A handheld continuous-flow real-time fluorescence qPCR system with a PVC microreactor

The Analyst ◽  
2020 ◽  
Vol 145 (7) ◽  
pp. 2767-2773 ◽  
Author(s):  
Bing Shi ◽  
Yuanming Li ◽  
Di Wu ◽  
Wenming Wu
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Biology ◽  
Real Time ◽  
Continuous Flow ◽  
Pathogen Detection ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Sensitivity Specificity

The polymerase chain reaction (PCR) has unique advantages of sensitivity, specificity and rapidity in pathogen detection, which makes it at the forefront of academia and application in molecular biology diagnosis.

scholarly journals Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit

Critical Care ◽  
2010 ◽  
Vol 14 (4) ◽  
pp. R159 ◽  
Author(s):  
Katsunori Yanagihara ◽  
Yuko Kitagawa ◽  
Masao Tomonaga ◽  
Kunihiro Tsukasaki ◽  
Shigeru Kohno ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Dna Detection ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pathogen Dna

scholarly journals 3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

2021 ◽  
Vol 22 (11) ◽  
pp. 6061
Author(s):  
Owen Higgins ◽  
Terry J. Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Detection ◽  
Pathogen Detection ◽  
Multiplex Detection ◽  
Chain Reaction ◽  
Single Base ◽  
Base Specificity ◽  
Pcr Technology ◽  
Polymerase Chain

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

Continuous-Flow Microfluidic Device for Real-Time Polymerase Chain Reaction

2016 ◽  
Vol 37 (11) ◽  
pp. 1878-1881
Author(s):  
Hanok Kim ◽  
Shinae Suk ◽  
Kwanseop Lim ◽  
Nokyoung Park ◽  
Jong Hoon Hahn
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Microfluidic Device ◽  
Continuous Flow ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Blood group antigen profile predicted by molecular biology - use of real-time polymerase chain reaction to genotype important KEL, JK, RHD, and RHCE alleles

2020 ◽  
Vol 18 (3) ◽  
pp. 59-64
Author(s):  
Fernando Manuel Ferreira Araújo ◽  
Christiana Pereira ◽  
Fátima Monteiro ◽  
Isabel Henriques ◽  
Elsa Meireles ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Biology ◽  
Real Time ◽  
Blood Group ◽  
Blood Group Antigen ◽  
Chain Reaction ◽  
Antigen Profile ◽  
Polymerase Chain

Real-Time Fluorescence Monitoring of the Polymerase Chain Reaction in a Novel Continuous Flow Reactor for Accurate DNA Quantification

2006 ◽  
Author(s):  
Michael B. Sayers ◽  
Tara M. Dalton ◽  
Mark R. Davies
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Continuous Flow ◽  
Flow Reactor ◽  
Chain Reaction ◽  
Continuous Flow Reactor ◽  
Capillary Tubing ◽  
Polymerase Chain ◽  
Thermal Cycler

Real-time Polymerase Chain Reaction (PCR) is the preferred method for quantification of gene expression levels due to its extreme sensitivity. Fluorescence based real-time PCR is commonly used for the quantification of the initial amount of a specific sequence of DNA. Real-time quantification may be achieved using fluorescent dyes, by optically monitoring the product formation as the PCR cycles progress. Stationary well based real-time quantification is quite common, however continuous flow real-time PCR which is the aim of this work is still in its infancy. A compact, high throughput continuous flow thermal cycler has been developed which allows for real-time fluorescent measurements to be obtained. The principle of operation of this device is that the three thermal zones required for a polymerase chain reactor are maintained on both sides of an aluminium block and bio-compatible FEP Teflon capillary tubing is then wrapped around these constant temperature blocks. The capillary tubing is wrapped around the device fifteen times which provides thirty PCR thermal cycles. The device has been designed and optimised to accurately monitor the product expression level using the double stranded DNA binding dye SYBR green I. Initially the PCR mixture is segmented into small nanoreactors, separated by an immiscible carrier fluid to eliminate cross contamination and reduce the likelihood of sample degradation due to contact with the capillary wall. These PCR nanoreactors are then cycled through the tubing and the DNA amplified. Fluorescent optical monitoring of these nanoreactors takes place where a water glycerine mixture, which is refractive index matched to the tubing, allows for improved fluorescent measurements of the nano-volume reactors to be obtained. Plasmid DNA, 240 base pairs long, has been successfully amplified using this device and the temperatures for the denaturation, annealing and extension phases have been accurately measured. Real-time fluorecence images of the flowing nano-volumes were recorded every second cycle using a CCD camera and from these images amplification curves have been successfully generated. Samples with various initial concentrations of DNA have been thermally cycled on the continuous flow reactor. The measured increase in fluorescence intensity from the flowing nano-volume reactors as they progressed through the thermal cycler demonstrated the effect of initial DNA template concentration.

scholarly journals Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

2004 ◽  
Vol 94 (10) ◽  
pp. 1075-1083 ◽  
Author(s):  
Katherine J. Hayden ◽  
David Rizzo ◽  
Justin Tse ◽  
Matteo Garbelotto
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Phytophthora Species ◽  
Phytophthora Ramorum ◽  
Plant Diseases ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
New Host ◽  
Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

scholarly journals Implementation of Case-Based Surveillance and Real-time Polymerase Chain Reaction to Monitor Bacterial Meningitis Pathogens in Chad

2019 ◽  
Vol 220 (Supplement_4) ◽  
pp. S182-S189 ◽  
Author(s):  
Marietou F Paye ◽  
Kadidja Gamougame ◽  
Sarah K Payamps ◽  
Alicia R Feagins ◽  
Daugla Doumagoum Moto ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Meningitis ◽  
Real Time ◽  
Pathogen Detection ◽  
Reference Laboratory ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Increased Sensitivity ◽  
Polymerase Chain ◽  
Case Based

Abstract Background Meningococcal serogroup A conjugate vaccine (MACV) was introduced in Chad during 2011–2012. Meningitis surveillance has been conducted nationwide since 2003, with case-based surveillance (CBS) in select districts from 2012. In 2016, the MenAfriNet consortium supported Chad to implement CBS in 4 additional districts and real-time polymerase chain reaction (rt-PCR) at the national reference laboratory (NRL) to improve pathogen detection. We describe analysis of bacterial meningitis cases during 3 periods: pre-MACV (2010–2012), pre-MenAfriNet (2013–2015), and post-MenAfriNet (2016–2018). Methods National surveillance targeted meningitis cases caused by Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Cerebrospinal fluid specimens, inoculated trans-isolate media, and/or isolates from suspected meningitis cases were tested via culture, latex, and/or rt-PCR; confirmed bacterial meningitis was defined by a positive result on any test. We calculated proportion of suspected cases with a specimen received by period, and proportion of specimens with a bacterial meningitis pathogen identified, by period, pathogen, and test. Results The NRL received specimens for 6.8% (876/12813), 46.4% (316/681), and 79.1% (787/995) of suspected meningitis cases in 2010–2012, 2013–2015, and 2016–2018, respectively, with a bacterial meningitis pathogen detected in 33.6% (294/876), 27.8% (88/316), and 33.2% (261/787) of tested specimens. The number of N. meningitidis serogroup A (NmA) among confirmed bacterial meningitis cases decreased from 254 (86.4%) during 2010–2012 to 2 (2.3%) during 2013–2015, with zero NmA cases detected after 2014. In contrast, proportional and absolute increases were seen between 2010–2012, 2013–2015, and 2016–2018 in cases caused by S. pneumoniae (5.1% [15/294], 65.9% [58/88], and 52.1% [136/261]), NmX (0.7% [2/294], 1.1% [1/88], and 22.2% [58/261]), and Hib (0.3% [1/294], 11.4% [10/88], and 14.9% [39/261]). Of specimens received at the NRL, proportions tested during the 3 periods were 47.7% (418), 53.2% (168), and 9.0% (71) by latex; 81.4% (713), 98.4% (311), and 93.9% (739) by culture; and 0.0% (0), 0.0% (0), and 90.5% (712) by rt-PCR, respectively. During the post-MenAfriNet period (2016–2018), 86.1% (678) of confirmed cases were tested by both culture and rt-PCR, with 12.5% (85) and 32.4% (220) positive by culture and rt-PCR, respectively. Conclusions CBS implementation was associated with increased specimen referral. Increased detection of non-NmA cases could reflect changes in incidence or increased sensitivity of case detection with rt-PCR. Continued surveillance with the use of rt-PCR to monitor changing epidemiology could inform the development of effective vaccination strategies.

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