A super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity

A fluorescent quantitative and comprehensive inspection technology, applied in the field of clinical diagnostic molecular biology, can solve the problems of high operator requirements, insufficient sensitivity, and long detection time, and achieve the effects of reducing background interference, ensuring specificity, and fast detection speed

Active Publication Date: 2021-06-18
SHANGHAI GENEPHARMA CO LTD
View PDF9 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, direct DNA sequencing is the gold standard for mutation detection, but this method has the following disadvantages: the detection sensitivity is not high enough, and if the content of the mutant gene accounts for less than 10% of the total genomic DNA, the mutation cannot be detected by direct sequencing The existence of samples; and the operation process is complicated, the detection time is long, and the requirements for operators are high; the non-closed tube operation involves the operation after PCR amplification, so it is easy to be contaminated, and the false negative rate is high, resulting in unsatisfactory results ; The interpretation of sequencing results is highly subjective; the number of samples tested in one experiment is limited, at most 8-24 cases
Therefore, direct sequencing is difficult to be widely used in clinical practice.
The ARMS-PCR method can achieve a sensitivity of 1%, which can meet the detection requirements for tumor tissue samples, but for blood samples that are convenient to sample, such as circulating tumor cells in plasma or blood, the tumor DNA content is often lower than 1%, ARMS-PCR The method is not enough to test the blood, which limits the judgment of clinicians on the efficacy of targeted drugs
In addition, the drug resistance mutations produced in the course of clinical drug use cannot be detected by previous methods due to insufficient sensitivity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity
  • A super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity
  • A super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1, the design of ultra-retardation fluorescent quantitative PCR primer probe

[0084] The primer-probe combination used to detect rare gene mutation sites consists of an upstream universal primer F, a downstream universal primer R, a general detection probe P, a specific amplification primer AP, and a locked nucleic acid blocking probe BL.

[0085] The upstream universal primer F, the downstream universal primer R and the general detection probe P sequences are all derived from the wild-type and mutant homology regions of the detection gene; the upstream universal primer F and the downstream universal primer R are 13-25nt in length , the TM value is 56-66°C; the length of the pass-through probe P is 12-30nt, and the TM value is 66-72°C; the pass-through probe P is modified by LNA or MGB; the LNA modification The number is 1-5, and the 4 base monomers of ATCG can be modified with LNA, and the 5' and 3' ends are respectively labeled with a fluorescent group and...

Embodiment 2

[0090] Embodiment 2, establishment of ultra-retardation fluorescent quantitative PCR detection method

[0091] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA.

[0092] 2. Using the cDNA obtained in step 1 as a template, carry out a control PCR reaction and a super-retardation PCR reaction;

[0093] Control PCR reaction system (take 20 μL system as an example): 10×PCR Buffer (Vazyme, catalog number: P122-d2) 2 μL, dNTP (Vazyme, catalog number: P031-01) 0.2 mM, MgCl 2 1.5mM, Universal Upstream Primer F 0.2μM-0.5μM, Universal Downstream Primer R 0.2μM-0.5μM, Universal Detection Probe P 0.2μM-0.5μM, 50×ROX (Invitrogen, catalog number: 12223-012) 0.4μL, Template 2 μL, make up to 20 μL with sterilized water.

[0094] Control PCR reaction program: 95°C for 3 minutes; 95°C for 15s, 62°C for 30s, 72°C for 30s, 40 cycles.

[0095] Fluorescent signals are detected during the reaction.

[0096] When the specific amplification primer is the dow...

Embodiment 3

[0102] Example 3, Superblocking Fluorescent Quantitative PCR for Rare Mutation of EGFR Gene T790M

[0103] 1. Detection method

[0104] 1. According to the method of Example 1, primer probes are designed for the EGFR gene T790M mutation (the EGFR gene wild-type target sequence is shown in sequence 6 of the sequence listing; the EGFR gene T790M mutant target sequence is shown in sequence 7 of the sequence listing), As follows:

[0105] Universal upstream primer EGFR-T790M-F: 5'-CCTCCAGGAAGCCTACGTGATGG-3' (SEQ ID NO: 1);

[0106] Universal downstream primer EGFR-T790M-R: 5'-CAGTTGAGCAGGTACTGGGAG-3' (SEQ ID NO: 2);

[0107] Specific amplification downstream primer EGFR-T790M-AP: 5'-AGGG+C+A+TGAGCTGCA-3' (sequence 3); wherein "+" is LNA modification, and "+" indicates subsequent base modification;

[0108] General detection probe EGFR-T790M-P: 5'-TGAGCTGCACGGTGGAGGTGA-3' (SEQ ID NO: 4); wherein the 5' end of the probe is marked with FAM, and the 3' end is marked with BHQ1;

[...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
melting pointaaaaaaaaaa
Login to view more

Abstract

The invention discloses a super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity. The invention adopts the technology of specific primers and probes, and can realize rapid detection of gene point mutations in blood samples. The advantages of the present invention include: (1) On the basis of conventional ARMS primers, LNA modification is added or additional mismatched bases are introduced at the last two to three positions of the 3', which does not affect the sensitivity of mutation detection and ensures the specificity of mutations (2) Introduce a modified blocking probe that is completely complementary to the wild type, and further reduce the background interference of the wild type on the basis of taking into account the amplification efficiency of the mutant type; (3) After introducing a highly specific On the basis of non-modified blocking probes, ARMS primers can detect different base mutations at the same site at the same time, so as to realize the merger detection of multiple sites in the same gene with only a few systems; (4) high sensitivity; (5) The detection speed is fast.

Description

technical field [0001] The invention relates to the field of clinical diagnostic molecular biology, in particular to a super-blocking fluorescent quantitative PCR method for detecting rare mutations with high sensitivity. Background technique [0002] With the continuous deepening of people's understanding of the process of tumorigenesis, it is currently believed that the occurrence of cancer is related to gene mutations, leading to uncontrolled proliferation of tumor cells. The abnormality can serve as a targeted reminder. Chemotherapy is currently one of the most important methods for clinical treatment of malignant tumors. However, with its widespread clinical use, the problem of drug resistance has become increasingly prominent. Tumor cells often develop resistance to chemotherapeutic drugs, resulting in patients no longer responding to treatment. Sensitive, which eventually leads to chemotherapy failure or even disease recurrence, in which EGFR gene T790M mutation acco...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6827C12N15/11
CPCC12Q1/6827C12Q2535/137C12Q2525/10C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 饶品彬石立立张佩琢
Owner SHANGHAI GENEPHARMA CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap