Probe sets, kits, reaction systems and systems for detecting t790m and c797s cis-trans mutation types

A C797S and T790M technology, applied in the field of digital PCR, can solve the problems of inability to calculate the mutation rate, long detection cycle, high cost, etc., and achieve the effects of simplified system, low detection limit and convenient transportation

Active Publication Date: 2020-05-12
SIMCERE DIAGNOSTICS CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the detection sensitivity of the NGS method is 0.2%-1%, and its detection period is long and the cost is high; the detection sensitivity of EGFR T790M and C797S mutation cis and trans forms based on the digital PCR method on the market is 0.1%, but this method cannot calculate the mutation rate and Inability to perform quality control of samples in the same reaction

Method used

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  • Probe sets, kits, reaction systems and systems for detecting t790m and c797s cis-trans mutation types
  • Probe sets, kits, reaction systems and systems for detecting t790m and c797s cis-trans mutation types
  • Probe sets, kits, reaction systems and systems for detecting t790m and c797s cis-trans mutation types

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1 probe and primer sequence design

[0120] Design probes and primers for detecting T790 and C797, among which, 6 probes are designed for C797, 1 probe is designed for T790, PCR primer pairs for amplification are designed for T790 and C797, and the number of probes and primers See Table 10 for specific sequence information, where the 5' end of the probe for C797 is marked with FAM, the 3' end is marked with a quencher group, the 5' end of the probe for T790 is marked with VIC, and the 3' end is marked with a quencher group .

[0121] Table 10 probe, primer sequence

[0122] probe name Numbering sequence C797S-0518-probe SEQ ID NO: 1 CTTCGGCTGCCTC C797S-0518-GC-probe SEQ ID NO: 2 CTTCGGCTCCCTC C797S-0518-TA-probe SEQ ID NO: 3 CTTCGGCAGCCTC ddPCR-C797S-WT-probe SEQ ID NO: 4 TTCGGCTGCCTCCTG ddPCR-C797S-TA-probe SEQ ID NO: 5 TTCGGCAGCCTCC ddPCR-C797S-GC-probe SEQ ID NO: 6 CTTCGGCTCCCTCCTG 0...

Embodiment 2

[0123] Embodiment 2 Detects the result of the selection of the probe of C797S

[0124] Use different probe combinations and concentration gradients to prepare different assay solutions, analyze the distribution of the scatter plot, and select the optimal probe and concentration:

[0125] C797S-0518-TA-probe probes use the final concentration of 0.25μM and 0.5μM, the dispersion of the scatter plot is the same, but the use of the final concentration of 0.5μM makes the fluorescence value increase (see Scatter Figure 1~4 ). Therefore, when detecting C797S (T>A), the C797S-0518-TA-probe probe (SEQ ID NO: 3) was selected, and the final concentration was selected to be 0.25 μM.

[0126] The final concentration of C797S-0518-GC-probe probe is 0.25 and 0.5μM, and the dispersion degree of the scatter plot is consistent (see Scatter Figure 5-8 ), the C797S mutation scatter point and the T790M mutation scatter point are too close, which is not conducive to the subsequent analysis of r...

Embodiment 3

[0128]This example proves that the compatibility probe can accurately reflect the copy number of the wild-type template and the copy number of the mutant template:

[0129] Use EGFR T790M assay (Reaction G) to detect normal human gDNA, H2228gDNA, HCC827gDNA, H1 975gDNA as a control, use C797S-0518-TA 0.25 assay (Reaction A) to detect normal human gDNA, H2228gDNA, HCC827gDNA, H1975gDNA as experimental group 1, use ddPCR-C797S-GC 0.25 assay (Reaction E) detected normal human gDNA, H2228 gDNA, HCC827 gDNA, H1975 gDNA as experimental group 2. Compared with the wild-type copy number of the control group, there was no significant difference in the wild-type copy number of the experimental group 1 and 2; there was no significant difference in the mutant-type copy number of the experimental group 1 and 2 compared with the wild-type copy number of the control; Probes (C797S Mu(T>A) probe and C797SMu(G>C) probe (the two probes are not mixed in the same reaction) can accurately detect bo...

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Abstract

The invention relates to a probe set used for detecting T790M and C797S cis-trans mutation types, a kit, a reaction system and a system. The probe set used for detecting the T790M and C797S cis-transmutation types comprises a probe 1, a probe 2 and a probe 3, wherein nucleotide sequences of the probe 1, the probe 2 and the probe 3 are shown in SEQ ID NO: 3, 6 and 7; quenching dyes and fluorescentdyes are correspondingly labeled at the two ends of the probe 1, the probe 2 and the probe 3; the fluorescent dye 1 is labeled on the probe 1; the fluorescent dyes 2 are labeled on both the probe 2 and the probe 3. The probe set used for detecting T790M and C797S cis-trans mutation types, the kit, the reaction system and the detection system, provided by the invention, have the advantages that T790M and C797S cis-trans mutations can be accurately detected, quantifiability, high sensitivity, good specificity and low detection limit are realized. In addition, the probe set used for detecting T790M and C797S cis-trans mutation types, the kit, the reaction system and the detection system, provided by the invention, can also directly control the quality of a sample to be detected in the case of not increasing the reaction number and other experiments.

Description

technical field [0001] The invention relates to the field of digital PCR, in particular to a probe set, a kit, a reaction system and a system for detecting T790M and C797S cis-trans mutation types. Background technique [0002] EGFR gene T790M and C797S mutations are gene mutation types found in tumor patients. EGFR gene T790M specifically refers to the 790th amino acid site of exon 20 of the epidermal growth factor receptor (EGFR) composed of threonine (T) is mutated to methionine (G), and the gene level is c.2369C>T; EGFR gene C797S specifically refers to the mutation of the 797th amino acid site of exon 20 of EGFR from cysteine ​​(C) to Serine (S), the gene level showed c.2389T>A or c.2390G>C. When T790M and C797S are mutated at the same time, T790M and C797S are located on the same allele, this configuration is called cis; T790M and C797S are located on different alleles, this configuration is called trans. The current research results show that T790M, C797S m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2531/113C12Q2563/159C12Q2561/101
Inventor 黄霖霆曹乾升俞莹李杜衡任用
Owner SIMCERE DIAGNOSTICS CO LTD
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