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Method for detecting a single nucleotide polymorphism (SNP) using lamp and blocking primers

A single nucleotide polymorphism, nucleic acid sequence technology, applied in the field of alleles, can solve problems such as low specificity prevention

Pending Publication Date: 2021-06-08
IMPERIAL INNOVATIONS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Addition of general QProbe (Ayukawa et al.Sci.Rep.7, 4253 (2017)) or utilization of Taq Mut enzyme (Lezhava, A. & Hayashizaki, Y. in 437–451 (2009). doi:10.1007 / 978-1 -60327-411-1_28) solve this problem to some extent, however, the need for melting analysis or low specificity prevent the use of these methods on PoC

Method used

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  • Method for detecting a single nucleotide polymorphism (SNP) using lamp and blocking primers
  • Method for detecting a single nucleotide polymorphism (SNP) using lamp and blocking primers
  • Method for detecting a single nucleotide polymorphism (SNP) using lamp and blocking primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0428] Example 1 - SNP C580Y and SNPs Y493H-specific USS-sbLAMP

[0429] Primer design for USS-sbLAMP specific for SNP C580Y and SNP Y493H.

[0430] The USS-sbLAMP method consists of a total of eight primers targeting ten different regions of the DNA template. The sbLAMP primer set consists of two outer primers (F3 and B3), two loop primers (LF and LB) and two inner primers (sbFIP and sbBIP), where F1c and B1c localize the SNP at the 5' end. The USS primer set consists of a forward blocking competitive primer (FB) and a reverse blocking competitive primer (BB).

[0431]Based on the gene K13 of Plasmodium falciparum, USS-sbLAMP primers for specific detection of C580Y and Y493H were designed. 从疟原虫基因组资源(PlasmoDB)提取来自所有人类感染性疟原虫物种的一致性参考基因组序列(PF3D7_1343700.1、PFIT_1342900、PKNH_1257700、PKNOH_S09541100、PVP01_1211100、PVX_083080、PmUG01_12021200、POcGH01_12019400),并使用Geneious 10.0.5软件 Alignment with the MUSCLE algorithm (Edgar et al. Nucleic Acids Res. 32, 1792–1797 (2004)) in (Kear...

Embodiment 2

[0478] Example 2 - Artemisinin Resistance SNPs Y493H

[0479] Validation of the USS-sbLAMP method with artemisinin resistance SNP Y493H

[0480] Following the USS-sbLAMP primer design guidelines created above, different sets of sbLAMP and USS primers were designed and tested to specifically detect the second K13 SNP Y439H (data not shown). were selected by 4.5 μM F1c 21 -B1c 17 +FB2 24 / BB1 26 Composed of USS MT -sbLAMP WT Primer set and F1c consisting of 4.5 μM 21 -B1c 17 +FB1 24 / BB0 26 Composed of USS WT -sbLAMP MT Primer sets are used for specific detection of WT and MT templates, respectively.

[0481] Ten-fold serial dilutions (5x10 4 , 5x10 3 , 5x10 2 and 5x10 1 copy / reaction) tested for sensitivity ( Figure 4 B and Table 5). For both alleles, the detection limit was 5x10 in 35 minutes 1 copy / react. R of standard curves generated for WT and MT templates, respectively 2 The values ​​are 0.994 and 0.996, respectively. Pure WT and MT templates at...

Embodiment 3

[0482] Example 3 - SNPs Using Clinical Isolates C580Y and SNPs Y493H

[0483] Cross-validation of the USS-sbLAMP method for detection of SNP C580Y and SNP Y493H using clinical isolates

[0484] The specificity of USS-sbLAMP for detection of SNP C580Y and SNP Y493H was obtained using human-infecting Plasmodium species (Plasmodium falciparum, Plasmodium ovale subsp. wallikeri, P. ovale subsp. curtisi, Plasmodium vivax, Plasmodium malariae). protozoa and Plasmodium knowlesi) gDNA samples were tested to demonstrate that there was no cross-reactivity between them. In addition, two samples of P. falciparum carrying mutations 539R and 580Y were also tested. There was no cross-reactivity with any human infectious Plasmodium species (Table 6), and samples with the 539R mutation were uniquely amplified by WT-specific reactions at 580C and 493Y, demonstrating the high specificity of the method.

[0485] Table 6. Cross-validation of the USS-sbLAMP method for detection of SNP C580Y...

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Abstract

The present application relates to methods for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence under isothermal conditions using primers specific for said first allele, in particular using Loop mediated isothermal amplification (LAMP), wherein the amplification of a second allele is prevented by using blocking primers.

Description

technical field [0001] The present application relates to a method for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence using primers under isothermal conditions. Background technique [0002] The emergence of drug resistance is a persistent threat to global public health, limiting the ability to effectively treat infectious diseases and compromising medical procedures. Therefore, the rapid detection of single nucleotide polymorphisms (SNPs) associated with drug-resistant phenotypes in infectious pathogens is the key to improving therapeutic efficacy and guiding the clinical application of drugs for the development of personalized medicine. However, detection of genetic markers remains a significant challenge for molecular-based diagnostics, especially those intended to be deployed as point-of-care (PoC). [0003] Currently, high-throughput methods such as next-generation sequencing or Sanger sequencing are considered the gold st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2527/143C12Q2531/101C12Q2537/161C12Q2549/119C12Q1/6876
Inventor 潘泰利斯·乔治乌肯尼·马尔帕蒂达·卡德纳斯余灵珊杰克·鲍姆尼古拉斯·米斯库里德斯耶苏斯·罗德里格兹·曼萨诺
Owner IMPERIAL INNOVATIONS LTD