CN103122387B - Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof - Google Patents
Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof Download PDFInfo
- Publication number
- CN103122387B CN103122387B CN201310045626.XA CN201310045626A CN103122387B CN 103122387 B CN103122387 B CN 103122387B CN 201310045626 A CN201310045626 A CN 201310045626A CN 103122387 B CN103122387 B CN 103122387B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- detection
- buffer
- washing
- use according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 49
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 27
- 238000003752 polymerase chain reaction Methods 0.000 title abstract description 26
- 206010020751 Hypersensitivity Diseases 0.000 title description 4
- 208000026935 allergic disease Diseases 0.000 title description 3
- 210000005266 circulating tumour cell Anatomy 0.000 title description 3
- 230000009610 hypersensitivity Effects 0.000 title description 3
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 41
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 40
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 40
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 27
- 238000000605 extraction Methods 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims description 47
- 238000000034 method Methods 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 210000005259 peripheral blood Anatomy 0.000 claims description 18
- 239000011886 peripheral blood Substances 0.000 claims description 18
- 239000011324 bead Substances 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 14
- 239000006174 pH buffer Substances 0.000 claims description 12
- 206010006187 Breast cancer Diseases 0.000 claims description 10
- 208000026310 Breast neoplasm Diseases 0.000 claims description 10
- 206010028980 Neoplasm Diseases 0.000 claims description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical group Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical group [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 8
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000013592 cell lysate Substances 0.000 claims description 7
- 239000003550 marker Substances 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 5
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 5
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 5
- 208000029742 colonic neoplasm Diseases 0.000 claims description 5
- 206010017758 gastric cancer Diseases 0.000 claims description 5
- 201000005202 lung cancer Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 239000002853 nucleic acid probe Substances 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000010839 reverse transcription Methods 0.000 claims description 5
- 201000011549 stomach cancer Diseases 0.000 claims description 5
- 102100034343 Integrase Human genes 0.000 claims description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 4
- 235000019270 ammonium chloride Nutrition 0.000 claims description 4
- 238000001917 fluorescence detection Methods 0.000 claims description 4
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 4
- 238000007885 magnetic separation Methods 0.000 claims description 4
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 210000003743 erythrocyte Anatomy 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 229940037001 sodium edetate Drugs 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 1
- 238000005336 cracking Methods 0.000 claims 1
- 230000003321 amplification Effects 0.000 description 13
- 238000003199 nucleic acid amplification method Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 101000998011 Homo sapiens Keratin, type I cytoskeletal 19 Proteins 0.000 description 6
- 238000012764 semi-quantitative analysis Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000003365 immunocytochemistry Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000007886 magnetic bead extraction Methods 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 208000011581 secondary neoplasm Diseases 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000490050 Eleutherococcus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- -1 Tris-HCl)) Chemical compound 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000037921 secondary disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides an application of a regent used in a detection method for preparing a kit for detecting specific markers of circulating tumor cells. The detection method comprises the following steps of: treating a sample; carrying out nucleic acid extraction on a treated product; and carrying out fluorescence PCR (Polymerase Chain Reaction) detection on a nucleic acid extractive. Furthermore, the invention further provides the reagent, the kit and the like in the application.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to application of Circulating Tumor Cells (CTCs) in a detection sample. In addition, the present invention relates to a reagent, a kit, and the like that can be used in the above-described applications.
Background
Statistically, about 90% of cancer patients die from secondary tumors, and metastasis of the primary tumor or release of Circulating Tumor Cells (CTCs) is often responsible for the formation of secondary tumors. As early as 1869, the scientist Ashworth first proposed the concept of CTCs. CTCs are currently defined as tumor cells released into the peripheral blood circulation from solid tumors or metastases, either spontaneously or as a result of a diagnostic procedure. Tumor cells which enter the circulation and are not cleared form tiny cancer emboli through migration, adhesion and mutual aggregation, and develop into metastases under certain conditions. Therefore, the detection of the CTCs in the peripheral blood indicates that the tumor metastasis is likely to occur, and the detection of the CTCs in the peripheral blood has important clinical application value and has great significance for clinical early screening and diagnosis, curative effect and prognosis. The U.S. Food and Drug Administration (FDA) has also approved a CTCs detection system for predicting progression-free survival (PFS) and total survival (OS) of metastatic breast, colorectal or prostate cancer.
For hundreds of millions of peripheral blood cells, the number of CTCs in the peripheral blood is rare, approximately every 105～107Only one CTCs is present in a single monocyte. The existing detection technologies for CTCs mainly include Immunocytochemistry (ICC), Flow Cytometry (FCM), and reverse transcription polymerase chain reaction (RT-PCR). The immunocytochemistry method has the defects that the sensitivity is low, and factors such as target antigen non-expression of poorly differentiated tumor cells, lymphocyte cross reaction and the like influence the detection specificity; flow cytometry is expensive, time consuming and low in sensitivity; the RT-PCR method is complicated in steps, time-consuming, long in time-consuming, easy to pollute and brings great inconvenience to clinical detection, and false positive and false negative can be caused by epithelial cell pollution, illegal transcription of markers, expression difference among individuals and the like during sampling.
Although there is a precedent of using fluorescence PCR to detect CTCs (see, for example, Jinxiangfeng, etc. non-small cell lung cancer patients peripheral blood CK19mRNA expression and significance, Qilu journal of medicine, 23 rd volume 3 th phase), it still suffers from false positive and false negative (especially false negative) results, and the interpretation results are not intuitive, and it is difficult to perform semi-quantitative analysis, so the sensitivity is very low, and it has to be judged by dividing "1 copy number per microliter is greater than or equal to the positive result, and 1 copy number per microliter is negative" (i.e. 1000 copy/ml), which has little value for popularization and use in the case that each milliliter of samples is only 1-100 copy in many practical patients, resulting in a lot of false negative results, so that doctors have no sense.
For this reason, the present inventors have made diligent and long-term efforts to find that the final detection results are not good using conventional RNA extraction kits, and thus improvement is required from the time of processing a sample (e.g., peripheral blood); it is also found that the improvement of partial primers and probes and their PCR amplification steps can make the interpretation result intuitive and can perform semi-quantitative analysis in a larger range; more surprisingly, the inventor abandons the research miszone of the prior art which pursues quantitative or semi-quantitative analysis, designs a primer pair and a probe which do not interfere with each other, and greatly increases the sensitivity and the reliability of qualitative analysis while keeping certain semi-quantitative analysis capability. Moreover, the method is easy to automate and scale, can reduce manual operation errors and pollution, saves operation time and cost, can be widely used for grading and prognosticating the diseases of patients with breast cancer, colon cancer, prostate cancer, lung cancer and gastric cancer, and provides a basis for selecting a treatment scheme and monitoring the treatment effect.
Disclosure of Invention
The invention aims to provide the application of reagents used in the novel detection method in the preparation of a kit for detecting circulating tumor cell molecule-specific markers. In addition, the invention also provides a reagent, a kit and the like which can be used in the application.
In particular, in a first aspect, the present invention provides the use of reagents used in a detection method for the manufacture of a kit for the detection of a marker specific for a cellular molecule of a circulating tumour (e.g. breast, colon, prostate, lung and/or stomach cancer, preferably breast cancer), wherein the detection method comprises:
(1) processing a sample (e.g., peripheral blood);
(2) extracting nucleic acid from the treated product obtained in the step (1); and,
(3) and (3) carrying out fluorescence PCR detection on the nucleic acid extract obtained in the step (2).
Preferably in the use of the first aspect of the invention, the detection of the circulating tumour cell molecule specific marker is also carried out by said detection method, although the circulating tumour cell molecule specific marker may be detected by other methods.
In the kit, different reagents may be separately contained in different containers, or several reagents which are stored for a long period of time without chemical reaction may be selected and stored in the same container. The container may be a bottle, a cartridge, a syringe, or the like capable of containing the above-mentioned reagents, such as containers conventionally used for containing PCR, enzyme, or nucleic acid reagents. Since some reagents are readily available, these reagents may not be included, e.g., only a portion of the reagents may be included in the kit. In addition, the kit may also contain a label or instructions indicating that the assay according to the first aspect of the invention is to be carried out. The label may be affixed to the container or printed directly onto the container, or separate instructions may be provided. The kit may be further packaged in a larger package as desired, such as for ease of transport and storage, and such products are also within the scope of the present invention. A preferred kit is the kit of the fourth aspect of the invention hereinafter.
As used herein, the term "sample" or "specimen" is used interchangeably to refer to an ex vivo sample, such as blood, blood product, urine or saliva, which potentially contains a target nucleic acid. Detection methods in the application of the present invention may be considered as an independent aspect of the present invention. The detection method is a double detection method, and aims at two CTCs markers respectively, so that the false positive of a qualitative result can be eliminated. Since whether the detected small amount or trace amount of CTCs can cause the secondary disease of the tumor or not can be judged by an experienced doctor according to the comprehensive conditions of the constitution, medical history, clinical symptoms, etc. of the corresponding person, the detection method in the application of the present invention is preferably a non-diagnostic method, i.e., a method in which the diagnosis result or health condition of the disease cannot be directly obtained.
Preferably, in the application of the first aspect of the present invention, the method for detecting fluorescence PCR in step (3) comprises: (3-1) mixing a nucleic acid extract, a reverse transcriptase, an RNase inhibitor, a DNA polymerase, dNTPs, a target nucleic acid primer pair and a probe labeled with a fluorophore and a quencher and having a detection wavelength different from that of the fluorophore labeled with the two probes in a single PCR reaction tube, wherein,
the target nucleic acid primer pair comprises:
AACGAGCCAGCCTATCTTGAATC，
TTCTGACTTGATCTGTGACCGGAG，
CTGAATCCAATCTTTGCTCACC, and
ATGCTTGAAGGCCAATATGA，
the target nucleic acid probe comprises:
CCGACATCGCCGCTCCGTAACAT, and
CCTCTATTCAGATCCAGCGGATA
(3-2) carrying out reverse transcription and PCR reaction, and detecting fluorescence with different wavelengths in real time; and,
(3-3) judging whether the marker target nucleic acid exists in the sample according to the Ct value calculated by the fluorescence detection result.
In this context, "single PCR reaction vessel" means that the reverse transcription and real-time PCR processes are technically carried out in the same vessel, without having to replace the vessel. In the real-time PCR method of the first aspect of the invention, the steps of cDNA synthesis, PCR amplification and real-time fluorescence detection can be completed in the same container, and the container does not need to be replaced in the whole process, so that the operation is convenient, the time is saved, and the error is reduced. The operator only needs to add the sample and the reaction reagent into the container, and the whole process is automatically completed by a common real-time fluorescence PCR instrument sold in the market.
Typically, each probe is labeled with a fluorescent group at the 5 'end and a quencher group at the 3' end of the nucleotide sequence. Preferably wherein each probe is labelled with a different fluorophore. Preferably, fluorescent groups and quenching groups thereof are commercially available, e.g., those which can be used And conventional products, which have different detection wavelengths, and can also be used to synthesize probes labeled with fluorescent labels, with purity meeting HPLC standard and without impurity bands, wherein the concentration of each target nucleic acid probe in the single PCR reaction vessel is greater than 5pmol/ml, preferably 6-12 pmol/ml, more preferably 7-10 pmol/ml, and most preferably 8 pmol/ml. In the embodiment of the present invention, the fluorescent-labeled probes labeled with different detection wavelengths used therein are synthesized by Shanghai Biotechnology engineering services Co., Ltd, and the preferred fluorescent group is any combination of two of FAM, VIC, NED, TexasRed and CY 5. Therefore, it may be further preferred that in the method of fluorescence PCR detection in step (3), each probe is labeled with a different fluorescent group, preferably a fluorescent group selected from FAM, VIC, NED, Texas Red and CY 5.
In the present invention, in order to balance the amplification conditions of different primer pairs and to expand the range capable of semiquantitation as much as possible, experiments have found that the optimal conditions are as follows: the conditions for each cycle of the PCR reaction were 94 ℃ for 10 seconds and 60 ℃ for 30 seconds. In a specific embodiment of the present invention, the PCR reaction is preceded by reverse transcription, i.e., denaturation at 42 ℃ for 30min and preferably at 94 ℃ for 10min, and then 45 cycles of the above conditions are performed.
The nucleic acid extraction may be performed by a conventional boiling method, a phenol-chloroform extraction method, a TRIzol method, a column method, or the like, and preferably the optimized magnetic bead extraction method of the present invention, that is, the method for nucleic acid extraction in step (2) is preferably a method for extracting RNA by a magnetic bead method. Such a method, in combination with the method of sample processing below, can effectively ameliorate the impact of processing/extraction failure present in the prior art. Preferred methods of nucleic acid extraction include:
adding a nucleic acid extract (preferably, the nucleic acid extract contains guanidinium isothiocyanate, sodium ethylenediaminetetraacetate, tween-20, sodium perchlorate, ethanol, and a pH buffer (e.g., Tris-HCl) to the treated product, incubating, adding magnetic beads (e.g.,magnetic beads), mixed well, magnetically separated, the supernatant discarded, washed (preferably twice, more preferably wherein the wash used for the first wash comprises sodium perchlorate and ethanol, and still more preferably wherein the wash used for the second wash comprises ethanol), and eluted (preferably wherein the eluent used for the elution comprises a pH buffer (e.g., Tris-HCl).
Although the sample can be directly subjected to nucleic acid extraction and/or fluorescence PCR detection, the present inventors have found that the final detection result can be effectively improved by performing a preliminary treatment before the nucleic acid extraction. The use of claim 1, wherein the method of treatment in step (1) comprises,
removing plasma from peripheral blood, lysing with cell lysate (e.g., ammonium chloride red blood cell lysate), adding anti-CD 45 immunomagnetic beads, mixing, incubating, magnetically separating, discarding supernatant, and washing (preferably with pH buffer (e.g., PBS)).
The invention also provides reagents and preferably reagents for use in the first aspect of the invention. For example, in a second aspect, the present invention provides a reagent for use in the first aspect of the present invention, which is the reagent for use in step (3) of the use, characterized in that it comprises a target nucleic acid primer pair and a probe, wherein the probe is labeled with a fluorophore and a quencher, and the detection wavelengths of the fluorophore labeled with the two probes are different, wherein,
the target nucleic acid primer pair comprises:
AACGAGCCAGCCTATCTTGAATC，
TTCTGACTTGATCTGTGACCGGAG，
CTGAATCCAATCTTTGCTCACC, and
ATGCTTGAAGGCCAATATGA，
the target nucleic acid probe comprises:
CCGACATCGCCGCTCCGTAACAT, and
CCTCTATTCAGATCCAGCGGATA。
preferably the reagent of the second aspect of the invention further comprises a reverse transcriptase, an RNase inhibitor, a DNA polymerase and dNTPs (including dATP, dCTP, dGTP and dTTP). Thus, a complete fluorescent PCR kit can be constructed.
Also preferably in the reagent of the second aspect of the present invention, each probe is labeled with a different fluorescent group, preferably a fluorescent group selected from FAM, VIC, NED, Texas Red and CY 5.
As another example, in a third aspect, the invention provides a reagent for use in the first aspect of the invention, which is the reagent used in steps (1) and (2) of the use, characterized in that it comprises a cell lysate (e.g., ammonium chloride red cell lysate), anti-CD 45 immunomagnetic beads and a wash solution (preferably the wash solution is a pH buffer (e.g., PBS)), and a nucleic acid extract (preferably the nucleic acid extract comprises guanidinium isothiocyanate, sodium edetate, tween-20, sodium perchlorate, ethanol and a pH buffer (e.g., Tris-HCl)), magnetic beads (e.g.,D-Beads magnetic Beads), a wash solution (preferably two wash solutions, more preferably one of the wash solutions comprises sodium perchlorate and ethanol, and even more preferably the other wash solution comprises ethanol), and an eluent (preferably the eluent comprises a pH buffer (e.g., Tris-HCl)). Thus, a kit for peripheral blood treatment and nucleic acid extraction can be constituted. The inventors have found that it is possible to obtain,the treatment of peripheral blood and nucleic acid extraction are beneficial to the detection result of fluorescence PCR.
In a fourth aspect, the invention provides a kit for detecting a marker specific for a cellular molecule of a circulating tumour (e.g. breast, colon, prostate, lung and/or stomach cancer, preferably breast cancer) comprising an agent of the second aspect of the invention and/or an agent of the third aspect of the invention. Preferably, it does not contain an internal control nucleic acid.
Preferably the kit of the fourth aspect of the invention comprises reagents according to the second aspect of the invention.
It is also preferred that the kit of the fourth aspect of the invention comprises the reagents of the third aspect of the invention.
It is also preferred that the kit of the fourth aspect of the invention comprises a reagent of the second aspect of the invention and a reagent of the third aspect of the invention.
In a fifth aspect, the invention provides a primer, probe mixture, which is a mixture of primers or probes selected from the group consisting of
AACGAGCCAGCCTATCTTGAATC，
TTCTGACTTGATCTGTGACCGGAG，
CTGAATCCAATCTTTGCTCACC，
ATGCTTGAAGGCCAATATGA，
CCGACATCGCCGCTCCGTAACAT, and
CCTCTATTCAGATCCAGCGGATA。
the mixture may be used in the application of the first aspect of the invention and may also be used to constitute a reagent according to the second aspect of the invention and/or a kit according to the fourth aspect of the invention.
The invention has the beneficial effects that: the rapid and hypersensitive CTCs detection is established, can be used for detecting whether CTCs exist in peripheral blood of patients with breast cancer, colon cancer, prostate cancer, lung cancer and gastric cancer, has high accuracy, greatly reduces the possibility of false positive and false negative, particularly almost eliminates the false negative result, and is suitable for practical popularization and use; the sensitivity is high, the lowest detection limit can detect 1 copy tumor cell in 7.5mL blood, and is improved by several orders of magnitude compared with the prior art; moreover, the method is convenient and quick to operate, easy to automate and scale, capable of shortening the detection time, reducing errors caused by manual operation, free of adding internal control primers and probes and capable of saving the detection cost.
Drawings
FIG. 1 is a fluorescent quantitative PCR amplification curve diagram (the diagram is a combined diagram of different copy number amplification detections) of a CK19 gene positive standard (the positive standard is a plasmid DNA containing a CK19 gene detection site, which can be constructed according to an article by the full information (non-small cell lung cancer patient peripheral blood CK19mRNA expression and significance. Qilu medical journal, 23 rd 3 th period)) detected by using the kit of the invention, wherein the diagram is from left to right and is respectively 105copy/ml、104copy/ml、103copy/ml、102copy/ml CK19 gene standard fluorescent quantitative PCR amplification curve.
FIG. 2 shows the results of the quantitative fluorescent PCR amplification test on samples of 8 female patients clinically and pathologically diagnosed with breast cancer (the pictures are drawn by combining the amplification tests of different samples, and the same color indicates the amplification curves of two probes of the same patient).
The figures are original amplification graphs of the ABI7500 fluorescence real-time quantitative PCR amplification instrument with software, wherein the abscissa in the graphs is 'PCR cycle number', and the ordinate is 'fluorescence value of amplification reaction'.
Detailed Description
The invention will be described herein below by means of specific examples. Unless otherwise specified, the method can be performed according to the methods listed in the experimental manuals such as "molecular cloning laboratory Manual" (third edition) (Cold Spring harbor laboratory Press), "cellular laboratory Manual" (science publishers, Beijing, China, 2001), "RNA experimental technical Manual" (science publishers, Beijing, China, 2004), "immunoassay technology" (science publishers, Beijing, China, 1991), and the references cited herein, which are well known to those skilled in the art. The probes and primers used herein may be synthesized by Shanghai Biotechnology engineering services, Inc.
Example 1 treatment of peripheral blood samples
The experimental material of the embodiment is that peripheral blood of a breast cancer patient collected and pathologically confirmed in a certain hospital is collected, 7 am is obtained, fasting is carried out, fresh blood is collected through an elbow vein, the blood is stored in an anticoagulation tube, the shaking is carried out uniformly, and the storage time at normal temperature is less than or equal to 4 hr.
1. Adding the whole blood sample in an anticoagulation tube into PBS (pH7.0) with the same volume, centrifuging at room temperature of 3000rpm for 5min, and removing the upper plasma;
2. adding equal volume of ammonium chloride erythrocyte lysate (from Biyun day) into the retained liquid, centrifuging at room temperature of 20rpm for 8min, mixing, centrifuging at 3000rpm at room temperature for 5min, and discarding the supernatant;
3. adding 1/10 volumes of anti-CD 45 immunomagnetic beads (available from Ningbo Liri antibody Biotechnology Co.), and mixing at room temperature for 20min on a shaker at 110 rpm;
4. after magnetic bead separation (discarding the supernatant) was performed using a magnetic separation rack, the magnetic beads were washed with PBS (pH 7.0), and the washings were combined to obtain a peripheral blood sample treatment solution.
Example 2 extraction of nucleic acids
The extraction of nucleic acid adopts a conventional magnetic bead extraction method, and comprises the following steps: peripheral blood obtained in example 1 at a volume ratio of 1: 100Adding nucleic acid extract (formula and final concentration: 1.2M guanidinium isothiocyanate, 10mM sodium ethylenediaminetetraacetate (pH8.0), tween-202% (W/W), 1M sodium perchlorate, 40% (V/V) ethanol, 10mM Tris-HCl (pH8.0)) into the sample treatment solution, bathing at 42 deg.C for 10min, and adding into the solution at a volume ratio of 10: 1Magnetic bead suspension (50 mg/mL, available from Beijing Eleutherococcus biotechnology, Inc.), shaking and mixing, removing supernatant after magnetic separation, adding appropriate amount of washing solution A (formula and final concentration: sodium perchlorate 1M, ethanol 25% (V/V)), washing, discarding washing solution A, adding appropriate amount of washing solution B (formula and final concentration: ethanol 70% (V/V)), washing, discarding washing solution B, adding eluent (formula and final concentration: Tris-HCl (pH8.0) 10 mM), and warm-bathing at 42 deg.C for 10min to obtain supernatant as nucleic acid extract, and diluting for detection in the following steps.
Example 3 fluorescent real-time quantitative PCR amplification
1, primer and Probe sequences
The following primer pairs and probes were synthesized, 6 nucleic acid sequences in total:
wherein the primer pair is as follows:
AACGAGCCAGCCTATCTTGAATC
TTCTGACTTGATCTGTGACCGGAG
CTGAATCCAATCTTTGCTCACC
ATGCTTGAAGGCCAATATGA
wherein the probe is
CCGACATCGCCGCTCCGTAACAT
CCTCTATTCAGATCCAGCGGATA
2, fluorescent labeling
The 5 'end of the probe is respectively marked with fluorescence labeling FAM and CY5, and the 3' end is marked with corresponding quenching groups.
3, PCR reaction conditions
The reverse transcription and the PCR amplification are carried out in the same reaction system, the total volume of the system is 60uL, and the final concentration of each component is as follows: 40uL of the nucleic acid extract obtained in example 2 or the CK19 gene positive standard at various dilutions, 15pmol/ml for each of the above 2 primer pairs, 8pmol/ml for each of the above 2 probes, 0.2mmol/ml for each of dNTP concentrations, and 10 XPCR buffer (available from TaKaRa, pH8.3, containing Mg2+) 6uL of Taq DNA polymerase, 5U of M-MLV reverse transcriptase, 20U of Rnasin5U and the balance of deionized water.
The steps of reverse transcription and PCR reaction are as follows: 30min at 42 ℃, 10min at 94 ℃ (10 s at 94 ℃, 30s at 60 ℃) for 45 cycles, and finally, using an ABI7500 real-time fluorescence PCR instrument to collect FAM and CY5 detection wavelengths by fluorescence.
4, interpretation of results criteria
Both the baseline and threshold values were automatically set by default for the ABI7500 fluorometer. If the Ct value of each fluorescence (FAM and/or CY 5) is less than or equal to 45, the CTCs exist in the sample and are judged to be positive; if the Ct value calculated by the fluorescence detection result is more than 45, the CTCs do not exist in the sample, and the sample is judged to be negative. Furthermore, if one primer pair is detected as positive by the corresponding probe, the amplification is qualitatively judged as positive regardless of whether or not the semi-quantitative analysis can be performed.
5, results
The amplification results of CK19 gene positive standard (as a control) at different dilutions are shown in FIG. 1, which indicates that the standard samples with different copy numbers can reflect accurate results and have very good readability of the results, which is 102To 105The linear relation of logarithm in the concentration range is good, and the semi-quantitative analysis can be carried out on the sample with the content of 100copy/ml or more, and the semi-quantitative range is suitable for carrying out the semi-quantitative analysisThe enclosure has advantages over the prior art.
The detection pattern for the actual patients is shown in FIG. 2, not only all can show the correct qualitative result of amplification, but also the two primer pairs and the probes do not interfere with each other, and CK19 with high copy number can be used for comparing with a control to obtain a semi-quantitative result.
In conclusion, the kit provided by the invention is rapid and high in sensitivity, the interpretation of the result is very clear and intuitive, and particularly, the (qualitative) result is accurate when repeatedly detecting samples of a large number of different actual patients, which shows that the reliability of the kit is high and internal control is not needed.
Sequence listing
<110> Huayimei Biotechnology Ltd, Suzhou
<120> fluorescent PCR (polymerase chain reaction) rapid hypersensitivity detection kit for Circulating Tumor Cells (CTCs) and application thereof
<130>CN
<160>6
<170>PatentIn version3.5
<210>1
<211>23
<212>DNA
<213> Artificial sequence
<400>1
aacgagccag cctatcttga atc 23
<210>2
<211>24
<212>DNA
<213> Artificial sequence
<400>2
ttctgacttg atctgtgacc ggag 24
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<400>3
ctgaatccaa tctttgctca cc 22
<210>4
<211>20
<212>DNA
<213> Artificial sequence
<400>4
atgcttgaag gccaatatga 20
<210>5
<211>23
<212>DNA
<213> Artificial sequence
<400>5
ccgacatcgc cgctccgtaa cat 23
<210>6
<211>23
<212>DNA
<213> Artificial sequence
<400>6
cctctattca gatccagcgg ata 23
Claims (19)
1. Use of reagents for use in a detection method for the manufacture of a kit for the detection of a marker specific for a circulating tumor cell molecule, wherein the detection method comprises:
(1) processing the sample;
(2) extracting nucleic acid from the treated product obtained in the step (1); and,
(3) performing fluorescent PCR detection on the nucleic acid extract obtained in the step (2), and the method for fluorescent PCR detection in the step (3) comprises:
(3-1) mixing a nucleic acid extract, a reverse transcriptase, an RNase inhibitor, a DNA polymerase, dNTPs, a target nucleic acid primer pair and a probe labeled with a fluorophore and a quencher and having a detection wavelength different from that of the fluorophore labeled with the two probes in a single PCR reaction tube, wherein,
the target nucleic acid primer pair comprises:
AACGAGCCAGCCTATCTTGAATC
TTCTGACTTGATCTGTGACCGGAG
CTGAATCCAATCTTTGCTCACC, and
ATGCTTGAAGGCCAATATGA
the target nucleic acid probe comprises:
CCGACATCGCCGCTCCGTAACAT, and
CCTCTATTCAGATCCAGCGGATA；
wherein,
each probe is marked with different fluorescent groups;
the conditions for each cycle of the PCR reaction were 94 ℃ for 10s and 60 ℃ for 30 s; and,
the concentration of each target nucleic acid probe in a single PCR reaction vessel was 8 pmol/ml;
(3-2) carrying out reverse transcription and PCR reaction, and detecting fluorescence with different wavelengths in real time; and,
(3-3) judging whether the marker target nucleic acid exists in the sample according to the Ct value calculated by the fluorescence detection result.
2. The use according to claim 1, wherein the tumor is breast cancer, colon cancer, prostate cancer, lung cancer and/or stomach cancer.
3. The use of claim 1, wherein the tumor is breast cancer.
4. The use of claim 1, wherein the sample is peripheral blood.
5. The use according to claim 1, wherein the fluorescent group is selected from FAM, VIC, NED, Texas Red and CY 5.
6. The use according to claim 1, wherein the nucleic acid extraction method in step (2) is a magnetic bead method for extracting RNA.
7. The use according to claim 6, wherein the nucleic acid extraction method comprises,
adding the nucleic acid extract into the treated product, keeping the temperature, adding magnetic beads, mixing uniformly, performing magnetic separation, discarding the supernatant, washing, and eluting.
8. The use of claim 7, wherein the nucleic acid extract comprises guanidinium isothiocyanate, sodium edetate, tween-20, sodium perchlorate, ethanol, and a pH buffer.
9. The use of claim 8, wherein the pH buffer is Tris-HCl.
10. Use as claimed in claim 7, wherein the magnetic beads areMagnetic beads.
11. Use as claimed in claim 7 wherein the washing is twice.
12. Use according to claim 11, wherein the washing liquid used in the first washing comprises sodium perchlorate and ethanol.
13. Use according to claim 11, wherein the wash liquor used for the second wash comprises ethanol.
14. The use according to claim 7, wherein the elution solution used comprises a pH buffer.
15. The use of claim 14, wherein the pH buffer is Tris-HCl.
16. The use of claim 1, wherein the method of treatment in step (1) comprises,
removing plasma of peripheral blood, cracking with cell lysate, adding anti-CD 45 immunomagnetic beads, mixing, keeping temperature, performing magnetic separation, discarding supernatant, and washing.
17. The use according to claim 16, wherein the cell lysate is ammonium chloride red blood cell lysate.
18. The use of claim 16, wherein the washing is with a pH buffer.
19. The use of claim 18, wherein the pH buffer is PBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045626.XA CN103122387B (en) | 2013-02-05 | 2013-02-05 | Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045626.XA CN103122387B (en) | 2013-02-05 | 2013-02-05 | Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103122387A CN103122387A (en) | 2013-05-29 |
| CN103122387B true CN103122387B (en) | 2015-02-04 |
Family
ID=48453528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310045626.XA Active CN103122387B (en) | 2013-02-05 | 2013-02-05 | Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103122387B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468828A (en) * | 2013-09-16 | 2013-12-25 | 上海卓润生物科技有限公司 | PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube |
| KR101619862B1 (en) * | 2014-09-30 | 2016-05-12 | 주식회사 녹십자 | Kit and Method of Measuring Titers of Protein Comprising Human Fc Using Indirect Enzyme-linked Immunosorbent Assay |
| EP3230744B1 (en) * | 2014-12-12 | 2021-05-12 | Exact Sciences Development Company, LLC | Compositions and methods for performing methylation detection assays |
| CN106282116A (en) * | 2016-08-08 | 2017-01-04 | 北京科迅生物技术有限公司 | The enrichment of a kind of colorectal cancer circulating tumor cell and the method for analysis |
| CN106987649A (en) * | 2017-05-31 | 2017-07-28 | 上海博慷生物科技有限公司 | A kind of primer sets and detection method for being used to detect glioma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101365800A (en) * | 2005-08-17 | 2009-02-11 | 麦蒂克西斯股份有限公司 | Composition and method for determination of CK19 expression |
| CN102272595A (en) * | 2008-11-04 | 2011-12-07 | 硅生物系统股份公司 | Method for identification, selection and analysis of tumour cells |
-
2013
- 2013-02-05 CN CN201310045626.XA patent/CN103122387B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101365800A (en) * | 2005-08-17 | 2009-02-11 | 麦蒂克西斯股份有限公司 | Composition and method for determination of CK19 expression |
| CN102272595A (en) * | 2008-11-04 | 2011-12-07 | 硅生物系统股份公司 | Method for identification, selection and analysis of tumour cells |
Non-Patent Citations (1)
| Title |
|---|
| Real-Time Quantification of CK-19 mRNA-Positive Cells in Peripheral Blood of Breast Cancer Patients Using the Lightcycler System;Aliki Stathopoulou et al;《clinical cancer》;20031101;第9卷;摘要、第5145页右栏第2段、第5146页左栏第2段-第5147页右栏第1段、表1、第5148页图2、第5150页左栏第2段、右栏第2段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103122387A (en) | 2013-05-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103122387B (en) | Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof | |
| CN109777877B (en) | Detection kit for auxiliary diagnosis of cerebral aneurysm based on PTBP1 methylation and application thereof | |
| CN107245531B (en) | Diarrhea pathogen multiple gene detection system and kit and application thereof | |
| CN112501268A (en) | Nanopore sequencing-based primer group and kit for rapidly identifying respiratory microorganisms and application of primer group and kit | |
| CN109457024A (en) | Shr gene polymorphism fluorescent PCR solubility curve detection kit and application | |
| CN104894118A (en) | Primer, probe and kit for detecting bovine viral diarrhea virus | |
| CN105803075A (en) | Alpha-thalassemia gene detecting kit | |
| CN108660215B (en) | Application of reagent for detecting circMAN1A2 and circRNF13 and kit | |
| CN106521023B (en) | A kind of kit and method detecting Chinese HPFH and δ β-thalassemia | |
| CN108796075B (en) | Application of reagent for detecting circRNF13 and LOC284454 and kit | |
| CN105838777A (en) | Method for monitoring secondary drug resistance of lung cancer patient to tyrosine kinase inhibitor through ddPCR technology | |
| CN108796074B (en) | Application of reagent for detecting circular RNA circRNF13 in preparation of tumor auxiliary diagnosis preparation and kit | |
| CN107083446B (en) | Diarrhea pathogenic bacteria multiple gene detection system and kit and application thereof | |
| CN111187842A (en) | Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer | |
| CN101864492A (en) | Nucleic acid detection kit for aided diagnosis of ankylosing spondylitis | |
| CN111394434B (en) | CHO host cell DNA residue detection kit adopting TaqMan probe method and application thereof | |
| CN111172273B (en) | Primer group, kit and detection method for SMN1 gene detection | |
| CN108660213B (en) | Application of reagent for detecting three non-coding RNAs and kit | |
| CN111057791A (en) | Kit for detecting HBV pgRNA in blood | |
| Tang et al. | Establishment and validation of a novel droplet digital PCR assay for ultrasensitive detection and dynamic monitoring of EGFR mutations in peripheral blood samples of non-small-cell lung cancer patients | |
| CN114427009A (en) | Multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus | |
| CN115717167A (en) | Novel marker combination and kit for early detection of multi-target liver cancer | |
| CN104946735A (en) | Kit and method for determining genotype of predetermined SNP site of DNA sample to be tested | |
| CN114410795A (en) | Liver cancer early detection based on miRNA (micro ribonucleic acid) feature marker | |
| CN108823308B (en) | Application of reagent for detecting circMAN1A2 and LOC284454 and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Rapid circulating tumor cell (CTCs) fluorescence PCR (Polymerase Chain Reaction) hypersensitivity detection kit and application thereof Effective date of registration: 20170621 Granted publication date: 20150204 Pledgee: Bank of Nanjing Limited by Share Ltd. Suzhou branch Pledgor: SUZHOU BACME BIOTECH Co.,Ltd. Registration number: 2017320010041 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230111 Granted publication date: 20150204 Pledgee: Bank of Nanjing Limited by Share Ltd. Suzhou branch Pledgor: SUZHOU BACME BIOTECH Co.,Ltd. Registration number: 2017320010041 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |