Quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients

A multiple myeloma, auxiliary diagnosis technology, applied in the direction of microbial determination/testing, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of lack of molecular targets for tumor-specific diagnosis of minimal residual disease detection and prognosis assessment, etc.

Inactive Publication Date: 2011-11-23
PEOPLES HOSPITAL PEKING UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] The development of molecular biology has made more and more tumor-related genes known. These important molecular markers have played a great role in the diagnosis and treatment of hematologic malignancies. Immunoglobulin gene rearrangement, C-MYC, RAS oncogene mutation and human telomerase reverse transcriptase mRNA overexpression, etc., but there is a lack of recognized molecular targets for tumor-related specific diagnosis, minimal residual disease detection and prognosis assessment

Method used

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  • Quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients
  • Quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients
  • Quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the design of specific primer pair and probe

[0021] The SSX-2 gene (NCBI gene bank sequence number: NM_175698 / 003147) is located on human chromosome Xp11.22, and a specific primer pair A (composed of upstream primer SSX-FP and downstream primer SSX-RP, amplified) was designed for the SSX-2 gene The product is 110bp) and probe A (probe SSX-T-probe):

[0022] Upstream primer SSX-FP (sequence 1 of the sequence listing): 5'-TAACCGTGGGAATCAGGTTGA-3'

[0023] Downstream primer SSX-RP (sequence 2 of the sequence listing): 5'-CCTCCGAATCATTTCCTTCCT-3'

[0024] Probe SSX-T-probe (5'→3'):

[0025] FAM-CCGAAGATCATGCCCAAGAAGCCAG-BHQ (the nucleotide sequence is sequence 3 in the sequence listing).

[0026] Specific primer pair B (composed of upstream primer ABL1-F and downstream primer ABL1_R, the amplified product is 124bp) and probe B (probe ABL1- T-probe):

[0027] Upstream primer ABL1-F (sequence 4 of the sequence listing): 5'-TGGAGATAACACTCTAAAGCATAACTAAAGGT...

Embodiment 2

[0032] Embodiment 2, the preparation of relevant plasmid

[0033] 1. Preparation of positive control plasmid (plasmid containing SSX-2 gene fragment)

[0034] With the cDNA of the bone marrow mononuclear cell of the multiple myeloma patient (volunteer) positive for SSX-2 gene expression as a template, carry out PCR amplification, the nucleotide sequence of the amplification product is as shown in the sequence 7 of sequence list (435bp ), the amplified product was purified and cloned into the pMD18-T plasmid, the recombinant plasmid was transformed into Escherichia coli DH5α competent, the positive clone was screened, the plasmid was extracted and purified, and sequenced. The results showed that the positive control plasmid D (backbone plasmid was pMD18 -T plasmid, insert the cDNA shown in sequence 7 at the ECOR V site).

[0035] The primer pairs used in PCR amplification are as follows:

[0036] Upstream primer: 5'-GTGCTCAAATACCAGAGAAGATC-3';

[0037] Downstream primer: 5'-TT...

Embodiment 3

[0045] Embodiment 3, detect the sensitivity detection of positive control plasmid

[0046] The internal reference control plasmid that embodiment 2 obtains is carried out 10 times gradient dilutions with sterilized double-distilled water for injection to obtain each dilution (each microliter contains respectively 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 copies of the ABL gene fragment); the copy number of the SSX-2 gene fragment or the ABL gene fragment was calculated by measuring the absorbance value). The specific primer pair B and probe B obtained in Example 1 were used for each internal control plasmid to perform RQ-PCR on a fluorescent real-time quantitative PCR instrument (type 7500-FAST from ABI Company, USA). For the internal control plasmid RQ-PCR fluorescence standard curve, see figure 1 (threshold is 0.082), the function is log 10 ABL1=(Ct-38.46) / -3.22, the correlation coefficient reaches above 0.99, and the sensitivity of detecting the internal reference ge...

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Abstract

The invention discloses a quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients, which comprises a specific primer pair A and a probe A, wherein the specific primer pair A is a primer pair composed of DNA (deoxyribonucleic acid) disclosed as Sequence 1 and DNA disclosed as Sequence 2 in the sequence table; and the nucleotide sequence of the probe A is disclosed as Sequence 3 in the sequence table. The kit disclosed by the invention not only can be used for qualitative diagnosis of multiple myeloma, but also can be used for detecting the expression level of the SSX-2 gene of the patient through the reference gene. The invention provides a quick, reliable and accurate new way for diagnosis of multiple myeloma, and provides references for observing curative effects and dynamically observing minimal residual disease. The invention can perform vital functions in the field of medical detection.

Description

technical field [0001] The invention relates to a quantitative detection kit for assisting diagnosis of multiple myeloma patients based on SSX-2 gene. Background technique [0002] Multiple myeloma (multiple myeloma, MM) is a life-threatening plasma cell malignancy, and its incidence rate ranks second among hematological malignancies. Although stem cell transplantation and new drug therapy can effectively control the disease, few patients can achieve complete remission or even cure, and the outcome of relapse and death is still inevitable. [0003] The development of molecular biology has made more and more tumor-related genes known. These important molecular markers have played a great role in the diagnosis and treatment of hematologic malignancies. Immunoglobulin gene rearrangement, C-MYC, RAS oncogene mutation, and human telomerase reverse transcriptase mRNA overexpression, etc., but there is a lack of recognized molecular targets for tumor-related specific diagnosis, mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 阮国瑞黄晓军刘开彦张瑶江滨陈珊珊刘艳荣李金兰
Owner PEOPLES HOSPITAL PEKING UNIV
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