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PCR (Polymerase Chain Reaction) detection method for mutation of human BRAF (v-raf Murine Sacoma Viral Oncogene Homolog) gene V600E, primer, probe and kit

A detection method and gene technology, applied in the field of detection of human BRAF gene V600E mutation status, can solve the problems of being difficult to apply to clinical detection, difficult to avoid cross-contamination, long detection period, etc., and achieve convenient and fast detection, fast and accurate detection. high sex effect

Inactive Publication Date: 2017-11-28
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of this method: longer detection cycle, expensive, non-closed tube operation, difficult to avoid

Method used

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  • PCR (Polymerase Chain Reaction) detection method for mutation of human BRAF (v-raf Murine Sacoma Viral Oncogene Homolog) gene V600E, primer, probe and kit
  • PCR (Polymerase Chain Reaction) detection method for mutation of human BRAF (v-raf Murine Sacoma Viral Oncogene Homolog) gene V600E, primer, probe and kit
  • PCR (Polymerase Chain Reaction) detection method for mutation of human BRAF (v-raf Murine Sacoma Viral Oncogene Homolog) gene V600E, primer, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 designs and synthesizes specific primer and probe

[0047] According to the DNA sequence of the human BRAF gene published in the NCBI database, the upstream and downstream primers and probes for specifically detecting the human BRAF V600E site were designed. Among them, the 3' end of the upstream primer is a mutation site (c.1799T>A), and LNA is marked on this site.

[0048] According to the DNA sequence of the human GAPDH gene published in the NCBI database, the upstream and downstream primers and probes for specific detection of the human GAPDH gene were designed.

[0049] The nucleotide sequences (5'-3') of the designed 4 primers and 2 probes are as follows:

[0050] BRAF upstream primer: GTGATTTTGGTCTAGCTACAG+A (SEQ ID NO: 1)

[0051] BRAF downstream primer: ATCCAGACAACTGTTCAAACTGATG (SEQ ID NO: 2)

[0052] BRAF probe: FAM-CTCGATGGAGTGGGTC-MGB (SEQ ID NO: 3)

[0053] GAPDH upstream primer: CTGCCAGCCTAGCGTTGAC (SEQ ID NO: 4)

[0054] GAPDH downstrea...

Embodiment 2

[0057] Specificity, sensitivity and linear regression experiment (based on fluorescence quantitative PCR platform) of embodiment 2 primers and probe

[0058] Genomic DNA containing only BRAF gene V600E wild-type and genomic DNA containing only BRAF gene V600E mutant were used as templates, and the specificity and sensitivity of the primers and probes for the above-mentioned BRAF gene V600E locus were detected by fluorescent quantitative PCR platform. The detection system is shown in Table 1:

[0059] Table 1 Specificity and Sensitivity Detection System of Primers and Probes (Fluorescence Quantitative PCR Platform)

[0060] Element

Volume (μL)

MgCl 2

0.4

KCl

2

(NH4) 2 SO 4

2

Tris-Hcl

2

ROX II

0.2

Taq enzyme

0.2

BRAF-F

0.4

BRAF-R

0.4

GAPDH-F

0.4

GAPDH-R

0.4

BRAF probe

0.3

GAPDH probe

0.3

template+water

11

total capacity

2...

Embodiment 3

[0078] Specificity, sensitivity and linear regression experiment (based on digital PCR platform) of embodiment 3 primers and probe

[0079] Genomic DNA containing only BRAF gene V600E wild-type and genomic DNA containing only BRAF gene V600E mutant were used as templates, respectively, using a digital PCR platform to detect the specificity and sensitivity of the primers and probes for the BRAF gene V600E site. The detection system is shown in Table 5:

[0080] Specificity and sensitivity detection system of table 5 primers and probes (digital PCR platform)

[0081] Element

Volume (μL)

2*ddPCR Supermix for Probes

10

BRAF-F

1.8

BRAF-R

1.8

GAPDH-F

1.8

GAPDH-R

1.8

BRAF probe

0.5

GAPDH probe

0.5

template+water

1.8

wxya 2 o

20

[0082] The detection method of specificity and sensitivity is as follows: prepare the digital PCR detection system according to Table 5, then trans...

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Abstract

The invention discloses a PCR detection method for human BRAF gene V600E mutation. The steps include: 1) extracting sample DNA and purifying it; 2) using the DNA as a template, adding an internal reference gene, and using specific detection method for human BRAF gene V600E site mutation The primer pair and probe of the status, as well as the primer pair and probe of the internal reference gene, are used for PCR reaction; 3) the mutation status of the V600E site of the sample BRAF gene is judged. The present invention also discloses a primer pair and a probe for specifically detecting the V600E site mutation status of the human BRAF gene for the above method and a kit comprising the above primer pair and the probe. The invention realizes rapid, accurate and sensitive detection of the V600E site mutation state by designing a primer pair and a probe for specifically detecting the V600E site of the human BRAF gene.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to the detection of human BRAF gene V600E mutation state. Background technique [0002] Murine sacoma viral oncogene homolog B1 (v-raf murine sacoma viral oncogenehomolog B1, BRAF) is a member of the rapidly accelerated fibrosarcoma (rapidly accelerated Fibrosarcoma, RAF) family, is a mitogen-activating protein The core molecule downstream of kirsten rat sarcoma viral oncogene (KRAS) in the kinase (mitogen-activated protein kinase, MAPK) signaling pathway is a key activator in the MEK / ERK activation pathway, which regulates cell growth important role in proliferation and apoptosis. [0003] The BRAF gene is located on human chromosome 7q34, contains 18 exons, and encodes a protein molecule with a total length of about 94Kd. BRAF mainly has three conserved regions, namely CR1, CR2 and CR3. Among them, the CR3 region is the structural domain of the kinase, containing a glycine r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/166C12Q2531/113C12Q2545/101C12Q2535/137C12Q2563/159
Inventor 徐祎春韩峻松袁箐周佳菁
Owner SHANGHAI BIOCHIP
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