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Nucleic acid sequence detection composition and detection method

A nucleic acid sequence and target nucleic acid sequence technology, applied in the field of molecular biology, can solve the problems of increasing detection cost and operational complexity

Pending Publication Date: 2019-12-20
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the method of Mariana et al. can improve the detection sensitivity to a certain extent, it requires additional end repair operations, which increases the detection cost and operational complexity

Method used

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  • Nucleic acid sequence detection composition and detection method
  • Nucleic acid sequence detection composition and detection method
  • Nucleic acid sequence detection composition and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] In this embodiment, a common nucleic acid variation is taken as an example to test the primer probe composition and detection system of the present invention. Specifically, the L858R mutation of the human EFGR gene was used as an example to simulate clinical samples to evaluate the performance of the detection system of the present invention.

[0079]EFGR is a common driver gene of non-small cell lung cancer, and its mutations mainly occur in exons 18 / 19 / 20 / 21, and more than 45% of EGFR driver mutations are encoded by the 858th amino acid of exon 21 from leucine Acid (L) is changed to arginine (R), and the above mutations cause continuous activation of EGFR downstream pathways, leading to tumorigenesis. At the same time, in the process of targeted therapy, tumors caused by the EGFR gene L858R mutation were found to be sensitive to the first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) drugs, so quantitative detection of EGFR gene L858R mutation and Continuous f...

Embodiment 2

[0113] In order to further verify the effects of the primers and methods of the present invention, another gene variation (human BRAF gene V600E mutation) was detected in this embodiment.

[0114] 1. Sample preparation:

[0115] Fragmented Colo 205 cell line DNA samples quantified by digital PCR, containing the BRAF gene V600E mutation with a mutation abundance of 65.8%, were used to simulate clinical circulating tumor DNA.

[0116] Specifically, with QIAGEN The DNA Mini Kit was used to extract the nucleic acid of the Colo 205 cell line containing the BRAF gene V600E mutation according to the operating instructions of the kit to obtain the genomic DNA of the BRAF gene V600E mutant cell line. Using KAPA Frag Kit, the extracted BRAF gene V600E mutant cell line DNA was digested and fragmented to obtain fragmented mutant DNA with a fragment length of about 120-130bp, which simulated the fragment size of clinical circulating tumor DNA.

[0117] The free DNA extracted from the pl...

Embodiment 3

[0143] In this embodiment, the DNA sample of NCI-H1650 cell line was used to test the detection effect of the primer probe composition and the detection method of the present invention on the deletion mutation for exon 19 mutation of human EGFR gene.

[0144] 1. Sample preparation:

[0145] The 293T cell line DNA purified by ultrasound was prepared, and it was confirmed by next-generation sequencing that it did not contain the EGFR gene 19del mutation, and it was used as a negative sample.

[0146] At the same time, the NCI-H1650 cell line DNA sample quantified by digital PCR was prepared. After fragmentation, the wild-type DNA sample was used to dilute the EGFR gene 19del mutation sample to a mutation abundance of 10% as a positive sample.

[0147] The blank control was Tris-EDTA buffer without DNA.

[0148] 2. Preparation of reaction system

[0149] 2.1 Primer probe composition

[0150] The primers and probes of the present invention are synthesized by Sangon Bioengineeri...

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Abstract

The invention discloses a high-sensitivity nucleic acid sequence detection composition and a detection method. The composition comprises a first upstream primer, a second upstream primer, a probe anda downstream primer, wherein the first upstream primer is sequentially composed of the following two parts from a 5' end to a 3' end of (1) a non-matching area, wherein complementary pairing does notoccur with a target nucleic acid sequence; the sequence of the non-matching area from the 5' end to the 3' end comprises the same sequence as the second upstream primer, and the same sequence as the probe; and (2) a matching area, wherein complementary pairing occurs with the target nucleic acid sequence. Compared with the prior art, the primer probe composition provided by the invention has the remarkable advantages of high sensitivity, high specificity, low cost and the like, is suitable for nucleic acid detection of various sample types, and has extremely high application value.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for improving detection sensitivity in nucleic acid sequence detection. Background technique [0002] The polymerase chain reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without the use of living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as gene cloning, phenotyping of experimental animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, diagnosis of infectious diseases, paternity testing, etc. . Due to its unparalleled ability to replicate and be precise, PCR is considered by molecular biologists to be the method of choice for nucleic acid detection. In the late 1990s, the real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) technology and related products launched by the American ABI comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 赵雨航王书芳葛志琪何辉煌李锦
Owner SICHUAN MACCURA BIOTECH CO LTD
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