CN113930498A - Method for detecting RASEF gene expression in nipple discharge and breast cancer screening kit thereof - Google Patents
Method for detecting RASEF gene expression in nipple discharge and breast cancer screening kit thereof Download PDFInfo
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- CN113930498A CN113930498A CN202010607683.2A CN202010607683A CN113930498A CN 113930498 A CN113930498 A CN 113930498A CN 202010607683 A CN202010607683 A CN 202010607683A CN 113930498 A CN113930498 A CN 113930498A
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Abstract
The invention aims to provide a method for detecting RASEF gene expression in nipple discharge, which utilizes the content of RASEF transcript mRNA or/and protein coded by RASEF gene in a nipple discharge biological sample as a report index of RASEF gene expression. By the method, an mRNA or protein detection kit can be constructed, the relationship between the expression change of the nipple discharge RASEF and the occurrence and development of the breast cancer is originally discovered and utilized, the mRNA or protein expression level of the RASEF in a nipple discharge sample of a subject is detected noninvasively, the occurrence and prognosis of the breast cancer are realized in an auxiliary prediction mode, and the method can be further used for research and development of medicines and treatment methods.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to RASEF gene expression detection.
Background
The breast cancer is the highest cancer of women all over the world, and the early screening and prediction are carried out in a low-invasive or non-invasive mode, so that the breast cancer diagnosis and prediction method is greatly helpful for saving the life of female breast cancer patients and reducing the social and economic burden caused by the cancer. In view of the potential association of abnormal nipple discharge with breast cancer, and by detecting an abnormal biomarker of nipple discharge, not only does biopsy sampling not need to be performed, but also continuous monitoring is possible, and no imaging radiation damage is caused, so that the method can be developed as a method for assisting in predicting the occurrence and prognosis of breast cancer, and has unique advantages compared with other screening technologies. However, the current biomarkers in breast discharge can be applied to members of breast cancer screening, and have ideal sensitivity and accuracy, which is very limited.
Disclosure of Invention
The early researches of the inventor discover for the first time that a new gene molecular marker RASEF has correlation between the expression and the occurrence, development and typing of breast cancer, so that the technical scheme of the invention provides a method and a kit product for detecting the mRNA and protein levels of RASEF by using nipple discharge as a sample.
One of the objects of the present invention is to provide a method and a kit for detecting the mRNA and protein levels of RASEF gene in nipple discharge.
The invention provides a method for detecting RASEF gene expression in nipple discharge, which utilizes the content of RASEF transcript mRNA or/and protein coded by RASEF gene in a nipple discharge biological sample as a report index of RASEF gene expression.
The invention also provides a method for detecting the expression of the RASEF gene in nipple discharge, and the protein coded by the RASEF gene is captured by the specific antibody and quantified by adopting an ELISA principle.
The invention also provides a method for detecting RASEF gene expression in nipple discharge, wherein the capture molecules of the mRNA of the RASEF transcript are primers and/or probes for specifically identifying cDNA corresponding to the RASEF gene transcript, and the sequence of the cDNA is shown as SEQ ID No. 7.
The invention also provides a method for detecting RASEF gene expression in nipple discharge, and the amino acid sequence of the coding protein is shown in SEQ ID No. 8.
The invention also provides a method for detecting RASEF gene expression in nipple discharge, and the primer sequence is any one or combination of SEQ ID No. 1-6.
The invention also provides an application of RASEF, and RASEF is used as a marker to be applied to preparation of a breast cancer screening kit.
The invention also provides an mRNA breast cancer screening kit based on RASEF, which comprises the capture molecules of the mRNA of the RASEF transcript, the capture molecules of the mRNA of the RASEF transcript are primers and/or probes for specifically identifying cDNA corresponding to the RASEF gene transcript, and the cDNA sequence is shown as SEQ ID No. 7.
The invention also provides an mRNA breast cancer screening kit based on RASEF, wherein the primer sequence is any one or combination of SEQ ID No. 1-6.
The invention also provides a protein breast cancer screening kit based on the RASEF gene coding, which comprises a specific antibody of the protein coded by the RASEF gene, and the amino acid sequence of the coded protein is shown as SEQ ID No. 8.
The invention also provides a protein breast cancer screening kit based on RASEF gene coding, the kit adopts competitive ELISA or RASEF detection kit based on double-antibody sandwich ELISA, the RASEF detection kit based on competitive ELISA comprises RASEF protein standard, anti-RASEF monoclonal antibody, enzyme-labeled secondary antibody and enzyme substrate; the RASEF detection kit based on the double-antibody sandwich ELISA comprises an anti-RASEF monoclonal antibody, a biotin-labeled RASEF antibody, an avidin-enzyme complex and an enzyme substrate.
The marker RASEF provided by the invention is also called RAS And EF-Hand Domain containment, And Ensembl has the identification ID of ENSG00000165105 And is mainly located on human chromosome 9. The invention provides two methods for detecting the gene marker in nipple discharge, which can collect the nipple discharge of a subject and detect the nipple discharge at two levels of mRNA and protein. The mRNA level of the gene in the nipple discharge can be detected by a reverse transcription Realtime PCR means, an RNA hybridization-based chip technology, an RNA sequencing-based sequencing technology and the like. The level of the gene protein in the nipple discharge can be detected by means of Western blot, ELISA, mass spectrum, high performance liquid and the like. In view of the fact that there are two transcripts for RASEF, RASEF-201(Ensembl ID: ENST00000340717.4) and RASEF-202(Ensembl ID: ENST 00000376447.4). The former transcription length is 1765bp, and the latter transcription length is 5585 bp. The former encoded about 189 amino acids, the latter about 740 amino acids.
Earlier studies of the present invention found that in basal cell-like breast cancer or triple negative breast cancer, the expression level of RASEF decreased compared to the normal population, while in non-triple negative breast cancer (e.g., Luminal a and Luminal B) patients, the expression increased compared to the normal population (see fig. 1). Whereas in Luminal A patients and non-Luminal patients positive for HER2, RASEF high expression is associated with adverse survival; in Luminal B patients, the opposite is true, and RASEF high expression survival is better; there was no significant association in survival in basal cell-like breast cancer or triple negative breast cancer. Therefore, the RASEF expression analysis of nipple discharge can be used for auxiliary screening of breast cancer; if breast cancer has been confirmed by biopsy, the expression level of RASEF can also be used to assist in predicting the prognostic progression of breast cancer in conjunction with breast cancer molecular typing.
The biological sample of the invention is female nipple discharge, namely a sample of an individual with positive normal breast discharge phenomenon.
The detection method included in the invention is a quantitative technology for mRNA or protein of RASEF in a liquid sample in any field, including but not limited to Realtime PCR after reverse transcription, a chip technology based on RNA hybridization, a sequencing technology based on RNA sequencing, Western blot, ELISA, mass spectrometry, high efficiency liquid phase and the like.
At the mRNA detection level, there are many methods for detecting mRNA expression, including but not limited to Polymerase Chain Reaction (PCR), real-time quantitative PCR (quantitative-time PCR), nucleic acid based amplification (NASBA), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR), thermal strand displacement (tSDA), and the like. According to the above detection method, a primer or probe that specifically recognizes the second type of cleavage of RASEF or its cDNA can be prepared to capture RASEF expression of nipple discharge. The invention preferably selects primers or probes which specifically recognize cDNA corresponding to mRNA of two exons of RASEF. As shown in the sequence SEQ ID 001-006.
At the protein level, as a preferred technical scheme, a RASEF detection kit based on competitive ELISA or double-antibody sandwich ELISA is adopted. The competitive ELISA kit at least comprises a RASEF protein standard, an anti-RASEF monoclonal antibody, an enzyme-labeled secondary antibody and an enzyme substrate: the method comprises the steps of coating an ELISA plate with RASEF standard substances with different concentrations, sequentially adding a nipple discharge sample to be detected and a monoclonal antibody to the plate for incubation, and detecting the amount of the anti-monoclonal antibody bound on the plate through an enzyme-labeled secondary antibody and an enzyme substrate, thereby determining the concentration of RASEF in the nipple discharge sample. The RASEF detection kit based on double-antibody sandwich ELISA at least comprises an anti-RASEF monoclonal antibody, a biotin-labeled RASEF antibody, an avidin-enzyme complex and an enzyme substrate: coating a flat plate with an anti-RASEF monoclonal antibody, sequentially adding a nipple discharge sample to be detected and a biotin-labeled anti-RASEF antibody to the flat plate for incubation, and reflecting the content of the anti-RASEF antibody on the flat plate through the color development of an avidin-enzyme complex and an enzyme substrate, thereby determining the concentration of RASEF in the nipple discharge sample.
The method has the advantages and the characteristics that by the method, an mRNA or protein detection kit can be constructed, the relationship between the expression change of the nipple discharge RASEF and the occurrence and the development of the breast cancer is originally discovered and utilized, the mRNA or protein expression level of the nipple discharge sample RASEF of a subject is detected noninvasively, the occurrence and the prognosis of the breast cancer are realized in an auxiliary prediction mode, and the method can be used for research and development of medicines and treatment methods. It is specifically stated that the claims do not limit the possible use of some of the techniques of the invention by physicians for disease diagnosis.
Drawings
FIG. 1 is a graph showing the relative expression level of mRNA in one example of the present invention.
Detailed Description
The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings.
Example 1 sequences
First primer forward primer pair of cDNA for RASEF (the latter number is the primer length)
TTCCCCTCAACCTCTAGGCTA 21
First primer pair and reverse primer pair of cDNA aiming at RASEF
CCTAGAGGTTGAGGGGAATGA 21
Second primer forward primer pair of cDNA aiming at RASEF
TTCCCCTCAACCTCTAGGCTA 21
Second primer reverse primer pair of cDNA for RASEF
CAACTTCACAATTTGTCCTCTGC 23
Third primer pair of cDNA forward primers for RASEF
CAGAGGGGATTTCAGAGGTCA 21
Third primer reverse primer pair of cDNA aiming at RASEF
AGACGGAAGCCACATCTTCTAA 22
cDNA sequence of RASEF
Amino acid sequence of RASEF
MEADGDGEELARLRSVFAACDANRSGRLEREEFRALCTELRVRPADAEAVFQRLDADRDGAITFQEFARGFLGSLRGGRRRDWGPLDPAPAVSEAGPETHDSEEDEGDEDAAAALATSCGPASPGRAWQDFQARLGDEAKFIPREEQVSTLYQNINLVEPRLIQPYEHVIKNFIREIRLQSTEMENLAIAVKRAQDKAAMQLSELEEEMDQRIQAAEHKTRKDEKRKAEEALSDLRRQYETEVGDLQVTIKKLRKLEEQSKRVSQKEDVAALKKQIYDLSMENQKVKKDLLEAQTNIAFLQSELDALKSDYADQSLNTERDLEIIRAYTEDRNSLERQIEILQTANRKLHDSNDGLRSALENSYSKFNRSLHINNISPGNTISRSSPKFIGHSPQPLGYDRSSRSSYVDEDCDSLALCDPLQRTNCEVDSLPESCFDSGLSTLRDPNEYDSEVEYKHQRGFQRSHGVQESFGGDASDTDVPDIRDEETFGLEDVASVLDWKPQGSVSEGSIVSSSRKPISALSPQTDLVDDNAKSFSSQKAYKIVLAGDAAVGKSSFLMRLCKNEFRENISATLGVDFQMKTLIVDGERTVLQLWDTAGQERFRSIAKSYFRKADGVLLLYDVTCEKSFLNIREWVDMIEDAAHETVPIMLVGNKADIRDTAATEGQKCVPGHFGEKLAMTYGALFCETSAKDGSNIVEAVLHLAREVKKRTDKDDSRSITNLTGTNSKKSPQMKNCCNG
Example 2
Collecting two groups of nipple discharge samples, wherein one group is a normal control (non-breast cancer), and the other group is breast cancer (both right breast infiltrating ductal carcinoma 2 stage, HER2 positive), extracting total RNA of the samples by using a Trizol method, and performing RNA quality detection; digesting ribosomal RNA by using TruSeq Stranded Total RNA with Ribo-Zero Gold kit, and adding an interrupting agent to break the RNA into short fragments; taking broken RNA as a template, synthesizing single-strand cDNA, preparing a double-strand synthesis reaction system to synthesize double-strand cDNA, replacing dTTP with dUTP during double-strand cDNA synthesis, connecting different joints, digesting one strand containing dUTP by using a UNG enzyme method, and only reserving one strand of cDNA connected with different joints of the strand; purifying one strand of cDNA by using a kit; carrying out end repair, tail A adding and sequencing joint connection on a purified cDNA chain, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed RNA library was qualified by Agilent 2100Bioanalyzer quality testing and then sequenced using an Illumina sequencer. After the original reads number is genome-matched, the expression level is expressed by a conventional FPKM method, and normalized by using a normal control group. Among them, mRNA expression of RASEF was significantly increased in breast cancer, as shown in fig. 1. Further, with respect to patients with confirmed breast cancer, three groups were divided into high, medium and low groups according to the mRNA level of RASEF, and survival analysis showed that the overall survival was significantly worse in the high and medium RASEF expression groups than in the low expression group (Log-rank test 7.91, P0.019).
Sequence listing
<110> Zhejiang province tumor hospital
<120> method for detecting RASEF gene expression in nipple discharge and breast cancer screening kit thereof
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cgggtgcggc cggccgacgc cgaggcagta ttccagcggc tggacgccga ccgtgacggc 180
gccatcacct tccaggagtt cgcgcgtggc ttcctcgggt ccctccgcgg ggggcggcgc 240
cgggactggg gtcctctgga tcccgcgccc gccgtgtctg aggcggggcc ggagacacac 300
gacagcgagg aggacgaagg cgacgaggac gcggcggcgg cgctggccac ctcgtgcggc 360
ccggcgagtc ccggccgggc ttggcaggat ttccaggcgc gacttgggga cgaagccaag 420
ttcattccca gagaagagca agttagtacc ttgtaccaaa acatcaacct tgtggagcca 480
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agcacagaaa tggaaaattt ggccattgcg gtgaagagag cccaggacaa ggcagctatg 600
cagttgagtg agttggaaga ggaaatggat cagaggattc aggctgcaga acataagaca 660
cggaaagacg aaaaacgcaa agctgaggaa gccctcagtg acctcagacg tcagtatgaa 720
actgaagtag gagatctgca ggtgaccatt aaaaagctaa gaaagctcga agaacaatca 780
aaacgcgtaa gtcaaaagga agatgtggct gcattgaaaa aacaaattta tgatttatca 840
atggaaaacc agaaagttaa gaaagacctt ttagaagcac agacaaacat agcctttctt 900
cagagtgagt tagatgcttt gaaaagtgat tatgctgatc agagtctgaa tactgaaagg 960
gatctggaaa taatccgagc atacacagaa gatcgaaata gtcttgagag gcaaattgaa 1020
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Met Glu Ala Asp Gly Asp Gly Glu Glu Leu Ala Arg Leu Arg Ser Val
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Phe Ala Ala Cys Asp Ala Asn Arg Ser Gly Arg Leu Glu Arg Glu Glu
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Phe Arg Ala Leu Cys Thr Glu Leu Arg Val Arg Pro Ala Asp Ala Glu
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Ala Val Phe Gln Arg Leu Asp Ala Asp Arg Asp Gly Ala Ile Thr Phe
50 55 60
Gln Glu Phe Ala Arg Gly Phe Leu Gly Ser Leu Arg Gly Gly Arg Arg
65 70 75 80
Arg Asp Trp Gly Pro Leu Asp Pro Ala Pro Ala Val Ser Glu Ala Gly
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Pro Glu Thr His Asp Ser Glu Glu Asp Glu Gly Asp Glu Asp Ala Ala
100 105 110
Ala Ala Leu Ala Thr Ser Cys Gly Pro Ala Ser Pro Gly Arg Ala Trp
115 120 125
Gln Asp Phe Gln Ala Arg Leu Gly Asp Glu Ala Lys Phe Ile Pro Arg
130 135 140
Glu Glu Gln Val Ser Thr Leu Tyr Gln Asn Ile Asn Leu Val Glu Pro
145 150 155 160
Arg Leu Ile Gln Pro Tyr Glu His Val Ile Lys Asn Phe Ile Arg Glu
165 170 175
Ile Arg Leu Gln Ser Thr Glu Met Glu Asn Leu Ala Ile Ala Val Lys
180 185 190
Arg Ala Gln Asp Lys Ala Ala Met Gln Leu Ser Glu Leu Glu Glu Glu
195 200 205
Met Asp Gln Arg Ile Gln Ala Ala Glu His Lys Thr Arg Lys Asp Glu
210 215 220
Lys Arg Lys Ala Glu Glu Ala Leu Ser Asp Leu Arg Arg Gln Tyr Glu
225 230 235 240
Thr Glu Val Gly Asp Leu Gln Val Thr Ile Lys Lys Leu Arg Lys Leu
245 250 255
Glu Glu Gln Ser Lys Arg Val Ser Gln Lys Glu Asp Val Ala Ala Leu
260 265 270
Lys Lys Gln Ile Tyr Asp Leu Ser Met Glu Asn Gln Lys Val Lys Lys
275 280 285
Asp Leu Leu Glu Ala Gln Thr Asn Ile Ala Phe Leu Gln Ser Glu Leu
290 295 300
Asp Ala Leu Lys Ser Asp Tyr Ala Asp Gln Ser Leu Asn Thr Glu Arg
305 310 315 320
Asp Leu Glu Ile Ile Arg Ala Tyr Thr Glu Asp Arg Asn Ser Leu Glu
325 330 335
Arg Gln Ile Glu Ile Leu Gln Thr Ala Asn Arg Lys Leu His Asp Ser
340 345 350
Asn Asp Gly Leu Arg Ser Ala Leu Glu Asn Ser Tyr Ser Lys Phe Asn
355 360 365
Arg Ser Leu His Ile Asn Asn Ile Ser Pro Gly Asn Thr Ile Ser Arg
370 375 380
Ser Ser Pro Lys Phe Ile Gly His Ser Pro Gln Pro Leu Gly Tyr Asp
385 390 395 400
Arg Ser Ser Arg Ser Ser Tyr Val Asp Glu Asp Cys Asp Ser Leu Ala
405 410 415
Leu Cys Asp Pro Leu Gln Arg Thr Asn Cys Glu Val Asp Ser Leu Pro
420 425 430
Glu Ser Cys Phe Asp Ser Gly Leu Ser Thr Leu Arg Asp Pro Asn Glu
435 440 445
Tyr Asp Ser Glu Val Glu Tyr Lys His Gln Arg Gly Phe Gln Arg Ser
450 455 460
His Gly Val Gln Glu Ser Phe Gly Gly Asp Ala Ser Asp Thr Asp Val
465 470 475 480
Pro Asp Ile Arg Asp Glu Glu Thr Phe Gly Leu Glu Asp Val Ala Ser
485 490 495
Val Leu Asp Trp Lys Pro Gln Gly Ser Val Ser Glu Gly Ser Ile Val
500 505 510
Ser Ser Ser Arg Lys Pro Ile Ser Ala Leu Ser Pro Gln Thr Asp Leu
515 520 525
Val Asp Asp Asn Ala Lys Ser Phe Ser Ser Gln Lys Ala Tyr Lys Ile
530 535 540
Val Leu Ala Gly Asp Ala Ala Val Gly Lys Ser Ser Phe Leu Met Arg
545 550 555 560
Leu Cys Lys Asn Glu Phe Arg Glu Asn Ile Ser Ala Thr Leu Gly Val
565 570 575
Asp Phe Gln Met Lys Thr Leu Ile Val Asp Gly Glu Arg Thr Val Leu
580 585 590
Gln Leu Trp Asp Thr Ala Gly Gln Glu Arg Phe Arg Ser Ile Ala Lys
595 600 605
Ser Tyr Phe Arg Lys Ala Asp Gly Val Leu Leu Leu Tyr Asp Val Thr
610 615 620
Cys Glu Lys Ser Phe Leu Asn Ile Arg Glu Trp Val Asp Met Ile Glu
625 630 635 640
Asp Ala Ala His Glu Thr Val Pro Ile Met Leu Val Gly Asn Lys Ala
645 650 655
Asp Ile Arg Asp Thr Ala Ala Thr Glu Gly Gln Lys Cys Val Pro Gly
660 665 670
His Phe Gly Glu Lys Leu Ala Met Thr Tyr Gly Ala Leu Phe Cys Glu
675 680 685
Thr Ser Ala Lys Asp Gly Ser Asn Ile Val Glu Ala Val Leu His Leu
690 695 700
Ala Arg Glu Val Lys Lys Arg Thr Asp Lys Asp Asp Ser Arg Ser Ile
705 710 715 720
Thr Asn Leu Thr Gly Thr Asn Ser Lys Lys Ser Pro Gln Met Lys Asn
725 730 735
Cys Cys Asn Gly
740
Claims (10)
1. The method for detecting the expression of the RASEF gene in the nipple discharge is characterized in that the content of the mRNA of the RASEF transcript or/and the protein coded by the RASEF gene in a nipple discharge biological sample is used as a report index of the expression of the RASEF gene.
2. The method for detecting the expression of the RASEF gene in nipple discharge of claim 1, wherein the protein encoded by the RASEF gene is captured by a specific antibody and quantified by ELISA.
3. The method for detecting the expression of the RASEF gene in nipple discharge of claim 1, wherein the capture molecules of the mRNA of the RASEF transcript are primers and/or probes that specifically recognize the cDNA corresponding to the RASEF gene transcript, and the sequence of the cDNA is represented by SEQ ID No. 7.
4. The method for detecting the expression of the RASEF gene in nipple discharge of claim 2, wherein the amino acid sequence of the encoded protein is shown in SEQ ID No. 8.
5. The method for detecting RASEF gene expression in nipple discharge of claim 3, wherein the primer sequence is any one or combination of SEQ ID Nos. 1-6.
6. The application of RASEF is characterized in that RASEF is used as a marker and applied to preparation of a breast cancer screening kit.
7. The kit for screening the mRNA for the breast cancer based on the RASEF is characterized by comprising capture molecules of the mRNA of the RASEF transcript, wherein the capture molecules of the mRNA of the RASEF transcript are primers and/or probes for specifically identifying cDNA corresponding to the mRNA of the RASEF gene transcript, and the cDNA sequence is shown as SEQ ID No. 7.
8. The RASEF-based mRNA breast cancer screening kit according to claim 7, wherein the primer sequence is any one or combination of SEQ ID Nos. 1-6.
9. A protein breast cancer screening kit based on RASEF gene coding is characterized by comprising a specific antibody of the protein coded by the RASEF gene, wherein the amino acid sequence of the coded protein is shown in SEQ ID No. 8.
10. The kit for screening the protein mammary cancer based on the RASEF gene code according to claim 9, wherein the kit is a RASEF detection kit based on competitive ELISA or double antibody sandwich ELISA, and the RASEF detection kit based on competitive ELISA comprises a RASEF protein standard, an anti-RASEF monoclonal antibody, an enzyme-labeled secondary antibody and an enzyme substrate; the RASEF detection kit based on the double-antibody sandwich ELISA comprises an anti-RASEF monoclonal antibody, a biotin-labeled RASEF antibody, an avidin-enzyme complex and an enzyme substrate.
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Citations (3)
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WO2012023290A1 (en) * | 2010-08-20 | 2012-02-23 | Oncotherapy Science, Inc. | Rasef as tumor marker and therapeutic target for cancer |
CN103842822A (en) * | 2011-02-10 | 2014-06-04 | 北达科他大学 | Breast cancer diagnosis using nipple discharge |
CN109061165A (en) * | 2018-08-27 | 2018-12-21 | 深圳市第二人民医院 | A kind of immune chromatography test paper, detection method and the application of nipple discharge CEA detection |
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2020
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WO2012023290A1 (en) * | 2010-08-20 | 2012-02-23 | Oncotherapy Science, Inc. | Rasef as tumor marker and therapeutic target for cancer |
CN103842822A (en) * | 2011-02-10 | 2014-06-04 | 北达科他大学 | Breast cancer diagnosis using nipple discharge |
CN109061165A (en) * | 2018-08-27 | 2018-12-21 | 深圳市第二人民医院 | A kind of immune chromatography test paper, detection method and the application of nipple discharge CEA detection |
Non-Patent Citations (1)
Title |
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