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Primer set, kit and method for detecting cardiovascular disease-related gene polymorphisms

A gene polymorphism and cardiovascular technology, which is applied in the field of reagent kits, biological molecular detection, and primer sets for detecting cardiovascular disease-related gene polymorphisms. It can solve the problems of high detection costs, high design costs, and lack of specificity. problems, to achieve high accuracy, low cost, and strong specificity

Active Publication Date: 2019-01-01
德诺杰亿(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of a single SNP site mainly includes sanger sequencing, fluorescent quantitative PCR, ARMs-PCR, etc., and the use of the above methods for the simultaneous detection of multiple SNP sites will increase the workload and cost; The detection of each SNP site mainly adopts the technology of gene chip or next generation sequencing, but the design cost of these two technologies is high and the detection cost is expensive
The existing technology lacks a method with good specificity, high sensitivity, low cost, and simultaneous detection of related gene polymorphisms at multiple sites, which has become an urgent problem to be solved

Method used

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  • Primer set, kit and method for detecting cardiovascular disease-related gene polymorphisms
  • Primer set, kit and method for detecting cardiovascular disease-related gene polymorphisms
  • Primer set, kit and method for detecting cardiovascular disease-related gene polymorphisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: the design of primer

[0093] Principles and considerations for primer design:

[0094] 1. Length: 15bp to 30bp, its effective length [Ln=2(G+C)+(A+T)] is generally not greater than 38, otherwise the optimum extension temperature of PCR will exceed the optimum action temperature of Taq enzyme, thus Reduce product specificity.

[0095] 2. G+C content: it should be between 40% and 60%. The annealing temperature in PCR amplification is generally the Tm value of the primer with a lower Tm value minus 5 to 10 degrees. When the primer length is less than 20, its Tm is always equal to 4×(G+C)+2×(A+T).

[0096] 3. The randomness of base distribution: more than 4 single bases in a row should be avoided. In particular, there should not be more than 3 consecutive Gs or Cs at the 3' end, otherwise the primers will be misprimed in the G+C-rich sequence region.

[0097] 4. The primer itself: it cannot contain a self-complementary sequence, otherwise a hairpin-like s...

Embodiment 2

[0102] Embodiment 2: Primer sequence screening detection

[0103] (1) Divide into 3 reaction tubes, Pool 1, Pool 2, and Pool 3, and add 10 ng of human genomic DNA (DNA sample of oral epithelial exfoliated cells) to each reaction tube.

[0104] (2) Using the DNA sample added in step (1) as a template, the PCR amplification primers (composed of SEQ ID NO: 1 to SEQ ID NO: 42) obtained in Example 1 were used for multiplex PCR amplification.

[0105] Wherein, the PCR amplification primers were combined in equal proportions (ie 1:1(v)) according to the method in Table 2, oscillated and mixed; and used after brief centrifugation.

[0106] Among them, the PCR amplification reaction system of each of Pool 1, Pool 2, and Pool 3 is: primer premix 10 μL, 2XPCR MIX 12.5 μL, template (positive quality control) 1 μL, ddH 2 Make up 25 μL of O, vortex and mix well, and perform multiple PCR amplification after brief centrifugation. The PCR reaction conditions include: stage 1: 95°C for 3 minut...

Embodiment 3

[0114] Embodiment 3: Primer sequence screening detection

[0115] The difference between this example and Example 2 is that the PCR amplification primers used in step (2) consist of SEQ ID NO:43 to SEQ ID NO:84. The detection result of this embodiment is as follows figure 2 shown.

[0116] From figure 1 and figure 2 The results obtained are shown in Table 4.

[0117] Table 4

[0118]

[0119] Depend on figure 1 and figure 2 As can be seen from Table 4, the primer sets detected in Examples 2 and 3 all obtained the typing information of all loci. Will figure 1 and figure 2 Comparing it can be seen that the peak height consistency between the sites of the primer set detected in Example 3 is better than that of the primer set detected in Example 2, so the primers consisting of SEQ ID NO:43 to SEQ ID NO:84 are recommended The set (set of primers shown in Table 1) is the preferred sequence information.

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Abstract

The invention discloses a primer set for detecting cardiovascular disease related gene polymorphism. The primer set comprises primers aiming at following SNP loci of SLCO1B1 521C, SLCO1B1 521T, SLCO1B1 463A, SLCO1B1 463C, SLCO1B1 388A, SLCO1B1 388G, ABCA1 656A, ABCA1 656G, CYP2C9 1075C, CYP2C9 1075A, ABCB1 1236T, ABCB1 1236C, ABCB1 2677A, ABCB1 2677G, ABCB1 2677T, ABCG2 421C, ABCG2 421A, CYP3A4 1334C, CYP3A4 1334T, CYP3A4 653G, CYP3A4 653C, SLC10A1 800 and SLC10A1 800C. The invention further discloses a kit comprising the primer set and a gene polymorphism detecting method utilizing the primerset. The primer set has the advantages of being high in specificity, high in detecting sensitivity, and high in accuracy. The detecting method is high in specificity, high in sensitivity and low in cost, and simultaneous detection of multiple loci can be achieved.

Description

technical field [0001] The invention belongs to the field of biological detection, specifically relates to biological molecular detection, and more specifically relates to a primer set, a kit and a method for detecting cardiovascular disease-related gene polymorphisms. Background technique [0002] Cardiovascular disease is not only the number one cause of death among many diseases in the world but also in my country. At present, there are about 290 million cardiovascular disease patients in my country, causing more than 3.5 million deaths every year. With the continuous development of my country's economy and the improvement of people's living standards, the proportion of fat in the diet continues to increase, coupled with bad habits such as smoking and alcoholism, the prevalence of cardiovascular diseases is gradually becoming younger, and the form is not optimistic. [0003] As humanity enters the era of precision medicine, genetic testing is becoming more common and che...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/172
Inventor 韩永刘瑜林英忠刘伶施莹曹健荣林小靖
Owner 德诺杰亿(北京)生物科技有限公司
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