scholarly journals Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252887
Author(s):  
Renate Schneider ◽  
Aline Lamien-Meda ◽  
Herbert Auer ◽  
Ursula Wiedermann-Schmidt ◽  
Peter L. Chiodini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Melting Curve ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Significant Rise ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

scholarly journals Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Liuyang Hu ◽  
Bing Han ◽  
Qin Tong ◽  
Hui xiao ◽  
Donglin Cao
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Melting Curve ◽  
Cost Effective ◽  
Culture Method ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Fluorescence Melting Curve Analysis

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

Quantitative Detection and Differentiation of Free-Living Amoeba Species Using SYBR Green–Based Real-Time PCR Melting Curve Analysis

2006 ◽  
Vol 53 (6) ◽  
pp. 506-509 ◽  
Author(s):  
Jonas Behets ◽  
Priscilla Declerck ◽  
Yasmine Delaedt ◽  
Lieve Verelst ◽  
Frans Ollevier
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Quantitative Detection ◽  
Melting Curve Analysis ◽  
Free Living ◽  
Free Living Amoeba

scholarly journals A FRET-ICT Dual-Modulated Ratiometric Fluorescence Sensor for Monitoring and Bio-Imaging of Cellular Selenocysteine

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 4999
Author(s):  
Zongcheng Wang ◽  
Chenhong Hao ◽  
Xiaofang Luo ◽  
Qiyao Wu ◽  
Chengliang Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Fluorescence Sensor ◽  
Quantitative Detection ◽  
Fluorescence Signal ◽  
Ratiometric Fluorescence ◽  
Sensitivity And Selectivity

Since the fluctuation of cellular selenocysteine (Sec) concentration plays an all-important role in the development of numerous human disorders, the real-time fluorescence detection of Sec in living systems has attracted plenty of interest during the past decade. In order to obtain a faster and more sensitive small organic molecule fluorescence sensor for the Sec detection, a new ratiometric fluorescence sensor Q7 was designed based on the fluorescence resonance energy transfer (FRET) strategy with coumarin fluorophore as energy donor and 4-hydroxy naphthalimide fluorophore (with 2,4-dinitrobenzene sulfonate as fluorescence signal quencher and Sec-selective recognition site) as an energy acceptor. The sensor Q7 exhibited only a blue fluorescence signal, and displayed two well distinguished emission bands (blue and green) in the presence of Sec with ∆λ of 68 nm. Moreover, concentrations ranging of quantitative detection of Sec of Q7 was from 0 to 45 μM (limit of detection = 6.9 nM), with rapid ratiometric response, high sensitivity and selectivity capability. Impressively, the results of the living cell imaging test demonstrated Q7 has the potentiality of being an ideal sensor for real-time Sec detection in biosystems.

scholarly journals Rapid detection of the factor XIII Val34Leu (163 G→T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis

2004 ◽  
Vol 42 (8) ◽  
Author(s):  
Amir H. Shemirani ◽  
László Muszbek
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Allele Frequency ◽  
Real Time Pcr ◽  
Factor Xiii ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Curve Analysis ◽  
Melting Curve Analysis

AbstractThe Val34Leu polymorphism in the A subunit of blood coagulation factor XIII (FXIII-A) is located in the activation peptide, just three amino acids upstream of the thrombin cleavage site. The Val→Leu replacement accelerates the rate of the proteolytic activation of FXIII and it seems to provide protection against myocardial infarction. Methods available for the assessment of the FXIII-A Val34Leu polymorphism are rather time-consuming, laborious and not easily applicable for large-scale studies. In this study a new method based on real-time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis was developed. The rapid, simple method was adapted to the widely used real-time PCR instrument, LightCycler (Roche Diagnostics). The results showed 100% coincidence with those obtained by the traditional PCR-restriction fragment length polymorphism (RFLP) assay and fluorescent DNA sequencing. Using this method, an allele frequency of 24.2% was obtained (n = 113), which well agrees with the allele frequency obtained by PCR-RFLP on a different group of the same ethnic Hungarian population (25.9%).

The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method

The Analyst ◽  
2018 ◽  
Vol 143 (14) ◽  
pp. 3292-3301 ◽  
Author(s):  
Huihui Mao ◽  
Guanghua Luo ◽  
Yuxia Zhan ◽  
Jun Zhang ◽  
Shuang Yao ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Probe Method ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve analysis, which might require only one pair of primers and one probe.

Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

Parasitology ◽  
2012 ◽  
Vol 139 (10) ◽  
pp. 1266-1272 ◽  
Author(s):  
O. SANPOOL ◽  
P. M. INTAPAN ◽  
T. THANCHOMNANG ◽  
P. SRI-AROON ◽  
V. LULITANOND ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
High Sensitivity ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Intermediate Hosts ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Schistosoma Mekongi

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

scholarly journals One-Step ARMS-PCR for the Detection of SNPs—Using the Example of the PADI4 Gene

2019 ◽  
Vol 2 (3) ◽  
pp. 63 ◽  
Author(s):  
Ehnert ◽  
Linnemann ◽  
Braun ◽  
Botsch ◽  
Leibiger ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Amplification Refractory Mutation System ◽  
Nucleotide Polymorphisms ◽  
Cellular Functions ◽  
Arms Pcr ◽  
One Step

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer’s disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.

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

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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