Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection

Lab on a Chip ◽  
2014 ◽  
Vol 14 (3) ◽  
pp. 562-568 ◽  
Author(s):  
Andrew C. Hatch ◽  
Tathagata Ray ◽  
Kelly Lintecum ◽  
Cody Youngbull
Keyword(s):  
Real Time ◽  
Continuous Flow ◽  
Fiber Optic ◽  
Quantitative Polymerase Chain Reaction ◽  
Conveyor Belt ◽  
Chain Reaction ◽  
Fluorescence Excitation ◽  
Droplet Emulsion ◽  
Polymerase Chain ◽  
Thermal Cycler

We have developed a ‘conveyor belt’ analog for real-time quantitative polymerase chain reaction (qPCR) in a continuous flow thermocycler. The device integrates droplet emulsion technology with LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve real-time fluorescence detection.

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 Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Quantification of Common Wheat Adulteration of Durum Wheat Pasta Using Real-Time Quantitative Polymerase Chain Reaction (PCR)

2002 ◽  
Vol 79 (4) ◽  
pp. 553-558 ◽  
Author(s):  
Rémi Alary ◽  
Arnaud Serin ◽  
Marie-Pierre Duviau ◽  
Philippe Jourdrier ◽  
Marie-Françoise Gautier
Keyword(s):  
Polymerase Chain Reaction ◽  
Durum Wheat ◽  
Real Time ◽  
Common Wheat ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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