	Negative
134	65.26743	0.33663	0.01499	0.00677
					135	20.86838	0.41363	Negative	0.02026
136	0.00513	Negative	Negative	Negative
					137	0.00006	Negative	Negative	Negative
138	0.00015	Negative	Negative	Negative

Carry out statistics as a result according to table 7, in MM patient, the positive rate of each genetic expression is in Table 8.

The positive rate of each genetic expression in table 8MM patient

Detected the expression of 138 routine MM Bone Marrow of Patients cell MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 gene, find that there is 92.8% patient (128/138) and at least express a kind of CT antigen gene, 70 examples just control and the recurrent intractable patient in this positive rate can reach 94.3% (66/70).Wherein MAGE-C1/CT7 is the CT antigen gene of common expression, just control and the recurrent intractable patient in positive rate reach 90% (63/70), the MAGE-A3 positive rate is 84.3% (59/70), the MAGE-C2 positive rate is 84.3% (59/70), the SSX-2 positive rate is 32.9% (23/70), and the positive rate of four kinds of CT antigen genes all significantly reduces in the effective patient for the treatment of.

Four kinds of CT antigen gene coexpression situations in above each Bone Marrow of Patients sample are analyzed, discovery 70 examples just control with the recurrent intractable patient in, the normal more CT antigen gene of coexpression, wherein four kinds of genes all the patient of positive expression account for 32.9%, the patient who expresses three kinds of genes accounts for 41.4%, express two kinds of genes, the patient that a kind of gene and four kinds of genes are not expressed only accounts for respectively 15.7%, 4.3% and 5.7%.

Two, the dependency of genetic expression and patient clinical index

Detect altogether the sample of bone marrow of 10 routine multiple myeloma patients (volunteer), the relative expression quantity of each gene (MAGE-C1/CT7/ABL, MAGE-A3/ABL, MAGE-C2/CT10/AB, SSX-2/ABL) detection method, with the step 3 of embodiment 4, is analyzed in conjunction with the multinomial clinical indices of each patient.The results are shown in Table 9.

The relation of table 9 genetic expression and patient clinical index

Result shows that the gene of four kinds of CT antigen coexpressions is more, and its state of an illness is more serious, expresses number and the multinomial clinical indices significant correlation of positive gene.The number of the CT antigen gene that can detect in prompting MM Bone Marrow of Patients can react the severity of disease to a certain extent.

Three, in patient's course of disease, the positive rate of each gene changes

Relative expression's quantity measuring method of each gene is with the step 3 of embodiment 4.If the relative expression quantity detected result is greater than 0, positive result; Otherwise negative result.

The 42 routine MAGE-C1/CT7 gene tests multiple myeloma of the positive are as a result just controlled patient (volunteer) and carried out tracing observation: 32 routine patients (76.2%) treatments effectively, this some patients were is after treatment effectively, and 16 routine patient (50%) MAGE-C1/CT7 gene test results are negative; 10 routine patient treatments invalid (23.8%), and MAGE-C1/CT7 gene test result is continuously the positive.

The 36 routine MAGE-A3 gene tests multiple myeloma of the positive are as a result just controlled patient (volunteer) and carried out tracing observation: 26 routine patients (72.2%) treatments effectively, this some patients were is after treatment effectively, and 13 routine patient (50%) MAGE-A3 gene test results are negative; 10 routine patient treatments invalid (27.8%), and MAGE-A3 gene test result is continuously the positive.

The 33 routine MAGE-C2/CT10 gene tests multiple myeloma of the positive are as a result just controlled patient (volunteer) and carried out tracing observation: 24 routine patients (72.7%) treatments effectively, this some patients were is at partial rcsponse or, after alleviating fully, 12 routine patient (50%) MAGE-C2/CT10 gene test results are negative; 9 routine patient treatments invalid (27.3%), and MAGE-C2/CT10 gene test result is continuously the positive.

The 18 routine SSX-2 gene tests multiple myeloma of the positive are as a result just controlled patient (volunteer) and carried out tracing observation: 13 routine patients (72.2%) treatments effectively, this some patients were is at partial rcsponse or after alleviating fully, 10 routine patient SSX-2 gene test results are continuously feminine gender, and 3 routine SSX-2 gene test results are continuously the positive; 5 routine patient treatments invalid (27.8%), wherein 3 routine SSX-2 gene test results are continuously feminine gender, and 2 routine SSX-2 gene test results are continuously the positive.

Result shows, MAGE-C1/CT7 gene, MAGE-A3 gene and MAGE-C2/CT10 gene continuous expression all in the patient that fails to respond to any medical treatment, these three kinds of genes of 50% continuous expression in the effective patient for the treatment of, these three kinds of genes of 100% continuous expression in the process of failing to respond to any medical treatment, the unstable expression of SSX-2 gene.

The dependency of embodiment 6, each gene expression amount and plasmocyte number and CD38+/CD138+ plasmocyte number

Relative expression's quantity measuring method of each gene is with the step 3 of embodiment 4.

The morphology passed through is added up the quantity per-cent that marrow plasmocyte in each patient's sample of bone marrow accounts for bone marrow nucleated cell.Account for the quantity per-cent of bone marrow nucleated cell by CD38+/CD138+ plasmocyte in each patient's of flow cytometer detection statistics sample of bone marrow.

130 routine MAGE-C1/CT7 gene tests as a result the multiple myeloma of the positive just control patient (volunteer) the results are shown in Figure 6.The horizontal marked positive correlation of the mrna expression of marrow plasmocyte number and MAGE-C1/CT7 gene (P<0.01, r=0.30).CD38+/CD138+ cell and the horizontal marked positive correlation of MAGE-C1/CT7 gene mRNA in marrow (P<0.01, r=0.27).

118 routine MAGE-A3 gene tests as a result the multiple myeloma of the positive just control patient (volunteer) the results are shown in Figure 7.The horizontal marked positive correlation of the mrna expression of marrow plasmocyte number and MAGE-A3 gene (P<0.01, r=0.18).CD38+/CD138+ cell and the horizontal marked positive correlation of MAGE-A3 gene mRNA in marrow (P<0.01, r=0.18).

117 routine MAGE-C2/CT10 gene tests as a result the multiple myeloma of the positive just control patient (volunteer) the results are shown in Figure 6.The horizontal marked positive correlation of the mrna expression of marrow plasmocyte number and MAGE-C2/CT10 gene (P<0.01, r=0.30).CD38+/CD138+ cell and the horizontal marked positive correlation of MAGE-C2/CT10 gene mRNA in marrow (P<0.01, r=0.33).

Embodiment 7, each gene and multiple myeloma ISS dependency by stages

Detection is the expression amount of each gene in multiple myeloma patients (volunteer) by stages in different I SS.Relative expression's quantity measuring method of each gene is with the step 3 of embodiment 4.If the relative expression quantity detected result is greater than 0, positive result; Otherwise negative result.The results are shown in Table 10 to table 13.

Expression amount and the multiple myeloma ISS relation by stages of table 10MAGE-C1/CT7 gene

ISS by stages

The positive patient number

Positive rate

Means standard deviation

I(n＝10)	10	70％	167.6±316.4
				II(n＝24)	22	91.7％	162.9±353.2
III(n＝31)	27	87.1％	1090.4±1945.7*

Expression amount and the multiple myeloma ISS relation by stages of table 11MAGE-A3

ISS by stages	The positive patient number	Positive rate	Means standard deviation
				I(n＝10)	7	70％	13.0±21.8
II(n＝24)	20	83.3％	3.5±8.6
				III(n＝31)	27	87.1％	4.8±9.2

Expression amount and the multiple myeloma ISS relation by stages of table 12MAGE-C2/CT10 gene

ISS by stages	The positive patient number	Positive rate	Means standard deviation
				I(n＝10)	90	90％	12.4±36.3
II(n＝24)	21	87.5％	2.6±7.2
				III(n＝31)	25	80.6％	11.8±33.9

Expression amount and the multiple myeloma ISS relation by stages of table 13SSX-2 gene

ISS by stages	The positive patient number	Positive rate	Means standard deviation
				I(n＝10)	2	20％	0.2±0.2
II(n＝24)	5	20.8％	0.3±0.5
				III(n＝31)	13	41.9％	0.7±2.1

ISS is clinical Staging System commonly used and prognostic system by stages, and good comparability and repeatability are arranged.MAGE-C1/CT7 gene expression dose and ISS be relevant (p=0.02, r=0.29, n=59) by stages, and ISS III phase patient organizes the MAGE-C1/CT7 gene expression dose of medullary cell apparently higher than I phase and II phase patient group.In ISS I phase, ISS II phase and ISS III phase Bone Marrow of Patients, the positive rate of MAGE-A3 gene test result increases successively.ISS III phase patient MAGE-C1/CT7 gene expression dose is apparently higher than ISS II phase (p<0.05).

The dependency of the expression amount of embodiment 8, each gene and patient's course of disease

107 parts of sample of bone marrow that obtain before and after some multiple myeloma patients (volunteer) treatment are analyzed.According to curative effect, be divided into treatment effective group and the group of failing to respond to any medical treatment.Treat effective group and comprise alleviation (CR) fully, do not alleviate the patient of (nCR) or partial rcsponse (PR) fully.The group of failing to respond to any medical treatment, comprise the patient of recurrence or progression of disease.Relative expression's quantity measuring method of each gene is with the step 3 of embodiment 4.The results are shown in Table 14.

The variation of each gene expression dose in the table 14 effective patient's group for the treatment of and the patient's group of failing to respond to any medical treatment

Annotate: the n value is sample size, and the p value is the test value of each gene expression dose of group before treating, after treatment.

Result shows, in the marrow of individual patient is followed the tracks of sample, the variation tendency of MAGE-C1/CT7 gene, MAGE-A3 gene and MAGE-C2/CT1 gene expression dose is consistent with this patient's clinical disease course.Just control the patient and obtain along with treatment gets involved while alleviating (CR) or partial rcsponse (PR) fully, above-mentioned three kinds of CT antigen gene expression levels descend.And the patient who fails to respond to any medical treatment, the stable gene high expression level, when recurrence or progression of disease, gene expression dose raises.Lapsing to of same patient disease state is consistent with the variation of three kinds of gene expression amounts, and especially the variation of MAGE-C1/CT7 is comparatively remarkable.The treatment effectively expression level of four kinds of CT antigen genes of group all obviously descends, and the decline of MAGE-C1/CT7 gene has statistical significance (p=0.05).Four kinds of genetic expressions of the group of failing to respond to any medical treatment all raise, and the rising of MAGE-C1/CT7 gene has statistical significance (p=0.03).

The dependency of the expression amount of embodiment 9, each gene and survival of patients time

Detect expression amount and the relation of survival of patients time of each gene in 75 routine multiple myeloma patients (volunteer) marrow.Method is with the step 3 of embodiment 4.If the relative expression quantity detected result is greater than 0, positive result; Otherwise negative result.The results are shown in Figure 7.

The survival time that relative expression's level of MAGE-C1/CT7 gene is greater than 40% patient significantly is less than the patient that relative expression's level of this gene is less than 40%.The survival time that relative expression's level of MAGE-A3 gene is greater than 1% patient significantly is less than the patient that relative expression's level of this gene is less than 1%.The survival time that relative expression's level of MAGE-C2/CT10 gene is greater than 1% patient significantly is less than the patient that relative expression's level of this gene is less than 1%.The patient's of SSX-2 gene masculine survival time significantly is less than the patient of this gene feminine gender.

Claims (6)

1. an assisting to diagnose multiple myeloma patient test kit, comprise primer probe compositions first, primer probe compositions second and primer probe compositions third; Described primer probe compositions first is comprised of first and probe first Auele Specific Primer; Described primer probe compositions second is comprised of second and probe second Auele Specific Primer; Described primer probe compositions third by Auele Specific Primer to third and probe third form; The primer pair that DNA shown in the sequence 2 of DNA and sequence table shown in the sequence 1 that described Auele Specific Primer is sequence table to first forms; The nucleotide sequence of described probe first is as shown in the sequence 3 of sequence table; The primer pair that DNA shown in the sequence 5 of DNA and sequence table shown in the sequence 4 that described Auele Specific Primer is sequence table to second forms; The nucleotide sequence of described probe second is as shown in the sequence 6 of sequence table; The primer pair that described Auele Specific Primer forms for DNA shown in the sequence 8 of DNA shown in the sequence 7 of sequence table and sequence table third; The nucleotide sequence of described probe third is as shown in the sequence 9 of sequence table.

2. test kit as claimed in claim 1, it is characterized in that: described test kit also comprises primer probe compositions fourth; Described primer probe compositions fourth is comprised of fourth and probe fourth Auele Specific Primer; The primer pair that DNA shown in the sequence 11 of DNA and sequence table shown in the sequence 10 that described Auele Specific Primer is sequence table to fourth forms; The nucleotide sequence of described probe fourth is as shown in the sequence 12 of sequence table.

3. test kit as claimed in claim 2, it is characterized in that: described test kit also comprises positive plasmid first, positive plasmid second, positive plasmid third and positive plasmid fourth; Described positive plasmid first is the recombinant plasmid of the MAGE-C1/CT7 gene fragment containing shown in the sequence 13 of ordered list; Described positive plasmid second is the recombinant plasmid of the MAGE-A3 gene fragment containing shown in the sequence 14 of ordered list; The recombinant plasmid that described positive plasmid third is the MAGE-C2/CT10 gene fragment containing shown in the sequence 15 of ordered list; Described positive plasmid fourth is the recombinant plasmid of the SSX-2 gene fragment containing shown in the sequence 16 of ordered list.

4. as arbitrary described test kit in claims 1 to 3, it is characterized in that: described test kit also comprises for the internal reference primer pair of reference gene and internal reference probe; Described reference gene is abl gene.

5. test kit as claimed in claim 4, is characterized in that: the primer pair that DNA shown in the sequence 18 of DNA and sequence table shown in the sequence 17 that described internal reference primer pair is sequence table forms; The nucleotide sequence of described internal reference probe is as shown in the sequence 19 of sequence table.

6. test kit as claimed in claim 5, it is characterized in that: described test kit also comprises the internal reference plasmid; Described internal reference plasmid is the recombinant plasmid of the abl gene fragment containing shown in the sequence 20 of ordered list.

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