Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor

2021 ◽  
Author(s):  
Qiuju Su ◽  
Xiang Zhou ◽  
Tianwen Wu ◽  
Kui Li ◽  
Wentao Xu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Lateral Flow ◽  
Genomic Fragment ◽  
Specific Pcr ◽  
Genotyping Method ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Multiplex Allele-Specific PCR with Optimized Locked Nucleic Acid Primers

BioTechniques ◽  
2003 ◽  
Vol 34 (6) ◽  
pp. 1150-1158 ◽  
Author(s):  
David Latorra ◽  
Deborah Hopkins ◽  
Krista Campbell ◽  
J. Michael Hurley
Keyword(s):  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Multiple SNPs detection for antimalarial pyrimethamine resistance via allele-specific PCR coupled with gold nanoparticles-based lateral flow biosensor

2020 ◽  
Author(s):  
Tingting Jiang ◽  
Yan Huang ◽  
Weijia Cheng ◽  
Yifei Sun ◽  
Wei Wei ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Genetic Diseases ◽  
Lateral Flow ◽  
Nucleotide Polymorphisms ◽  
Multiple Allele ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Molecular genotyping holds tremendous potential to detect antimalarial drug resistance (ADR) related to single nucleotide polymorphisms (SNPs). However, it suffers from complicated procedures and expensive instruments. Thus, rapid point-of-care testing (POCT) molecular tools are urgently needed for field survey and clinical use. Herein, a POCT platform consisted of multiple allele-specific PCR (AS-PCR) and gold nanoparticles (AuNPs) based lateral flow biosensor was designed and developed for SNPs detection of Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene related to pyrimethamine resistance. The multiple AS-PCR utilized 3' terminal artificial antepenultimate mismatch and double phosphorothioate-modified allele-specific primers. The duplex PCR amplicons with 5' terminal labeled with biotin and digoxin, respectively, could be recognized by streptavidin (SA)-AuNPs on the conjugate pad and then captured by anti-digoxin antibody through immunoreactions on the test line to produce a golden red line for detection. The system was applied to analyze SNPs in Pfdhfr N51I, C59R, and S108N of 98 clinical isolates from uncomplicated P. falciparum malaria patients. Compared with the results of nested PCR followed Sanger DNA sequencing, the sensitivity is all 97.96% (96/98) for the N51I, C59R, and S108N. For specificity, there were 100% (98/98), 95.92% (94/98), and 100% (98/98) for N51I, C59R, and S108N, respectively. The limit of detection is approximately 200 fg/μl for plasmid DNA as the template and 100 parasites/μl for blood filter paper. The established platform not only offers a powerful tool for molecular surveillance of ADR but also is easily extended to interrelated SNP profiles for infectious diseases and genetic diseases.

Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR

2016 ◽  
Vol 38 (10) ◽  
pp. 1709-1714 ◽  
Author(s):  
Cong Wang ◽  
Xiaomin Chen ◽  
Yuying Wu ◽  
Hao Li ◽  
Yu Wang ◽  
...  
Keyword(s):  
Visual Detection ◽  
Lateral Flow ◽  
Lateral Flow Strip ◽  
Ras Mutations ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals A Rapid Robust Method for Subgrouping Non-NF2 Meningiomas According to Genotype and Detection of Lower Levels of M2 Macrophages in AKT1 E17K Mutated Tumours

2020 ◽  
Vol 21 (4) ◽  
pp. 1273 ◽  
Author(s):  
Claire L. Adams ◽  
Emanuela Ercolano ◽  
Sara Ferluga ◽  
Agbolahan Sofela ◽  
Foram Dave ◽  
...  
Keyword(s):  
Immune Cell ◽  
Tumour Microenvironment ◽  
M2 Macrophages ◽  
Who Grade ◽  
Specific Pcr ◽  
Genotyping Method ◽  
Mutational Status ◽  
Hla Dr ◽  
Allele Specific ◽  
Allele Specific Pcr

The majority of meningiomas are grade I, but some grade I tumours are clinically more aggressive. Recent advances in the genetic study of meningiomas has allowed investigation into the influence of genetics on the tumour microenvironment, which is important for tumorigenesis. We have established that the endpoint genotyping method Kompetitive Allele Specific PCR (KASP™) is a fast, reliable method for the screening of meningioma samples into different non-NF2 mutational groups using a standard real-time PCR instrument. This genotyping method and four-colour flow cytometry has enabled us to assess the variability in the largest immune cell infiltrate population, M2 macrophages (CD45+HLA-DR+CD14+CD163+) in 42 meningioma samples, and to suggest that underlying genetics is relevant. Further immunohistochemistry analysis comparing AKT1 E17K mutants to WHO grade I NF2-negative samples showed significantly lower levels of CD163-positive activated M2 macrophages in meningiomas with mutated AKT1 E17K, signifying a more immunosuppressive tumour microenvironment in NF2 meningiomas. Our data suggested that underlying tumour genetics play a part in the development of the immune composition of the tumour microenvironment. Stratifying meningiomas by mutational status and correlating this with their cellular composition will aid in the development of new immunotherapies for patients.

Enhanced allele-specific PCR discrimination in SNP genotyping using 3? locked nucleic acid (LNA) primers

2003 ◽  
Vol 22 (1) ◽  
pp. 79-85 ◽  
Author(s):  
David Latorra ◽  
Krista Campbell ◽  
Andreas Wolter ◽  
J. Michael Hurley
Keyword(s):  
Nucleic Acid ◽  
Snp Genotyping ◽  
Locked Nucleic Acid ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals High-Sensitivity Detection of the A3243G Mutation of Mitochondrial DNA by a Combination of Allele-Specific PCR and Peptide Nucleic Acid-Directed PCR Clamping

2004 ◽  
Vol 50 (11) ◽  
pp. 2045-2051 ◽  
Author(s):  
Michiyo Urata ◽  
Yui Wada ◽  
Sang Ho Kim ◽  
Worawan Chumpia ◽  
Yuzo Kayamori ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Quantitative Detection ◽  
Wild Type ◽  
Specific Pcr ◽  
Wide Range ◽  
A3243g Mutation ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation. Methods: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3′ end matched to nucleotide position 3243 of the mutant. Results: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5–100 ng; annealing temperature, 61–66 °C; and PNA, 1.5–3.5 μmol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed <0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant. Conclusions: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.

Warfarin genotyping with hybridization-induced aggregation on a poly(ethylene terephthalate) microdevice

The Analyst ◽  
2017 ◽  
Vol 142 (2) ◽  
pp. 366-374 ◽  
Author(s):  
Hillary S. Sloane ◽  
Morgan B. Carter ◽  
Alexa E. C. Cecil ◽  
Delphine Le Roux ◽  
Daniel L. Mills ◽  
...  
Keyword(s):  
Ethylene Terephthalate ◽  
Specific Pcr ◽  
Genotyping Method ◽  
Poly Ethylene Terephthalate ◽  
Allele Specific ◽  
Poly Ethylene ◽  
Induced Aggregation ◽  
Allele Specific Pcr

A novel genotyping method is established, using allele-specific PCR followed by hybridization-induced aggregation (HIA) of microbeads on a multiplexed microdevice.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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