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Probe and kit for detecting SNP (Single Nucleotide Polymorphism) site by utilizing recombinase mediated isothermal amplification method based on probe guidance

A technology of isothermal amplification and recombinant enzymes, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems with high requirements and achieve rapid results

Active Publication Date: 2018-05-22
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the RAA detection method has high sensitivity and specificity, the traditional RAA detection method relies on the design of a pair of amplification primers and a probe, and avoids non-specific amplification of primers and non-specific combination of primers and probes. This has high requirements for primers and probes, and there is no report on using this method to study SNP sites

Method used

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  • Probe and kit for detecting SNP (Single Nucleotide Polymorphism) site by utilizing recombinase mediated isothermal amplification method based on probe guidance
  • Probe and kit for detecting SNP (Single Nucleotide Polymorphism) site by utilizing recombinase mediated isothermal amplification method based on probe guidance
  • Probe and kit for detecting SNP (Single Nucleotide Polymorphism) site by utilizing recombinase mediated isothermal amplification method based on probe guidance

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The flanking sequence of the MTHFRA1298C SNP site was obtained through the dbSNP database of the National Center for Biotechnology Information (NCBI), and a primer and two primers suitable for this method were designed according to the design principles of the RAA method using Oligo7.37 software. probe. At the same time, in addition to the design principles of RAA, we require that the SNP site should be located just before the THF site of the probe, such as figure 1 shown. All primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd. and purified by high performance liquid chromatography. PRAA primers and probes are listed in Table 1.

[0049] Table 1 PRAA primers and probes

[0050]

[0051] a. Forward primers used in A and C reactions

[0052] b. Specific probes for A reactions

[0053] c. Specific probes for C reactions

[0054] d. Modification of the probe: FAM, 6-Carboxyfluorescein (6-carboxyfluorescein); THF, tetrahydrofuran (tetrahy...

Embodiment 2

[0056] 1. Collection of clinical specimens

[0057] From 2016 to 2017, 150 children with CHD hospitalized in Children's Cardiac Surgery in Hebei Province were collected, including 72 males and 78 females, aged 1 month to 14 years. The whole blood DNA of the specimen was extracted with the Tiangen Biochemical Blood Genomic DNA Extraction Kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., the genomic DNA was dissolved in 150 μL of nuclease-free water, and stored at -20°C until use. The DNA concentration of the obtained samples ranged from 10–75 ng / μL. This study was approved by the Medical Ethics Committee of Hebei Children's Hospital, and the parents of the children signed the informed consent.

[0058] 2. Perform PRAA detection on 150 clinical samples

[0059] The RAAExo kit produced by Jiangsu Qitian Gene Company was used. Both A and C reactions were carried out in a 0.2ml reaction tube, which included a pre-lyophilized enzyme mixture (SSB, UvsX, DNA polym...

Embodiment 3

[0068] In order to further explore the effect of sample concentration on the specificity of the PRAA method, we constructed two plasmids carrying homozygous AA and CC homozygotes at this site, and made a series of dilutions, including 10 7 ,10 6 ,10 5 ,5*10 4 ,10 4 ,5*10 3 ,10 3 ,5*10 2 ,10 2 The copies / reactions were then tested separately using the PRAA method (A and C reactions).

[0069] Use the AA type plasmid to explore the sensitivity of the A reaction and the specificity of the C reaction, and use the CC type plasmid to explore the specificity of the A reaction and the sensitivity of the C reaction. For plasmids, we found that the lower limit of templates that can be detected by reactions A and C are both 5*10 3 copy each reaction, ( Figure 3-1 with Figure 3-1 ). Non-specific amplification will occur when the amount of mismatched template reaches 105 copies per reaction ( Figure 3-3 with Figure 3-4 ). It is suggested that the PRAA method may be locate...

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Abstract

The invention provides a probe and kit for detecting an SNP (Single Nucleotide Polymorphism) site by utilizing a recombinase mediated isothermal amplification method based on probe guidance and belongs to the technical field of SNP site detection. According to the probe for detecting the SNP site by utilizing the recombinase mediated isothermal amplification method based on the probe guidance, onthe basis of the probe for the recombinase mediated isothermal amplification method, the probe is used for carrying out enzymolysis on a next-site basic group of a tetrahydrofuran replacement SNP siteor a next-site basic group of an SNP site complementary site by an excision enzyme III until tetrahydrofuran site cutting is finished; a cut-off fluorescence group modified basic group generates a fluorescence signal in a system and residual probes are partially used as downstream primers. A simplified detection process of the probe needs a downstream primer component so that nonspecific amplification between the probe and the primer is reduced. The invention provides the kit for detecting the SNP site and the kit comprises an upstream primer and the probe. The kit can be used for detecting various SNP sites.

Description

technical field [0001] The invention belongs to the technical field of SNP site detection, and in particular relates to a probe and a kit for detecting SNP sites by a probe-guided recombinase-mediated isothermal amplification method. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level, accounting for more than 90% of all known polymorphisms. SNP sites are single nucleotide variations on the genome, including transitions, transversions, deletions, and insertions, forming genetic markers with a large number and rich polymorphisms. With the completion of the Human Genome Project and the HapMap Project, single nucleotide polymorphism (SNP) has become the third generation of genetic markers. Many phenotypic differences in the human body, susceptibility to drugs or diseases, etc. may be related to SNPs, and timely underst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2521/507
Inventor 马学军段素霞王佶张益申辛欣何小周
Owner 中国疾病预防控制中心病毒病预防控制所
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