scholarly journals Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis

2020 ◽  
Vol 8 (6) ◽  
pp. 818 ◽  
Author(s):  
Marcello Ceccarelli ◽  
Aurora Diotallevi ◽  
Gloria Buffi ◽  
Mauro De Santi ◽  
Edith A. Fernández-Figueroa ◽  
...  
Keyword(s):  
High Resolution ◽  
Diagnostic Algorithm ◽  
Leishmania Species ◽  
Species Discrimination ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Hrm Analysis ◽  
Mucocutaneous Leishmaniasis ◽  
Geographical Regions ◽  
Etiological Agents

Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

scholarly journals Tracing Heterozygosity in the Vvlexp1 Locus in Grapevine by Sequencing and High-resolution Melt Analysis

2013 ◽  
Vol 138 (2) ◽  
pp. 120-124
Author(s):  
Ulrike C.M. Anhalt ◽  
Katharina Martini ◽  
Ernst-Heinrich Ruehl ◽  
Astrid Forneck
Keyword(s):  
Sequence Analysis ◽  
High Resolution ◽  
Vitis Vinifera ◽  
Segregation Analysis ◽  
High Resolution Melt ◽  
Codominant Marker ◽  
High Resolution Melt Analysis ◽  
Hrm Analysis ◽  
Multiple Loci ◽  
Traditional Breeding

Multiple loci in a continuously asexually reproducing genome such as vegetatively propagated grapevine (Vitis vinifera) can be heterozygote. The methodology to analyze heterozygous loci is manifold ranging from traditional breeding and studying segregating offspring, codominant marker analyses to whole sequence analysis. Results of heterozygosity studies on challenging loci need to be carefully confirmed to ensure accuracy and avoid misinterpretation. One of these methods is high-resolution melt (HRM) analysis in combination with sequencing and segregation analysis. We present first the adoption of HRM analyses for grapevine and its potential to confirm heterozygotic markers with low or no sequence size differences.

scholarly journals High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran

2021 ◽  
Author(s):  
Shima Fayaz ◽  
Pezhman Fard-Esfahani ◽  
Fariborz Bahrami ◽  
Parviz Parvizi ◽  
Soheila Ajdary
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Leishmania Species ◽  
Old World ◽  
Hrm Analysis ◽  
Etiologic Agents ◽  
Pcr Method ◽  
Etiological Agents ◽  
Rapid Discrimination ◽  
Low Sensitivity

Background and Objectives: The three old world Leishmania species i.e., L. major, L. tropica, and L. infantum are considered as potential etiological agents of the various clinical forms of leishmaniasis in Iran. Different species co-exist in some areas. Accurate differentiation between the species is essential for choosing an appropriate therapy. Conventional and gold standard methods for the detection and characterization of parasites are time-consuming, laborious, and have low sensitivity. A polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis has been employed for detection and species identification. Most of the studies suffer from the use of multiple targets and/ or requiring more than one reaction to identify a single sample. The present study aimed to design a PCR method based on the amplification of kinetoplast DNA minicircles (kDNA) and HRM analysis of the amplicons for rapid discrimination of the three mentioned species. Materials and Methods: DNA from reference strains including L. major, L. tropica, and L. infantum and fifty-eight strains subjected to PCR-HRM analysis targeting kDNA. All the samples were also analyzed by conventional kDNA- PCR. Results: The PCR-HRM analysis allowed discrimination between the three Old World species. The normalized HRM curves for the amplicons of kDNA indicated a unique and repeatable melting plot for each species, even in combination with human and mouse genomic DNA. Conventional kDNA- PCR could not properly discriminate L. tropica from L. infantum. Conclusion: PCR- HRM analysis of kDNA proved to be fast and accurate for discrimination of L. major, L. tropica, and L. infantum.

scholarly journals High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6

2013 ◽  
Vol 14 (8) ◽  
pp. 913-922 ◽  
Author(s):  
Greyson P Twist ◽  
Roger Gaedigk ◽  
J Steven Leeder ◽  
Andrea Gaedigk
Keyword(s):  
High Resolution ◽  
Homologous Gene ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Sequence Variations

A rapid method for sex identification in Cannabis sativa using High Resolution Melt (HRM) analysis.

Botany ◽  
2021 ◽  
Author(s):  
Erin Jacqueline Gilchrist ◽  
Daniela Hegebarth ◽  
Shumin Wang ◽  
Teagen D. Quilichini ◽  
Jason Sawler ◽  
...  
Keyword(s):  
High Resolution ◽  
Rapid Method ◽  
X Chromosome ◽  
Cannabis Sativa ◽  
Sex Identification ◽  
Male Plant ◽  
High Resolution Melt ◽  
Male And Female ◽  
High Resolution Melt Analysis ◽  
Plant Sex

We report the identification of two SNPs in Cannabis sativa that are associated with female and male plant sex phenotypes, and are located on the top arm of the X chromosome. High Resolution Melt analysis was used to develop and validate a novel, rapid method for sex identification in medical/recreational cannabis as well as in hemp. This method can distinguish between dioecious male (XY) and dioecious female (XX) cannabis plants with 100% accuracy, and can also be used to differentiate between male and female Humulus lupulus (hop) plants.

High-Resolution Melt Analysis

2013 ◽  
pp. 409-422 ◽  
Author(s):  
Einar Berg ◽  
Tania Nolan
Keyword(s):  
High Resolution ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis

scholarly journals Detecting and Differentiating Phytoplasmas Belonging to Subgroups 16SrIV-A and 16SrIV-D Associated With Lethal Declines of Palms in Florida Using qPCR and High-Resolution Melt Analysis (HRMA)

Plant Disease ◽  
2017 ◽  
Vol 101 (8) ◽  
pp. 1449-1454 ◽  
Author(s):  
Brian W. Bahder ◽  
Ericka E. Helmick ◽  
Nigel A. Harrison
Keyword(s):  
High Resolution ◽  
Sequence Data ◽  
Management Strategies ◽  
Economic Losses ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Melting Temperatures ◽  
Detection And Identification ◽  
Lethal Yellowing ◽  
Sufficient Variation

Lethal yellowing (LY) and Texas Phoenix palm decline (TPPD) are two important phytoplasma diseases of palms in Florida. Both have been responsible for major economic losses historically and remain a constant threat to the sustainability of palm production in the landscaping and nursery industries in Florida. These two diseases cause rapid, lethal decline in afflicted palms, so rapid detection and identification is crucial to implement appropriate management strategies to reduce further spread and losses. In this study, a qPCR assay was developed to detect and identify the causal agents of LY and TPPD. Based on sequence data of the 16S gene for the 16SrIV-A phytoplasma (LY) and the 16SrIV-D phytoplasma (TPPD), two regions were identified in the gene that possessed sufficient variation to yield amplicons with measurable differences in melting temperature based on high resolution melt analysis (HRMA). One region was in the 5′ region and the other was located in the 3′ region of the gene. Products from both regions yielded amplicons with significantly different melting temperatures between the two phytoplasma strains. This research allows for the detection and identification of phytoplasmas in palms rapidly by eliminating many lengthy and post-PCR steps commonly used in phytoplasma identification.

Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

Transfusion ◽  
2020 ◽  
Vol 60 (11) ◽  
pp. 2702-2713
Author(s):  
Tomoya Hayashi ◽  
Ryota Aminaka ◽  
Hiroyuki Ishii ◽  
Yoshihiko Tani ◽  
Yoshihiro Fujimura ◽  
...  
Keyword(s):  
High Resolution ◽  
Blood Donors ◽  
Human Platelet ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis ◽  
Platelet Antigen ◽  
Human Platelet Antigen
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