Composition for nucleic acid detection

A composition and nucleic acid technology, applied in the field of molecular biology, can solve the problems of lower detection sensitivity, extremely high requirements for primer specificity, and high cost, and achieve the effects of improving primer specificity, avoiding false positive signals, and improving sensitivity

Pending Publication Date: 2022-03-29
SICHUAN MACCURA BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Among them, ①TaqMan probe method usually uses competitive probes for mutant type and wild type respectively. The two probes are competitive probes similar to point mutation detection, or a specific probe is designed on the gene wild type template. , design a universal probe that can simultaneously indicate the wild-type and mutant templates in other conserved regions of the gene, and use the concentration difference measured by the two probes to quantify the concentration of the mutant template. Amplification primers are designed upstream and downstream, resulting in longer amplicons. Due to the highly random fragmentation distribution of ctDNA, shorter ctDNA fragments may be missed, reducing detection sensitivity. At the same time, when there are many types of mutations in a certain gene For different mutation types, it is necessary to design a variety of different mutant probes, which is expensive and is not conducive to large-scale promotion and use.
②The primer-specific discrimination method is to design different mutant primers to specifically distinguish different mutation types, design a probe and downstream primer in the conserved region downstream of it, and design a probe and downstream primer in other conserved regions of the gene to simultaneously indicate wild-type and Universal probes for mutant templates and their upstream and downstream primers. This method still has the problem of long amplicons, which is not conducive to the detection of short ctDNA. In addition, the specificity requirements of primers are extremely high, and cross-reactions are prone to occur.
③The method of blocking wild-type amplification often uses expensive modifications such as Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA) to block the amplification of wild-type templates, so as not to generate false positive signals, but When the difference between some wild-type sequences and mutant sequences is too small or the mutant region is a wild-type repetitive sequence, the added inhibitor will not be able to distinguish the two sequences and affect the signal detection of the mutant

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  • Composition for nucleic acid detection
  • Composition for nucleic acid detection
  • Composition for nucleic acid detection

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Experimental program
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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, EGFR exon 20 insertion mutation was used as an example to simulate clinical samples to evaluate the performance of the detection system of the present invention.

[0079] 1. Sample preparation:

[0080] Prepare fragmented 293T cell line DNA samples, quantify them with a Qubit fluorescence quantitator, and dilute to 10 ng / μL as negative control samples.

[0081] At the same time, an artificially synthesized plasmid sample containing the EGFR gene exon 20 insertion mutation (COSM12377) sequence was prepared, and after fragmentation, the negative sample was used for dilution and mixing as a positive sample.

[0082] Use Tirs-EDTA buffer without any DNA as blank control sample.

[0083] 2. Preparation of reaction system

[0084] 2.1 Primer probe composition

[0085] The primers and probes of the pres...

Embodiment 2

[0104] In order to reflect the effects of the present invention, this embodiment uses oligonucleotides F1C of different lengths to evaluate the impact on detection specificity.

[0105] 1. Sample preparation:

[0106] Prepare fragmented 293T cell line DNA samples, quantify them with a Qubit fluorescence quantitator, and dilute to 10 ng / μL as negative control samples.

[0107] At the same time, an artificially synthesized plasmid sample containing another mutant type sequence (COSM12378) of exon 20 insertion mutation of the EGFR gene was prepared. After fragmentation, the negative sample was used for dilution and mixing as a positive sample.

[0108] 2. Preparation of reaction system

[0109] 2.1 Primer probe composition

[0110] The specific sequence of the primer probe composition is shown in Table 3 below.

[0111] table 3:

[0112]

[0113] Among the above primer probes, the oligonucleotide F1C-2-12 is 12bp in length, and can be complementary to the 12bp at the 3' en...

Embodiment 3

[0130] In order to further verify the effects of the primers and methods of the present invention, another gene variation (EGFR gene T790M mutation) was detected in this embodiment.

[0131] 1. Sample preparation:

[0132] Prepare fragmented 293T cell line DNA samples, quantify them with a Qubit fluorescence quantitator, and dilute to 10 ng / μL as negative control samples.

[0133] At the same time, an artificially synthesized plasmid sample containing the T790M mutation (COSM6240) sequence of exon 20 of the EGFR gene was prepared. After fragmentation, the negative sample was used for dilution and mixing as a positive sample.

[0134] Use Tirs-EDTA buffer without any DNA as blank control sample.

[0135] 2. Preparation of reaction system

[0136] 2.1 Primer probe composition

[0137] The specific sequence of the primer probe composition is shown in Table 5 below.

[0138] table 5:

[0139]

[0140]

[0141] Primers and probes were synthesized by Sangon Bioengineering...

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Abstract

The invention discloses a high-sensitivity and high-specificity composition for nucleic acid detection and application of the composition in a detection kit. The composition comprises a first upstream primer F1, a second upstream primer F2, a probe P, a downstream primer R and oligonucleotide F1C, the first upstream primer consists of the following two parts in sequence from the 5'end to the 3 'end: (1) a mismatching region which is not complementarily paired with a target nucleic acid sequence; the sequence of the mismatching region comprises a sequence which is the same as the sequence of the second upstream primer and a sequence which is the same as the sequence of the probe from the 5'end to the 3 'end; and (2) a matching region: performing complementary pairing with a target nucleic acid sequence. Compared with the prior art, the primer probe composition provided by the invention has the remarkable advantages of high sensitivity, good 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 primer probe composition for improving the sensitivity and specificity of nucleic acid detection and its application in PCR reaction. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848C12Q1/6858C12N15/11
CPCC12Q1/6848C12Q1/6858C12Q2531/113
Inventor 赵雨航葛志琪何辉煌
Owner SICHUAN MACCURA BIOTECH CO LTD
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