scholarly journals Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

2020 ◽  
Vol 48 (7) ◽  
pp. e42-e42 ◽  
Author(s):  
Justin C Rolando ◽  
Erik Jue ◽  
Jacob T Barlow ◽  
Rustem F Ismagilov
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Rapid Diagnostics ◽  
High Resolution Melt ◽  
Specific Amplification ◽  
The Impact

Abstract Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella

2017 ◽  
Vol 41 (6) ◽  
Author(s):  
Faramarz Masjedian Jazi ◽  
Reza Mirnejad ◽  
Vahhab Piranfar ◽  
Noor Amir Mozafari ◽  
Taghi Zahraei Salehi ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Serum Antibody ◽  
Clinical Samples ◽  
Species Differentiation ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Antibody Titers ◽  
Polymerase Chain ◽  
Animal Isolates

AbstractBackground:It is of great importance to quickly and accurately detectMethods:The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared.Results:The mean range [cycles threshold±standard deviation (CConclusions:The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing

scholarly journals Rapid Identification and Discrimination ofBrucellaIsolates by Use of Real-Time PCR and High-Resolution Melt Analysis

2010 ◽  
Vol 48 (3) ◽  
pp. 697-702 ◽  
Author(s):  
Jonas M. Winchell ◽  
Bernard J. Wolff ◽  
Rebekah Tiller ◽  
Michael D. Bowen ◽  
Alex R. Hoffmaster
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Identification ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis

scholarly journals Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 757
Author(s):  
Sara Franco Ortega ◽  
Ilenia Siciliano ◽  
Simona Prencipe ◽  
Maria Lodovica Gullino ◽  
Davide Spadaro
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Screening Method ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

scholarly journals Chloroplast Genome Sequence ofArabidopsis thalianaAccession Landsbergerecta, Assembled from Single-Molecule, Real-Time Sequencing Data

2016 ◽  
Vol 4 (5) ◽  
Author(s):  
Kai Bernd Stadermann ◽  
Daniela Holtgräwe ◽  
Bernd Weisshaar
Keyword(s):  
Arabidopsis Thaliana ◽  
High Resolution ◽  
Chloroplast Genome ◽  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Sequencing Data ◽  
Data Set ◽  
Chloroplast Genomes ◽  
Chloroplast Genome Sequence

A publicly available data set from Pacific Biosciences was used to create an assembly of the chloroplast genome sequence of theArabidopsis thalianagenotype Landsbergerecta. The assembly is solely based on single-molecule, real-time sequencing data and hence provides high resolution of the two inverted repeat regions typically contained in chloroplast genomes.

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

2011 ◽  
Vol 64 (12) ◽  
pp. 2453-2459 ◽  
Author(s):  
G. N. van Blerk ◽  
L. Leibach ◽  
A. Mabunda ◽  
A. Chapman ◽  
D. Louw
Keyword(s):  
Surface Water ◽  
High Resolution ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Gene Target ◽  
Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

scholarly journals Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern Uganda by high‐resolution melt analysis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kennedy Kassaza ◽  
Anna C. Long ◽  
Jennifer M. McDaniels ◽  
Mharlove Andre ◽  
Wasswa Fredrickson ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
High Resolution ◽  
Clinical Samples ◽  
Resistance Mutations ◽  
High Resolution Melt ◽  
Blood Spots ◽  
Group 2 ◽  
The Impact ◽  
Southwestern Uganda ◽  
Group 1

Abstract Background Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries. Methods Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda. Results The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10− 5 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2). Conclusions The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

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