scholarly journals Melting curve analysis to differentiate Rickettsia spp. and Rickettsia felis

2021 ◽  
Vol 948 (1) ◽  
pp. 012079
Author(s):  
D Widiastuti ◽  
Agustiningsih ◽  
S P M Wijayati ◽  
E Lestari
Keyword(s):  
Melting Curve ◽  
Diagnostic Procedures ◽  
Laboratory Diagnosis ◽  
Melting Curve Analysis ◽  
Quantitative Real Time Pcr ◽  
Rickettsia Felis ◽  
Rickettsia Species ◽  
Cat Flea ◽  
Pcr Product ◽  
Pcr Targeting

Abstract Rickettsiosis, caused by Rickettsia species, is one of the old arthropod-borne illness that commonly found in humans and animals. One of the barriers to rickettsiosis control is the intricacy and time-consuming nature of rickettsiosis laboratory diagnosis. This study aimed to establish quantitative real-time PCR targeting the gltA gene for the DNA differentiation of Rickettsia spp. and Ricketsia felis. The collection of cat flea was extracted to acquire the DNA of Rickettsia. Primers were designed based on the analysis of Rickettsia gltA gene sequences. The confirmation of R. felis was performed by sequencing of PCR product. BLAST analysis was done to confirm the closest similarity of the sequences. Results of this study highlighted the melting temperature was reached at 78,5 °C for Rickettsia spp. and 76.5+0.5 °C for Rickettsia felis. The melting peak temperatures were significantly different between Rickettsia spp. and R. felis (p<0.05). The findings of this work are crucial in the development of powerful diagnostic procedures for detecting and distinguishing Rickettsia spp. and R. felis species.

Fluorescent Melting Curve Analysis Compatible With a Flowing Polymerase Chain Reactor

2005 ◽  
Author(s):  
Tara M. Dalton ◽  
David J. Kinahan ◽  
Mark R. Davies
Keyword(s):  
Base Pair ◽  
Double Helix ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Pair Composition ◽  
Pcr Product ◽  
Pcr Products ◽  
Dna Strand ◽  
Thermal Cycler

A primary tool for analysing PCR product is the Fluorescent Melting Curve Analysis (FMCA). The temperature at which a double helix DNA strand denatures depends both on its length and base pair composition. Accurate measurement of this melting temperature using fluorescence allows estimations be made regarding DNA product length and composition. Current progress in development of PCR thermal cyclers has been primarily aimed at micro-channel based flowing devices. This paper addresses the challenges associated with performing FMCA analysis which is compatible with the output from a flowing PCR thermocycler. Two PCR products of significantly different lengths and base pair composition are compared using space domain FMCA. Results allow for differentiation of the PCR product, and compare favourably with results from a commercial thermal cycler. The successful application of FMCA within a channel shows its potential for use in high throughput flow based total analysis systems (μTAS).

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.

PITX2 and NEURL1 SNP polymorphisms in Hungarian atrial fibrillation patients determined by quantitative real-time PCR and melting curve analysis

2019 ◽  
Vol 299 ◽  
pp. 44-49
Author(s):  
Krisztina Szirák ◽  
Beáta Soltész ◽  
Orsolya Hajas ◽  
Réka Urbancsek ◽  
Edina Nagy-Baló ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Quantitative Real Time Pcr

scholarly journals Dynamic gene expression in salivary glands of the cat flea during Rickettsia felis infection

2021 ◽  
Vol 79 (5) ◽  
Author(s):  
Monika Danchenko ◽  
Hanna J Laukaitis ◽  
Kevin R Macaluso
Keyword(s):  
Gene Expression ◽  
Salivary Glands ◽  
Dynamic Change ◽  
Human Pathogens ◽  
Rickettsia Felis ◽  
Rickettsia Species ◽  
Cat Flea ◽  
Cat Fleas ◽  
Specific Antigens ◽  
And Control

ABSTRACT The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection.

scholarly journals Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15) mRNA in Various Human Cell Types

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Sang Mee Hwang ◽  
Mi Jung Kim ◽  
Ho Eun Chang ◽  
Yun Ji Hong ◽  
Taek Soo Kim ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Platelet ◽  
Human Fibroblasts ◽  
Leukemia Cell ◽  
Embryonic Stem ◽  
Melting Curve ◽  
Leukemia Cell Line ◽  
Melting Curve Analysis ◽  
Platelet Antigen ◽  
Human Platelet Antigen

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

scholarly journals Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

2005 ◽  
Vol 43 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Kijeong Kim ◽  
Juwon Seo ◽  
Katherine Wheeler ◽  
Chulmin Park ◽  
Daewhan Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis

Diagnosis of neuroschistosomiasis by antibody specificity index and semi-quantitative real-time PCR from cerebrospinal fluid and serum

2014 ◽  
Vol 63 (2) ◽  
pp. 309-312 ◽  
Author(s):  
Georg Härter ◽  
Hagen Frickmann ◽  
Sebastian Zenk ◽  
Dominic Wichmann ◽  
Bettina Ammann ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Symptoms ◽  
Diagnostic Procedures ◽  
Lower Limbs ◽  
Antibody Specificity ◽  
Quantitative Real Time Pcr ◽  
Specific Antibodies ◽  
Routine Diagnosis

We describe the case of a 16-year-old German male expatriate from Ghana who presented with obstipation, dysuria, dysaesthesia of the gluteal region and the lower limbs, bilateral plantar hypaesthesia and paraesthesia without pareses. A serum–cerebrospinal fluid (CSF) Schistosoma spp. specific antibody specificity index of 3.1 was considered highly suggestive of intrathecal synthesis of anti-Schistosoma spp. specific antibodies, although standardization of this procedure has not previously been described. Diagnosis was confirmed by detection of Schistosoma DNA in CSF by semi-quantitative real-time PCR at 100-fold concentration compared with serum. Accordingly the two diagnostic procedures, which have not previously been applied for routine diagnosis, appear to be useful for the diagnosis of neuroschistosomiasis. Clinical symptoms resolved following anthelmintic and anti-inflammatory therapy.

scholarly journals Diagnosis of α-1-Antitrypsin Deficiency: An Algorithm of Quantification, Genotyping, and Phenotyping

2006 ◽  
Vol 52 (12) ◽  
pp. 2236-2242 ◽  
Author(s):  
Melissa R Snyder ◽  
Jerry A Katzmann ◽  
Malinda L Butz ◽  
Ping Yang ◽  
D Brian Dawson ◽  
...  
Keyword(s):  
Laboratory Testing ◽  
Null Allele ◽  
Melting Curve ◽  
Curve Analysis ◽  
Laboratory Evaluation ◽  
Melting Curve Analysis ◽  
Genotype Assay ◽  
Antitrypsin Deficiency ◽  
Phenotype Analysis ◽  
A1at Deficiency

Abstract Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

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