Development of a portable low-cost real-time PCR system

2017 ◽  
Author(s):  
K. S. Chong ◽  
K. B. Gan ◽  
Sue-Mian Then
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

2015 ◽  
Author(s):  
Rainer Gransee ◽  
Tristan Schneider ◽  
Deniz Elyorgun ◽  
Xenia Strobach ◽  
Tobias Schunck ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Melting Curve ◽  
Lab On A Chip ◽  
Curve Analysis ◽  
Melting Curve Analysis

Low-Cost Monitoring of Campylobacter in Poultry Houses by Air Sampling and Quantitative PCR

2014 ◽  
Vol 77 (2) ◽  
pp. 325-330 ◽  
Author(s):  
M. S. R. SØNDERGAARD ◽  
M. H. JOSEFSEN ◽  
C. LÖFSTRÖM ◽  
L. S. CHRISTENSEN ◽  
K. WIECZOREK ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Air Sampling ◽  
Quantitative Risk Assessment ◽  
Quantitative Real Time Pcr ◽  
Airborne Particle ◽  
Air Samples ◽  
Particle Count ◽  
Poultry Houses

The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 μm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 104 and 105 CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative real-time PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter.

scholarly journals Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

2009 ◽  
Vol 107 (5) ◽  
pp. 1433-1439 ◽  
Author(s):  
K.L. Leung ◽  
C.W. Yip ◽  
W.F. Cheung ◽  
A.C.T. Lo ◽  
W.M. Ko ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Method

scholarly journals Confirmation of multi-parallel quantitative real-time PCR as the gold standard for detecting soil-transmitted helminths in stool

2021 ◽  
Author(s):  
Marina Papaiakovou ◽  
Nils Pilotte ◽  
Julia Dunn ◽  
David TJ Littlewood ◽  
Rubén O Cimino ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Low Cost ◽  
Strongyloides Stercoralis ◽  
Diagnostic Methods ◽  
Quantitative Real Time Pcr ◽  
Soil Transmitted Helminths ◽  
Stool Samples ◽  
Kappa Agreement

AbstractDue to its simplicity and cost-effectiveness, microscopy has seen extensive field-use as the diagnostic standard for the detection of soil-transmitted helminths (STH) in stool samples. However, the sensitivity of microscopy-based detection is inadequate in reduced-transmission settings where worm burden is oftentimes low. Equally problematic, eggs of closely related species oftentimes have indistinguishable morphologies, leading to species misidentification. In light of these shortcomings, the purpose of this study was to demonstrate multi-parallel quantitative real-time PCR (qPCR) as the new “gold standard” for STH detection. Accordingly, stool samples from non-endemic participants were spiked with limited numbers of eggs or larvae (1 to 40) of five different species of STH. DNA extracts were tested using two unique multi-parallel real-time PCR-based diagnostic methods. These methods employed different target sequences (ribosomal internal transcribed spacer, or highly repetitive non-coding regions), to evaluate the detection of DNA from as little as one egg per sample. There was a statistically significant kendall correlation between egg/larvae counts and qPCR from both methods for Trichuris trichiura (0.86 and 0.872 for NHM and Baylor assays) and a strong correlation (0.602 and 0.631 for NHM and Baylor assays, respectively) for Ascaris lumbricoides. Less strong but still significant was the Kendall Tau-b value for A. duodenale (0.408 for both) and for S. stercoralis (0.483 and 0.653, respectively). In addition, using field stool samples from rural Argentina both assays had fair to moderate kappa agreement (0.329-0.454), except for Strongyloides stercoralis (0.121) that both assays had slight agreement. In spite of the small cohort of samples, both qPCR assays, targeting of two independent genomic regions, provided reproducible results and we believe that, low cost multi-parallel quantitative real-time PCR-based diagnostics should supplant microscopy as the new gold standard for stool-based detection of soil transmitted helminths in public-health and community settings.

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

scholarly journals Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR

2015 ◽  
Vol 142 (5) ◽  
pp. 555 ◽  
Author(s):  
BVishnu Bhat ◽  
DBenet Bosco Dhas ◽  
AHiasindh Ashmi ◽  
SubashChandra Parija ◽  
N Banupriya
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Low Cost ◽  
Snp Genotyping ◽  
Cycle Threshold ◽  
Allele Specific

scholarly journals Multiple Compact Camera Fluorescence Detector for Real-Time PCR Devices

Sensors ◽  
2021 ◽  
Vol 21 (21) ◽  
pp. 7013
Author(s):  
Seul-Bit-Na Koo ◽  
Hyeon-Gyu Chi ◽  
Jong-Dae Kim ◽  
Yu-Seop Kim ◽  
Ji-Sung Park ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Performance ◽  
Signal To Noise Ratio ◽  
Low Cost ◽  
Optical Element ◽  
Dna Amplification ◽  
Biological Research ◽  
Fluorescence Detector ◽  
Fluorescence Excitation

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.

scholarly journals Detection of the Brachyspina mutation in Uruguayan Holstein cows using real time PCR and melting curve analysis

2021 ◽  
Vol 51 (9) ◽  
Author(s):  
María Teresa Federici Rodriguez ◽  
Rody Artigas ◽  
Sofía Guerra ◽  
Andrea Branda Sica ◽  
Noelia Vázquez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Stop Codon ◽  
Low Cost ◽  
Melting Curve ◽  
Premature Stop Codon ◽  
Curve Analysis ◽  
Assay Method ◽  
Melting Curve Analysis ◽  
Group I

ABSTRACT: Brachyspina syndrome (BS) is a rare monogenic autosomal recessive hereditary disorder of the Holstein Fresian breed caused by a deletion of 3.3Kb in the Fanconi anemia complementation group I (FANCI) gene on BTA-21, which leads to a frame-shift and premature stop codon. Some of the consequences of BS are the reduction of the fertility rate and milk production. This study developed a simple, sensitive, rapid cost- effective assay method based on real time PCR and melting curve analysis for the detection of BS carrier animals. Sixty-eight normal homozygous and four heterozygous carrier genotypes were detected and confirmed through traditional PCR- electrophoresis analysis. We concluded that the assay we have developed proved to be a reliable, highly precise and low-cost tool, which could be used to monitor the presence of the BS mutation in uruguayan Holstein breed.

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