CN110982886A - Human K-ras gene mutation detection kit and application thereof - Google Patents

Human K-ras gene mutation detection kit and application thereof Download PDF

Info

Publication number
CN110982886A
CN110982886A CN201911395744.7A CN201911395744A CN110982886A CN 110982886 A CN110982886 A CN 110982886A CN 201911395744 A CN201911395744 A CN 201911395744A CN 110982886 A CN110982886 A CN 110982886A
Authority
CN
China
Prior art keywords
ras gene
gene mutation
probe
primer
human
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.)
Pending
Application number
CN201911395744.7A
Other languages
Chinese (zh)
Inventor
赵平锋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan Optical Valley United Medical Laboratory Co Ltd
Original Assignee
Wuhan Optical Valley United Medical Laboratory Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Wuhan Optical Valley United Medical Laboratory Co Ltd filed Critical Wuhan Optical Valley United Medical Laboratory Co Ltd
Priority to CN201911395744.7A priority Critical patent/CN110982886A/en
Publication of CN110982886A publication Critical patent/CN110982886A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a human K-ras gene mutation detection kit and application thereof, wherein the kit comprises a PCR reaction solution, a primer probe mixed solution, a positive quality control product and a negative quality control product, the primer probe mixed solution comprises a forward primer, a reverse primer, a fluorescent probe and a locked nucleic acid probe, and the concentration ratio of the primer probe is optimized. The invention combines the locked nucleic acid technology and the fluorescent quantitative PCR technology to detect K-ras gene mutation, is simple and easy to implement, has high specificity and sensitivity as high as 0.1 percent, and provides an effective means for prognosis judgment after tumor screening.

Description

Human K-ras gene mutation detection kit and application thereof
Technical Field
The invention relates to the field of gene detection, in particular to a human K-ras gene mutation detection kit and application thereof.
Background
The formation, progression and metastasis of malignant tumors are associated with irreversible accumulation of mutations and dysregulation of oncogenes and tumor suppressor genes. Among them, Ras gene is a protooncogene found in rat sarcoma virus and is the first oncogene to be isolated and identified from human tumor cells. It encodes a 21kD immune-related protein, designated p21s protein or RAS protein. RAS proteins are GDP/GTP-binding proteins that act as intracellular signal transducers. The main role of RAS proteins in wild-type cells is to regulate proliferation, differentiation and senescence. Researches show that the Ras proto-oncogene is involved in the occurrence and development processes of many kinds of cancers, and the Ras proto-oncogene becomes an oncogene through activation, and the common activation mode comprises point mutation, high expression level, insertion mutation, translocation mutation and the like.
Ras gene family and human tumor associated genes including K-Ras, N-Ras and H-Ras 3. Ras gene mutation can occur in various tumors, K-Ras mutation mainly occurs in colorectal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer and endometrial cancer, N-Ras mutation is commonly seen in melanoma and hematological malignancy, H-Ras mutation is commonly seen in liver cancer, thyroid cancer and bladder cancer, and the K-Ras gene mutation is most widely and commonly involved. 17% -25% of tumors have activated K-ras gene mutation, and the key regions for activation are codons 12,13, 59, 61 and 63, wherein the K-ras gene mutation sites are almost concentrated at codons 12 and 13, and the common hot spot mutations include 7, namely six mutation types of codon 12 (GGT > GAT, GGT > GCT, GGT > GTT, GGT > AGT, GGT > CGT, GGT > TGT) and one mutation type of codon 13 (GGC > GAC). The K-ras gene mutation varies among different types of tumors, and different mutation sites thereof affect the prognosis of patients to some extent. Therefore, the mutation detection and analysis of the K-ras gene in different tumors can provide theoretical basis for selecting targeted drugs, selecting postoperative treatment schemes and formulating individualized treatment strategies.
At present, the main method for detecting K-ras gene mutation is a traditional sanger sequencing method, the detection is time-consuming, the operation process is complex, pollution is easily caused, the sensitivity is low, the detection rate is low particularly when a template is mixed, and meanwhile, the detection cost is high.
Disclosure of Invention
In view of the above, the invention provides a human K-ras gene mutation detection kit with strong specificity, high sensitivity and simple and rapid operation and application thereof.
The technical scheme of the invention is realized as follows:
in a first aspect, the present invention provides a primer probe composition for detecting human K-ras gene mutation, comprising a forward primer, a reverse primer and a fluorescent probe.
The sequence of the forward primer is shown as SEQ ID NO.1, 5'-CTGGTGGAGTATTTGATAGTGTATT-3';
the sequence of the reverse primer is shown as SEQ ID NO.2, 5'-CAAGATTTACCTCTATTGTTGGA-3';
the sequence of the fluorescent probe is shown in SEQ ID NO.3, 5'-CTGCTGAAAATGACTGAA-3', the 5 'end of the fluorescent probe is marked with a fluorescent group FAM, and the 3' end of the fluorescent probe is marked with a quenching group BHQ 1.
Based on the above technical scheme, preferably, the primer probe composition further comprises a locked nucleic acid probe, wherein the sequence of the locked nucleic acid probe is shown as SEQ ID No.4, 5'-CTACGCCACCAGCT-3', and the 3 ' end of the locked nucleic acid probe is connected with a PO4 group.
In addition to the above technical means, preferably, the locked nucleic acid probe sequence is modified with LAN at 3 rd, 6 th, 7 th, 9 th, 10 th, and 11 th bases.
In a second aspect, the invention provides a human K-ras gene mutation detection kit, which comprises a PCR reaction solution, a primer probe mixed solution, a positive quality control product and a negative quality control product, wherein the primer probe mixed solution comprises the primer probe composition of the first aspect of the invention.
On the basis of the technical scheme, preferably, the human K-ras gene mutation detection kit is applied to fluorescent quantitative PCR reaction, and the concentration ratio of forward and reverse primers, a fluorescent probe and a locked nucleic acid probe in a primer probe mixed solution in a reaction system is 2 (1-2) to (3-4).
On the basis of the technical scheme, preferably, the positive quality control product is plasmid DNA containing K-ras gene mutation fragments.
In a third aspect, the invention provides the use of the human K-ras gene mutation detection kit of the second aspect of the invention in detecting the K-ras gene mutation ratio.
In the fourth aspect, the invention also provides a method for detecting human K-ras gene mutation, which adopts the human K-ras gene mutation detection kit in the second aspect of the invention and takes the nucleic acid of a sample to be detected as a template to carry out fluorescent quantitative PCR detection.
Compared with the prior art, the human K-ras gene mutation detection kit and the application thereof have the following beneficial effects:
(1) the invention designs and synthesizes primers for amplifying K-ras gene segments by PCR, utilizes the characteristics of locked nucleic acid, designs and synthesizes probes marked by the locked nucleic acid around the 12 th and 13 th codons of the K-ras, takes the locked nucleic acid as a complex of the probe and a DNA chain, has high thermal stability, is sensitive to base mismatching, and the melting temperature of the DNA can be greatly reduced by the mismatching of one base;
(2) the specific primer probe composition provided by the kit can detect whether the K-ras gene is mutated or not at high sensitivity, the locked nucleic acid probe technology is combined with the real-time fluorescence PCR technology, the wild type and the mutant type can be completely separated by one-time reaction, and meanwhile, the processes of glue preparation, electrophoresis, sequencing and the like are reduced due to direct closed-tube operation, so that the pollution probability is greatly reduced, the false positive rate is reduced, the whole process is simple and easy to implement, and the detection sensitivity can reach 0.1%.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the amplification of wild-type and mutant templates of K-ras genes No. 12 and 13 in example 2.
FIG. 2 is a gel electrophoresis photograph showing the amplification products of wild-type template and mutant template of K-ras gene Nos. 12 and 13 in example 2, in which lanes 1 to 7 are amplification products of mutant template, No. 8 is amplification product of wild-type template, No. 9 is amplification product of TE buffer as template, and M represents DNA marker.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified, and reference is made to the molecular cloning protocols (fourth edition). The primers and probes used in the examples were synthesized by the firm of Biotechnology engineering (Shanghai) Ltd.
Example 1 human K-ras Gene mutation detection kit
S1, design and synthesis of primer and probe
The primer probe sequences for K-ras gene mutation detection are as follows:
a forward primer: 5'-CTGGTGGAGTATTTGATAGTGTATT-3', respectively;
reverse primer: 5'-CAAGATTTACCTCTATTGTTGGA-3', respectively;
a fluorescent probe: 5 '-FAM-CTGCTGAAAATGACTGAA-BHQ 1-3';
locked nucleic acid probe: 5' -CTACGCCACCAGCT-PO 4-3', in which bases 3, 6, 7, 9, 10, 11 of the sequence are labeled with LAN.
S2 and other reagents
(1) The 2 × PCR reaction solution contains: Tris-HCl buffer (pH8.3)20mmol/L, MgCl24mmol/L, KCl100mmol/L, dNTPs 2mmol/L, Taq DNA polymerase 0.08U/μ L;
(2) the positive quality control product is an artificial plasmid containing K-ras gene mutation fragments;
(3) the negative quality control material is TE buffer solution without nucleic acid and artificial plasmid containing K-ras wild type gene.
Example 2 detection of mutations at codons 12 and 13 of K-ras Gene by fluorescent quantitative PCR
S1, artificial plasmid synthesis and extraction
A plasmid containing K-ras gene codons 12 and 13 and homologous sequences of 200bp at the upstream and the downstream of the codons is synthesized by a biological engineering (Shanghai) corporation, and simultaneously 6 mutations (GGT > GAT, GGT > GCT, GGT > GTT, GGT > AGT, GGT > CGT, GGT > TGT) of the codon 12 and 1 mutation (GGC > GAC) of the codon 13 are respectively introduced into the plasmid to finally obtain a wild type plasmid and 7 mutant plasmids. The K-ras wild-type and 7 mutant artificial plasmids were purified using a plasmid extraction kit (Shanghai Biotech) according to the protocol. The purified 8 plasmids were quantitated using a NanoDrop 2000 ultramicro spectrophotometer and diluted to 1000 copies/. mu.L with deionized water. Meanwhile, the mutant plasmid and the wild type plasmid are mixed according to the proportion of 1:1000, and the content of the mutant plasmid in the finally obtained mixed plasmid is 0.1 percent.
S2 fluorescent quantitative PCR
Respectively taking 7 mutant plasmids as templates to carry out fluorescence quantitative PCR amplification, and preparing a PCR reaction system: 2 XPCR reaction solution 12.5. mu.L, primer probe mixture 2. mu.L, DNA template 2. mu.L, and complement ddH2O to 25. mu.L. In the amplification reaction system, the concentration of forward and reverse primers is 0.2 mu mol/L, the concentration of fluorescent probes is 0.2 mu mol/L, and the concentration of locked nucleic acid probes is 0.3 mu mol/L. TE buffer without nucleic acid and pure wild-type template were used as controls.
The PCR conditions were set on the PCR machine as shown in Table 1, and the fluorescent quantitative PCR reaction was completed.
TABLE 1 fluorescent quantitative PCR reaction conditions
Figure BDA0002346255200000051
Observing the PCR reaction result, as shown in FIG. 1, the amplification curves using the mutant plasmids as templates are S-shaped, and Ct values are between 20 and 30, so that the products can be effectively amplified; the amplification curves of the control group with TE buffer without nucleic acid and pure wild-type template were both flat and Ct values greater than 36, indicating that neither amplification was efficient. Furthermore, the PCR products were detected by gel electrophoresis, as shown in FIG. 2, it was found that the amplification products using TE buffer solution containing no nucleic acid and pure wild-type plasmid as templates were very few and invisible on the gel, while the amplification products using mutant plasmid as template were numerous and consistent in size. It is demonstrated that the kit of the present invention can detect mutations in the mixture as low as 0.1%.
S3 optimization of primer probe proportion
Selecting a recombinant plasmid containing a K-ras gene 13 codon mutant and a wild type plasmid as templates, and performing multiple amplifications according to the same method of S2: when the concentrations of the forward and reverse primers, the fluorescent probe and the locked nucleic acid probe in the PCR reaction system are respectively 0.2 mu mol/L, 0.2 mu mol/L and 0.3 mu mol/L, the Ct value of the amplification result of the mutant plasmid is 25.73-28.25, and the Ct value of the amplification result of the wild plasmid is more than 36; when the concentrations of the forward and reverse primers, the fluorescent probe and the locked nucleic acid probe in the PCR reaction system are respectively 0.2 mu mol/L, 0.1 mu mol/L and 0.4 mu mol/L, the Ct value of the amplification result of the mutant plasmid is 23.18-25.86, and the Ct value of the amplification result of the wild plasmid is more than 36; when the concentrations of the forward and reverse primers, the fluorescent probe and the locked nucleic acid probe in the PCR reaction system are respectively 0.2 mu mol/L, 0.2 mu mol/L and 0.4 mu mol/L, the Ct value of the amplification result of the mutant plasmid is 26.69-29.55, and the Ct value of the amplification result of the wild plasmid is more than 36.
Example 3 detection of K-ras Gene mutation in Paraffin tissue samples
S1, extraction and purification of tissue sample genome DNA
200 patients with non-small cell lung cancer are selected as subjects, 10% neutral formalin-fixed paraffin-embedded non-small cell lung tumor tissue sections are used, a paraffin-embedded tissue DNA extraction kit of TIANGEN company is used for extracting genomic DNA from the sample paraffin-embedded tissue sections, and the operation steps are detailed in the kit specification. The extracted genomic DNA was quantified using a NanoDrop 2000 ultramicro spectrophotometer and diluted to a concentration of 20 ng/. mu.L with deionized water.
S2 fluorescent quantitative PCR
The extracted and purified genomic DNA of the tissue sample was assayed by the procedure of step S2 in example 2, using any one of the 7 mutant artificial plasmids prepared in example 2 as a positive quality control substance and TE buffer solution containing no nucleic acid as a negative quality control substance; and simultaneously detecting the DNA by using a DNA direct sequencing method. The results are shown in Table 2.
TABLE 2 results of tissue samples tested by different methods
Detection method K-ras mutations K-ras wild type Rate of positive detection
The detection method of the invention 122 78 61％
DNA sequencing 107 93 53.5％
As shown in Table 2, the positive detection rate of the kit for tissue samples is 61%, which is obviously higher than that of direct DNA sequencing, because the wild type DNA is mixed in the actual tissue samples, the mutant concentration is relatively low, and the direct DNA sequencing result is interfered, and the kit for detection of the tissue samples can inhibit the amplification of the wild type DNA, enrich the mutant amplification and improve the sensitivity and specificity of detection.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan Guanggu valley combined medical laboratory GmbH
<120> human K-ras gene mutation detection kit and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>25
<212>DNA
<213> (Artificial sequence)
<400>1
ctggtggagt atttgatagt gtatt 25
<210>2
<211>23
<212>DNA
<213> (Artificial sequence)
<400>2
caagatttac ctctattgtt gga 23
<210>3
<211>18
<212>DNA
<213> (Artificial sequence)
<400>3
ctgctgaaaa tgactgaa 18
<210>4
<211>14
<212>DNA
<213> (Artificial sequence)
<400>4
ctacgccacc agct 14

Claims (8)

1. A primer probe composition for detecting human K-ras gene mutation, characterized in that: the kit comprises a forward primer, a reverse primer and a fluorescent probe, wherein the sequence of the forward primer is shown as SEQ ID NO.1, the sequence of the reverse primer is shown as SEQ ID NO.2, and the sequence of the fluorescent probe is shown as SEQ ID NO. 3.
2. The primer probe composition of claim 1, wherein: the kit also comprises a locked nucleic acid probe, wherein the sequence of the locked nucleic acid probe is shown as SEQ ID NO.4, and the 3' end of the locked nucleic acid probe is connected with a PO4 group.
3. The primer probe composition of claim 2, wherein: the 3 rd, 6 th, 7 th, 9 th, 10 th and 11 th bases of the locked nucleic acid probe sequence are subjected to LAN modification.
4. A human K-ras gene mutation detection kit comprises a PCR reaction solution, a primer probe mixed solution, a positive quality control product and a negative quality control product, and is characterized in that: the primer probe mixture includes the primer probe composition according to claim 2.
5. The human K-ras gene mutation detection kit of claim 4, wherein: the method is applied to fluorescent quantitative PCR reaction, and the concentration ratio of forward and reverse primers, fluorescent probes and locked nucleic acid probes in the primer probe mixed solution in a reaction system is 2 (1-2) to 3-4.
6. The human K-ras gene mutation detection kit of claim 4, wherein: the positive quality control product is plasmid DNA containing K-ras gene mutation fragments.
7. The use of the human K-ras gene mutation detection kit of claim 4 in detecting the K-ras gene mutation ratio.
8. A method for detecting human K-ras gene mutation, which is characterized by comprising the following steps: the human K-ras gene mutation detection kit of claim 4, which is used for carrying out fluorescent quantitative PCR detection by using a nucleic acid of a sample to be detected as a template.
CN201911395744.7A 2019-12-30 2019-12-30 Human K-ras gene mutation detection kit and application thereof Pending CN110982886A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911395744.7A CN110982886A (en) 2019-12-30 2019-12-30 Human K-ras gene mutation detection kit and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911395744.7A CN110982886A (en) 2019-12-30 2019-12-30 Human K-ras gene mutation detection kit and application thereof

Publications (1)

Publication Number Publication Date
CN110982886A true CN110982886A (en) 2020-04-10

Family

ID=70078909

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911395744.7A Pending CN110982886A (en) 2019-12-30 2019-12-30 Human K-ras gene mutation detection kit and application thereof

Country Status (1)

Country Link
CN (1) CN110982886A (en)

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110207143A1 (en) * 2008-12-19 2011-08-25 Abbott Laboratories Diagnostic test for mutations in codons 12-13 of human k-ras
CN102367478A (en) * 2011-10-25 2012-03-07 浙江大学 ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method
CN102747158A (en) * 2012-07-16 2012-10-24 武汉海吉力生物科技有限公司 Human K-ras (K-rat sarcoma) gene mutation parting type fluorescent quantitation PCR (polymerase chain reaction) detecting reagent kit and detecting method
CN102888466A (en) * 2012-10-30 2013-01-23 武汉友芝友生物制药有限公司 KRAS gene mutation detection kit and detection method
CN103695555A (en) * 2013-12-25 2014-04-02 上海星耀医学科技发展有限公司 Fluorescent genotyping detection kit and detection method for eight K-ras gene mutations
CN104694622A (en) * 2014-12-17 2015-06-10 上海长海医院 Probe for detecting K-ras gene mutation, primer, kit and method
CN105567837A (en) * 2016-02-02 2016-05-11 河南中医学院第一附属医院 Primer and probe system, method and kit for detecting K-ras gene mutation
CN106148498A (en) * 2015-04-09 2016-11-23 上海济远生物科技有限公司 KRAS gene mutation detection kit and application thereof
CN107164495A (en) * 2017-06-12 2017-09-15 安徽安龙基因医学检验所有限公司 The kit and its detection method detected for mankind's KRAS gene mutation
CN108220448A (en) * 2018-03-29 2018-06-29 浙江大学 A kind of K-Ras detection method of gene mutation and its application
CN108220444A (en) * 2018-02-28 2018-06-29 无锡禾盛医疗器械有限公司 A kind of primer combination of probe of KRAS gene mutation detection and its application

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110207143A1 (en) * 2008-12-19 2011-08-25 Abbott Laboratories Diagnostic test for mutations in codons 12-13 of human k-ras
CN102367478A (en) * 2011-10-25 2012-03-07 浙江大学 ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method
CN102747158A (en) * 2012-07-16 2012-10-24 武汉海吉力生物科技有限公司 Human K-ras (K-rat sarcoma) gene mutation parting type fluorescent quantitation PCR (polymerase chain reaction) detecting reagent kit and detecting method
CN102888466A (en) * 2012-10-30 2013-01-23 武汉友芝友生物制药有限公司 KRAS gene mutation detection kit and detection method
CN103695555A (en) * 2013-12-25 2014-04-02 上海星耀医学科技发展有限公司 Fluorescent genotyping detection kit and detection method for eight K-ras gene mutations
CN104694622A (en) * 2014-12-17 2015-06-10 上海长海医院 Probe for detecting K-ras gene mutation, primer, kit and method
CN106148498A (en) * 2015-04-09 2016-11-23 上海济远生物科技有限公司 KRAS gene mutation detection kit and application thereof
CN105567837A (en) * 2016-02-02 2016-05-11 河南中医学院第一附属医院 Primer and probe system, method and kit for detecting K-ras gene mutation
CN107164495A (en) * 2017-06-12 2017-09-15 安徽安龙基因医学检验所有限公司 The kit and its detection method detected for mankind's KRAS gene mutation
CN108220444A (en) * 2018-02-28 2018-06-29 无锡禾盛医疗器械有限公司 A kind of primer combination of probe of KRAS gene mutation detection and its application
CN108220448A (en) * 2018-03-29 2018-06-29 浙江大学 A kind of K-Ras detection method of gene mutation and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIQING LIN等: "Hairpin LNA biosensor with enzyme tagged AuNPs as tracer foramperometric detection of K-ras mutation gene", vol. 108, pages 808 - 813, XP028757695, DOI: 10.1016/j.electacta.2013.07.042 *
倪润芳等: "锁核酸修饰探针熔解曲线分析在Kras基因突变检测中的应用", vol. 58, no. 58, pages 817 - 825 *
章伟等: "应用锁核酸探针技术检测肿瘤样本中 K- ras 基因突变", vol. 48, no. 48, pages 86 - 89 *

Similar Documents

Publication Publication Date Title
EP0655090B1 (en) Detection of gene sequences in biological fluids
US8679788B2 (en) Methods for the detection of nucleic acid differences
CN105861678B (en) Design method of primer and probe for amplifying low-concentration mutation target sequence
US20130149695A1 (en) Method for detecting genetic mutation by using a blocking primer
EP1881079A1 (en) Method for the detection of EGFR mutations in blood samples
CN110438223B (en) Primer and probe for detecting Kras gene point mutation, kit and detection method thereof
CN101899496A (en) K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof
KR20140010093A (en) Kit and method for sequencing a target dna in a mixed population
CN113151519B (en) Multiple fluorescence PCR reagent for simultaneously detecting helicobacter pylori and drug-resistant gene thereof and application thereof
WO2011087709A2 (en) Eml4-alk translocations in lung cancer
CN104328164A (en) Kit for detecting human EGFR gene mutation by using fluorescence probe hybridization method
CN105039514A (en) Detection kit for human BRAF gene V600E mutation
CN108456721B (en) Method for synchronously detecting gene mutation and methylation and application thereof
CN108642153B (en) High-sensitivity mutation site detection system, method and application
CN112824535A (en) Primer composition for gene mutation multiplex detection and kit thereof
CN110511984B (en) Rapid fluorescence detection method for EGFR gene exon 19 deletion mutation and application
CN101875971A (en) Rapid detection of BRAF (Block-Repeated Active Flag) gene mutation
CN113789388B (en) Esophageal cancer gene methylation level detection reagent and application thereof
CN104694622A (en) Probe for detecting K-ras gene mutation, primer, kit and method
CN101875970A (en) Rapid detection of APC (Adenomatous Polyposis Coli) gene mutation
CN111304329A (en) Kit for detecting mutation of BRAF gene V600E site
CN109136367B (en) Method for improving diagnosis efficiency of BRAF gene V600E mutation
CN110373454A (en) A kind of kit and method of joint-detection EGFR genetic mutation
CN114134208B (en) Fluorescent quantitative PCR kit, reaction system and nucleic acid quantitative detection method
CN110982886A (en) Human K-ras gene mutation detection kit and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20200410