scholarly journals CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9113 ◽  
Author(s):  
Nuttachat Wisittipanit ◽  
Chaiwat Pulsrikarn ◽  
Sudarat Srisong ◽  
Rungthiwa Srimora ◽  
Nattinee Kittiwan ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Learning Algorithm ◽  
Food Poisoning ◽  
Hospital Setting ◽  
Epidemiological Studies ◽  
Detection And Identification ◽  
Appropriate Treatment ◽  
Signature Sequences ◽  
Evolutionary Trajectories

Background Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans. Method A small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis. Results CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles. Conclusion CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

scholarly journals Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

2013 ◽  
Vol 79 (24) ◽  
pp. 7837-7845 ◽  
Author(s):  
Thu Nguyet Phung ◽  
Domenico Caruso ◽  
Sylvain Godreuil ◽  
Nicolas Keck ◽  
Tatiana Vallaeys ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Limit Of Detection ◽  
High Resolution Melting Analysis ◽  
23S Rrna ◽  
Economic Losses ◽  
Causal Organism ◽  
Content Type ◽  
Detection And Identification ◽  
Melting Analysis

ABSTRACTMycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species ofMycobacteriumhave been reported to infect fish. Among them,Mycobacterium marinum,M. fortuitum, andM. chelonaeare generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate severalMycobacteriumspecies involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purifiedM. marinumandM. fortuitumDNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacteriumspecies, thereby demonstrating their specificity for the genusMycobacterium.

High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone

2019 ◽  
Vol 75 (1) ◽  
pp. 106-109 ◽  
Author(s):  
Leshan Xiu ◽  
Chi Zhang ◽  
Yamei Li ◽  
Feng Wang ◽  
Junping Peng
Keyword(s):  
High Resolution ◽  
Neisseria Gonorrhoeae ◽  
Internal Control ◽  
High Resolution Melting ◽  
Direct Detection ◽  
High Specificity ◽  
Dual Therapy ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Clinical Samples

Abstract Objectives Increased awareness of the international spread of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone, which threatens recommended dual therapy, is essential. The objective of the present study was to develop and evaluate a rapid, simple and cost-effective method based on high-resolution melting (HRM) analysis for direct detection of the FC428 clone from clinical isolates and specimens. Methods The singleplex HRM assay was designed to identify the FC428 clone by using specific primers, which flank the alteration A311V in the penA-60.001 allele. Analytical performance was initially evaluated by testing 623 isolates and a panel of non-gonococcal strains. To ensure the method can be directly applied in clinical samples, two internal control targets (opa and porA) were also designed and included in the final multiplex HRM assay. Two hundred and eighty-two clinical samples (94 urine and 188 urethral/genital swabs) were then analysed using this multiplex HRM assay. Results The FC428 clone was easily differentiated from the non-mosaic alleles and other mosaic alleles without A311 mutations by comparing the differences in melt curves. Cross-reactivity was not observed for the penA-60.001 allele when testing 15 non-gonococcal Neisseria strains. When applied to the 623 isolates, the HRM assay successfully characterized one isolate as an FC428 clone (MLST1903, NG-MAST3435, NG-STAR233). Our data show that the multiplex HRM assay with high specificity can be directly applied in clinical samples. Conclusions This method can generate results within 90 min at a cost of less than US$0.5 per isolate or sample, making this assay an ideal tool for large epidemiological studies to enhance surveillance of the internationally transmitted ceftriaxone-resistant N. gonorrhoeae FC428 clone.

scholarly journals A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Aline Lamien-Meda ◽  
Hans-Peter Fuehrer ◽  
David Leitsch ◽  
Harald Noedl
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Limit Of Detection ◽  
Plasmodium Species ◽  
Malaria Diagnosis ◽  
Epidemiological Studies ◽  
Taqman Probe ◽  
Species Differentiation ◽  
Resource Limited ◽  
Highly Sensitive

Abstract Background The use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease. Methods A qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum. Results Out of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0 %) single infections, 12 (10.6 %) mixed infections, and 5 (4.4 %) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2 %. The assay limit of detection was varied from 21.47 to 46.43 copies /µl, equivalent to 1–2.11 parasites/µl. All P. falciparum infections were confirmed with the associated Taqman probe. Conclusions Although the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

scholarly journals MeltingPlot: a user-friendly online tool for epidemiological investigation using High Resolution Melting data

2020 ◽  
Author(s):  
Matteo Perini ◽  
Gherard Batisti Biffignandi ◽  
Domenico Di Carlo ◽  
Ajay Ratan Pasala ◽  
Aurora Piazza ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Epidemiological Studies ◽  
Supplementary Information ◽  
Web Interface ◽  
Web Tool ◽  
Online Tool ◽  
Link Type ◽  
Surveillance Programs ◽  
User Friendly

AbstractSummaryMeltingPlot is an open source web tool for pathogen typing and epidemiological investigations using High Resolution Melting (HRM) data. The tool implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. MeltingPlot also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological studies. HRM technique allows pathogen typing in less than 5 hours with ~5 euros per sample. MeltingPlot is the first tool specifically designed for HRM-based epidemiological studies and it can analyse hundreds of isolates in a few seconds. Thus, the use of MeltingPlot makes HRM-based typing suitable for large surveillance programs as well as for rapid outbreak reconstructions.Availability and implementationMeltingPlot is implemented in R.The web interface is available at https://skynet.unimi.it/index.php/tools/meltingplot.The source code is also available at https://github.com/MatteoPS/[email protected] informationSupplementary data are available at Bioinformatics online.

Detection and identification of β-thalassemia 3.5 kb deletion by SYBR Green1 and high resolution melting analysis

2009 ◽  
Vol 82 (2) ◽  
pp. 159-160 ◽  
Author(s):  
Prathom Prathomtanapong ◽  
Sakorn Pornprasert ◽  
Arunee Phusua ◽  
Sudjai Suanta ◽  
Rattika Saetung ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Detection And Identification ◽  
Melting Analysis
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