CN113846159B - Special chip for detecting expression of breast cancer related gene - Google Patents
Special chip for detecting expression of breast cancer related gene Download PDFInfo
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- CN113846159B CN113846159B CN202110110056.2A CN202110110056A CN113846159B CN 113846159 B CN113846159 B CN 113846159B CN 202110110056 A CN202110110056 A CN 202110110056A CN 113846159 B CN113846159 B CN 113846159B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention relates to a special chip for detecting breast cancer related gene expression, which takes a pair of primers of RT-PCR reaction as a probe pair to be fixed on a substrate, utilizes the gene chip to complete simultaneous detection of multiple gene expression profiles at one time, and can rapidly complete detection of breast cancer specific gene expression conditions by utilizing an RNA sample.
Description
Technical Field
The invention relates to the field of biochips, in particular to a special chip for detecting breast cancer related gene expression.
Background
The biochip technology has been widely applied to a plurality of fields such as clinical disease diagnosis, health management, drug research and development, animal and plant quarantine, food detection, environmental monitoring, scientific research, forensic medicine detection and the like, and has wide application prospect and great market demand.
The gene chip is one kind of biochip, which is prepared through planting serial probes with known sequence on the chip substrate, and may be used in the hybridization detection of specific mark nucleic acid and reporting the nucleic acid information in the detected object through identification and information processing.
New cancer cases occur in 1929 in the world in 2020, wherein 1006 ten thousand cases are male, 923 ten thousand cases are female; 996 ten thousand cases of cancer death worldwide in 2020, of which 553 thousand in men and 443 ten thousand in women. The number of new breast cancer cases is up to 226 ten thousand, which exceeds 220 ten thousand of lung cancer cases, and the breast cancer replaces the lung cancer, becomes the first cancer in the world, and has become the first killer threatening the life of women.
Currently, the detection of breast cancer is mainly based on imaging and immunohistochemistry, assisted by gene sequencing based on instruments and equipment, and simultaneously, the expression profile analysis and adjuvant drug analysis of breast cancer-related genes are performed by quantitative PCR, such as: the expression level of the breast cancer 21 gene is used for judging the prognosis condition of a patient, and whether auxiliary chemotherapy is needed or not is determined according to the detection result, and whether endocrine auxiliary treatment can be performed by using tamoxifen or not is determined.
The current main method is to extract RNA from a tissue slice of a patient, a series of complex operations such as dehydration, hydration and the like are required to be completed to obtain the purified RNA, the obtained RNA is respectively added into each PCR tube according to a test target, each PCR tube completes quantitative PCR reaction of one gene (RNA), the operation is very complicated, and the RNA can be completed by professional staff, so that the automation integration is inconvenient.
Disclosure of Invention
The invention aims to solve the defects in the prior art, utilizes an enzymatic synthesis technology of nucleic acid synthesis in a biological system, converts RNA information of a tumor into an extended chain sequence of a probe through DNA polymerase, completes all detection processes on one chip, can complete the detection of the expression condition of breast cancer related genes in one step by automation of the detection, and provides an effective judgment basis for the prognosis judgment and subsequent treatment of the breast cancer.
The invention has the following inventive concept: the primers for amplifying the target RNA are fixed on the substrate, so that a plurality of RNAs can be amplified synchronously; meanwhile, DNA polymerase is used for converting and integrating RNA sequence information into the extended strand of the probe, and the related information of the RNA can be obtained by detecting the extended strand of the probe; finally, the Click is used to mark dNTP(s) integrated into the probe extension chain, so as to obtain the position of the probe (positive result) for completing the PCR reaction and convert to obtain the RNA expression level of the breast cancer related gene.
In order to solve the problems, the technical scheme of the invention is as follows: a special chip for detecting breast cancer related gene expression is characterized by comprising the following steps:
s1, screening genes related to breast cancer occurrence and genes related to breast cancer prognosis according to a breast cancer related guide, selecting a pair of probes for each gene according to a specific mRNA sequence of each gene, and ensuring that the group of probes can complete RT-PCR amplification of the specific mRNA sequence;
s2, completing chemical synthesis of probe pairs, and simultaneously ensuring that a connecting arm is added to the 5' tail end of each probe;
s3, mixing each group of probe pairs together, and fixing each probe on a substrate through a covalent bond by a connecting arm to manufacture a special chip capable of detecting the expression of the breast cancer related genes;
s4, adding total RNA to be detected to a special chip, assembling an RT-PCR amplification system on the special chip, combining a specific mRNA sequence with a probe matched with the specific mRNA sequence, starting RT-PCR amplification, and converting specific mRNA sequence information into a probe extension chain;
s5, after finishing RT-PCR amplification, fully washing and removing various components of an RT-PCR reaction system on the special chip, ensuring that only a probe and a probe extension chain are on the special chip, and ensuring that a detection system has no background interference;
s6 in the RT-PCR reaction system, the marked dNTP is taken as a substrate, the marked dNTP is integrated in a probe extension chain in the RT-PCR reaction process, and the detection result of the special chip for expressing the breast cancer related gene is finally obtained based on the signal of the marked dNTP and the sample application position of each group of probe pairs.
Preferably, a specific mRNA sequence is detected by directly performing an RT-PCR amplification reaction using a pair of primers (probe pair) immobilized on a chip.
Preferably, the RT-PCR amplification reaction is completed on a very small chip, and is not required to be divided into a plurality of PCR tubes, one chip can simultaneously complete hundreds of detections, and the RT-PCR reaction of the other pair of primers (probe pair) cannot be influenced by the fixed pair of primers (probe pair).
Preferably, one nucleotide substrate of the RT-PCR amplification reaction is a labeled dNTP which is integrated into the extension strand of the probe during the RT-PCR amplification reaction.
Preferably, the labeled dNTPs do not affect the extension of the probe strand by the action of the DNA polymerase, and can further complete detectable signal modification.
Preferably, the number of labeled dNTPs that can be incorporated into each probe extension strand is quantitatively calculated from the target mRNA sequence.
Preferably, the labeled dNTPs on the probe extension chain can complete detectable information modification by utilizing a Click reaction, and further complete analysis of related indexes of the breast cancer through the copy number ratio of mRNA of genes corresponding to fluorescence intensity of each site.
Compared with the prior art, the special chip for detecting the expression of the breast cancer related genes can simultaneously detect tens of thousands of gene expression profiles on one chip, can finish the rapid detection of a small sample amount, is very convenient for the expression screening of the breast cancer related genes, can realize the multiple screening with low cost, and can find the expression change of the breast cancer genes and treat the breast cancer genes in time. In the invention, dNTP capable of generating signals is integrated into the probe extension chain, the label is stable, quantitative analysis can be realized, the inconvenience that fluorescence detection is required for each amplification in the traditional quantitative PCR is not needed, and the signals are obtained once after the reaction is finished, so that the detection time is effectively shortened.
Detailed Description
The present invention is further described in detail below with reference to specific examples. The following examples are illustrative only, not limiting, and are not intended to limit the scope of the invention. The chemical reagents and instruments used in the present invention are commercially available without specific reference.
Example 1
A special chip for detecting breast cancer related gene expression is characterized by comprising the following steps:
s1, designing corresponding amplification primers according to a mature mRNA sequence of a breast cancer 21 gene specified in a breast cancer guideline, which is specifically shown in the following table:
TABLE 1 primer for detecting Gene expression of Breast cancer 21
Screening genes related to breast cancer occurrence and genes related to breast cancer prognosis according to a breast cancer treatment related guideline, selecting a pair of probes for each gene according to a specific mRNA sequence of each gene, and ensuring that the group of probes can complete RT-PCR amplification of the specific mRNA sequence;
s2, completing chemical synthesis of probe pairs, and simultaneously ensuring that a connecting arm consisting of 6 ethylene glycol polymer chains is added at the 5' tail end of each probe, and the tail end of each probe is connected with and decorated with an amino group for connecting with a substrate;
s3, mixing a group of probes for amplifying a specific mRNA sequence of the same gene together to form probe pairs, fixing the probe pairs on a substrate by amino, ensuring that each group of probe pairs is fixed on the substrate in a covalent bond mode through a connecting arm, and recording the corresponding relation between the positions of the probes and the sequences of the probe pairs to manufacture a special chip capable of detecting the expression of the breast cancer 21 gene;
s4, obtaining total RNA by using a sample obtained by micro-needle puncture through a special kit (Saimei Fei company), adding the total RNA to a special chip capable of detecting the expression of the breast cancer 21 gene, assembling an RT-PCR amplification system (related RT-PCR is purchased from Saimei Fei company) on the special chip, wherein one dNTP substrate is ethinyl-labeled uridine (EdU) deoxyribose triphosphate, combining a probe matched with a specific mRNA sequence, starting RT-PCR amplification, converting the specific mRNA sequence information into an extension chain (cDNA) taking the probe as a primer, and enabling the EdU to enter the probe extension chain according to the corresponding thymine nucleotide position;
s5, after finishing RT-PCR amplification, fully washing and removing various components of an RT-PCR reaction system on the special chip, ensuring that only a probe and a probe extension chain are on the special chip, and ensuring that a detection system has no background interference;
s6 in an RT-PCR reaction system, the marked EdU is integrated in a probe extension chain in the RT-PCR reaction process, the EdU can utilize Click reaction to finish fluorescence marking, all the EdU in each probe extension chain can be directly marked with fluorescence molecules (such as cy 3) on a chip, and because each EdU base on the probe extension chain is marked with cy3 molecules, the fluorescence signal of the probe is greatly improved, and the positive signal detection efficiency is effectively improved;
s7, judging the qualitative expression of the breast cancer specific gene according to the probe position information and the existence of the fluorescent signal, converting the fluorescent signal according to the specific RNA sequence and the corresponding probe pair sequence, and performing the copy number comparison analysis of different mRNAs; s8, the RS score calculation formula in the breast cancer guideline is shown in the table
RS score calculation
RS score calculation is carried out, and reference basis can be provided for breast cancer prognosis judgment.
The above description is only for the purpose of creating a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
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Claims (1)
1. A kit for detecting breast cancer related gene expression is characterized by comprising a special chip and an RT-PCR amplification system;
the preparation method of the special chip comprises the following steps:
s1, screening genes related to breast cancer occurrence and genes related to breast cancer prognosis according to a breast cancer related guideline, selecting a pair of probes for each gene according to a specific mRNA sequence of each gene, and ensuring that the group of probes can complete RT-PCR amplification of the specific mRNA sequence;
s2, completing chemical synthesis of probe pairs, and simultaneously ensuring that a connecting arm is added to the 5' tail end of each probe;
s3, mixing each group of probe pairs together, and fixing each probe on a substrate through a covalent bond by a connecting arm to manufacture a special chip capable of detecting the expression of the breast cancer related genes;
the sequence of the probe is shown in SEQ ID NO. 1-42;
the connecting arm is composed of 6 ethylene glycol polymer chains;
one dNTP substrate in the RT-PCR amplification system is ethinyl-labeled deoxyribose triphosphate uridine EdU, the EdU enters a probe extension chain according to the position of corresponding thymine nucleotide, fluorescence labeling is completed by utilizing Click reaction, and all the EdU in each probe extension chain are directly labeled with fluorescence molecules on a chip.
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| CN102337327A (en) * | 2010-07-21 | 2012-02-01 | 其昌達生物高科技(上海)有限公司 | 28-gene array detection system for breast cancer prognosis |
| CN110129435A (en) * | 2019-02-22 | 2019-08-16 | 深圳塔歌生物技术有限公司 | 21 gene Multiple detection kit of breast cancer |
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| US20040067492A1 (en) * | 2002-10-04 | 2004-04-08 | Allan Peng | Reverse transcription on microarrays |
| AU2003302264A1 (en) * | 2002-12-20 | 2004-09-09 | Biotrove, Inc. | Assay apparatus and method using microfluidic arrays |
| CN1291037C (en) * | 2004-01-19 | 2006-12-20 | 中国科学院上海微系统与信息技术研究所 | In-situ polymerase chain reaction approach for detecting target gene on chip |
| CN101008034A (en) * | 2006-12-13 | 2007-08-01 | 南开大学 | Breast carcinoma transfer related gene chip establishment and its establishing method |
| CN101921858B (en) * | 2010-08-23 | 2013-08-28 | 广州益善生物技术有限公司 | Liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level |
| US9902990B2 (en) * | 2011-05-27 | 2018-02-27 | The University Of British Columbia | Microfluidic cell trap and assay apparatus for high-throughput analysis |
| US11091803B2 (en) * | 2014-04-14 | 2021-08-17 | W2 Biosolutions, Llc | Nucleic acid quantification method |
| CN107058523A (en) * | 2017-03-21 | 2017-08-18 | 温州迪安医学检验所有限公司 | A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application |
| CN106977498B (en) * | 2017-03-29 | 2020-05-22 | 华南师范大学 | Light click probe for fluorescence response labeled DNA in cell and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337327A (en) * | 2010-07-21 | 2012-02-01 | 其昌達生物高科技(上海)有限公司 | 28-gene array detection system for breast cancer prognosis |
| CN110129435A (en) * | 2019-02-22 | 2019-08-16 | 深圳塔歌生物技术有限公司 | 21 gene Multiple detection kit of breast cancer |
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