scholarly journals Loop-mediated isothermal amplification–fluorescent loop primer assay for the genotyping of a single nucleotide polymorphism at position 2254 in the viral DNA polymerase gene of equid alphaherpesvirus 1

2019 ◽  
Vol 31 (4) ◽  
pp. 640-644 ◽  
Author(s):  
Koji Tsujimura ◽  
Hiroshi Bannai ◽  
Manabu Nemoto ◽  
Hiroshi Kokado
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Polymerase Gene ◽  
Nucleotide Polymorphism ◽  
Viral Dna ◽  
Single Nucleotide ◽  
Loop Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Annealing Curve

We developed a loop-mediated isothermal amplification (LAMP)–fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5’-FAM–labeled backward loop primer (FLB) and 3’-dabcyl–labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.

scholarly journals A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

2018 ◽  
Vol 5 (4) ◽  
Author(s):  
Abu Naser Mohon ◽  
Didier Menard ◽  
Mohammad Shafiul Alam ◽  
Kevin Perera ◽  
Dylan R Pillai
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Dynamic Range ◽  
Developed Countries ◽  
Isothermal Amplification ◽  
Test Method ◽  
Nucleotide Polymorphism ◽  
In Vitro Susceptibility ◽  
Single Nucleotide ◽  
Loop Mediated Isothermal Amplification

Abstract Background Artemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing. Methods We developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy. Results The SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9–98.3) and 91.9% specific (95% CI, 82.6–96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9–100) and 97.3% specific (95% CI, 89.7–99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries. Conclusions Single-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient.

scholarly journals Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification

2021 ◽  
Vol 85 (2) ◽  
pp. 359-368
Author(s):  
Satoru Michiyuki ◽  
Norihiro Tomita ◽  
Yasuyoshi Mori ◽  
Hidetoshi Kanda ◽  
Kosuke Tashiro ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Promoter Region ◽  
Predictive Biomarker ◽  
Isothermal Amplification ◽  
Host Immune System ◽  
Nucleotide Polymorphism ◽  
Multiple Cancer ◽  
Single Nucleotide ◽  
Loop Mediated Isothermal Amplification ◽  
Quenching Probe

ABSTRACT Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

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