Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values

2017 ◽  
Vol 132 ◽  
pp. 41-45 ◽  
Author(s):  
Kazuya Tone ◽  
Ryuichi Fujisaki ◽  
Takashi Yamazaki ◽  
Koichi Makimura
Keyword(s):  
Melting Temperature ◽  
Melting Curve ◽  
Curve Analysis ◽  
Isothermal Amplification ◽  
Inorganic Pyrophosphatase ◽  
Melting Curve Analysis ◽  
Loop Mediated Isothermal Amplification

scholarly journals Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis

Diagnostics ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Supriya Sharma ◽  
Sandeep Kumar ◽  
Md.Zohaib Ahmed ◽  
Nitin Bhardwaj ◽  
Jaskirat Singh ◽  
...  
Keyword(s):  
Point Of Care ◽  
Melting Curve ◽  
Lateral Flow ◽  
Curve Analysis ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Loop Mediated Isothermal Amplification ◽  
Malaria Species ◽  
Flow Detection

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

scholarly journals Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel

2021 ◽  
pp. 153-160
Author(s):  
Dyah Widiastuti ◽  
Agustiningsih Agustiningsih ◽  
Ihda Zuyina Ratna Sari ◽  
Tri Ramadhani
Keyword(s):  
Aedes Aegypti ◽  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Pyrethroid Resistance ◽  
Melting Curve ◽  
Previous Method ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Wild Type

Detection of V1016G mutation is important for identifying the mechanism of  synthetic pyrethroid resistance in Aedes aegypti population. The previous method has described an allele specific polymerase chain reaction (AS-PCR) using conventional PCR to detect the mutation. Although the method has great differentiating power and reproducibility, faster and more sensitive genotyping method is essential to accurately detect the mutation. This study evaluate the used of SYBR® Green real-time PCR and melting curve analysis (MCA) to identify the V1016G mutation. The collection of homozygous 1016G, heterozygous, and wild type (1016 V) mosquitoes DNA genome was extracted using genomic DNA mini kit. The SsoAdvanced™ Universal SYBR® Green Supermix was used to identify alleles by real-time PCR followed melting curve analysis of the amplicons. Melting curve analysis produced reproducible results for the loci tested. The melting temperature was reached at 78.5 oC for homozygous 1016G mosquito and at 86 oC for wild type mosquito. Meanwhile, the heterozigous mosquito revealed two peaks of melting temperature at both 78.5 oC and 86 oC. These easily interpretable and distinguishable melting curve results were consistent with AS-PCR results obtained for the same alleles. The described MCA application for screening V1016G mutation is fast and widely accessible also could be implemented under field conditions

scholarly journals Web-based LinRegPCR: application for the visualization and analysis of (RT)-qPCR amplification and melting data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Andreas Untergasser ◽  
Jan M. Ruijter ◽  
Vladimir Benes ◽  
Maurice J. B. van den Hoff
Keyword(s):  
Melting Temperature ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Amplification Product ◽  
Web Based ◽  
Amplification Curve ◽  
Qpcr Data ◽  
Melting Curves ◽  
The Individual

Abstract Background The analyses of amplification and melting curves have been shown to provide valuable information on the quality of the individual reactions in quantitative PCR (qPCR) experiments and to result in more reliable and reproducible quantitative results. Implementation The main steps in the amplification curve analysis are (1) a unique baseline subtraction, not using the ground phase cycles, (2) PCR efficiency determination from the exponential phase of the individual reactions, (3) setting a common quantification threshold and (4) calculation of the efficiency-corrected target quantity with the common threshold, efficiency per assay and Cq per reaction. The melting curve analysis encompasses smoothing of the observed fluorescence data, normalization to remove product-independent fluorescence loss, peak calling and assessment of the correct peak by comparing its melting temperature with the known melting temperature of the intended amplification product. Results The LinRegPCR web application provides visualization and analysis of a single qPCR run. The user interface displays the analysis results on the amplification curve analysis and melting curve analysis in tables and graphs in which deviant reactions are highlighted. The annotated results in the tables can be exported for calculation of gene-expression ratios, fold-change between experimental conditions and further statistical analysis. Web-based LinRegPCR addresses two types of users, wet-lab scientists analyzing the amplification and melting curves of their own qPCR experiments and bioinformaticians creating pipelines for analysis of series of qPCR experiments by splitting its functionality into a stand-alone back-end RDML (Real-time PCR Data Markup Language) Python library and several companion applications for data visualization, analysis and interactive access. The use of the RDML data standard enables machine independent storage and exchange of qPCR data and the RDML-Tools assist with the import of qPCR data from the files exported by the qPCR instrument. Conclusions The combined implementation of these analyses in the newly developed web-based LinRegPCR (https://www.gear-genomics.com/rdml-tools/) is platform independent and much faster than the original Windows-based versions of the LinRegPCR program. Moreover, web-based LinRegPCR includes a novel statistical outlier detection and the combination of amplification and melting curve analyses allows direct validation of the amplification product and reporting of reactions that amplify artefacts.

scholarly journals Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene

2000 ◽  
Vol 38 (7) ◽  
pp. 2756-2759 ◽  
Author(s):  
Johanna Pietilä ◽  
Qiushui He ◽  
Jarmo Oksi ◽  
Matti K. Viljanen
Keyword(s):  
Borrelia Burgdorferi ◽  
Melting Temperature ◽  
Melting Curve ◽  
Sensu Stricto ◽  
Curve Analysis ◽  
Reca Gene ◽  
Melting Curve Analysis ◽  
Pcr Product ◽  
Pcr Products ◽  
Borrelia Burgdorferi Sensu Stricto

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recAgene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured fromIxodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2°C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

scholarly journals Genotyping of Entamoeba histolytica by Real-Time Polymerase Chain Reaction with Sybr Green I and Melting Curve Analysis

1970 ◽  
Vol 4 (1) ◽  
pp. 53-60
Author(s):  
SMM Rahman ◽  
R Haque ◽  
S Roy ◽  
MMH Mondal
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Entamoeba Histolytica ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Sybr Green I ◽  
Melting Curve Analysis ◽  
Intestinal Amoebiasis

In this study, for the detection of distinct genotype of E. histolytica of human, a nested Real-Time PCR amplification of SREHP gene using SYBR Green I and melting curve analysis was done. A total of 60 specimens (stool and liver aspirate specimens), which were found Entamoeba histolytica positive by E. histolytica specific ELISA and ssrRNA gene PCR, were selected and the experiment was conducted during the period of July 2003 to June 2004. After melting curve analysis of amplified PCR products from these isolates of stool and liver aspirate specimens, 5 genotypes were found belonging to the melting temperatures 84°C, 83°C, 82°C, 81°C and 79°C. All these 5 genotypes were present in intestinal amoebiasis patients and when the genotypes from intestinal amoebiasis patients were compared with the genotypes of amoebic liver amoebiasis patients, the genotype 84°C melting temperature was found to be absent in amoebic liver amoebiasis patients. For both the cases of intestinal and amoebic liver amoebiasis patients the genotype belonging to 83°C melting temperature was more prevalent than the other genotypes which suggest that this genotype is more responsible for the development of amoebiasis. In comparison to conventional PCR method where we found 23 different banding patterns, the Real-Time PCR and melting curve analysis method was found to be more reliable for the detection of distinct genotypes of E. histolytica because with this method we found only 5 genotypes. In conclusion, this Real-Time PCR using SYBR Green I and melting curve analysis for the genotyping of E. histolytica, excludes the need of post PCR manipulations and would be helpful for the rapid detection and screening of E. histolytica genotypes among endemic population and also for the epidemiological study. Key words: Entamoeba histolytica, genotype, diarrhoea, Real-Time PCR, melting curve analysis doi:10.3329/bjvm.v4i1.1526 Bangl. J. Vet. Med. (2006). 4 (1): 53-60

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