共查询到20条相似文献,搜索用时 46 毫秒
1.
This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic
chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed
on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic
structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated,
transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the
hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro
heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform
precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips,
a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required
an operation voltage of 12VRMS at a frequency of 3 KHz for digital microfluidic actuation and 9VDC for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less
sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip
(LOC) by utilizing EWOD-based digital microfluidics. 相似文献
2.
Ahmad F Seyrig G Tourlousse DM Stedtfeld RD Tiedje JM Hashsham SA 《Biomedical microdevices》2011,13(5):929-937
Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially
in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system
for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging
capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system
is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82
dye and real time fluorescence loop-mediated isothermal amplification referred here as microRTf-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRTf-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD
exposure of 5 s to 105 starting DNA copies of microRTf-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument.
Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRTf-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the
exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for
rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips. 相似文献
3.
Ilija Pješčić Collin Tranter Patrick L. Hindmarsh Niel D. Crews 《Biomedical microdevices》2010,12(2):333-343
In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature
gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined.
The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative
design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition,
thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 μl/min induced no measurable
change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol,
even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility
of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient
cycles in 15 min at a 2 μl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture
filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (ΦX174), images taken
after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of
a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves
of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized
as a function of spatial redundancy and acquisition time. 相似文献
4.
Song-I Han Ki-Ho Han A. Bruno Frazier Jerome P. Ferrance James P. Landers 《Biomedical microdevices》2009,11(4):935-942
This paper presents an automated micro-SPE device for DNA extraction using monolithically integrated high-pressure microvalves.
The automated micro-SPE device was fabricated through glass-to-glass thermal bonding and microfluidic system interface technologies.
To increase the DNA extraction efficiency, silica beads were packed in the extraction microchannel involving two weir structures.
Experimental results show that the DNA extraction efficiency using the automated micro-SPE device containing bare silica beads
was 75.87% in the first 8 μl of solution eluted by automated SPE procedure. In addition, the reproducibility of the DNA extraction
was evaluated by ten successive measurements. Genomic DNA extracted from human WBCs had an absorbance ratio of DNA to protein
(A260/A280) of 1.56. The applicability of this automated micro-SPE device to genetic sample preparation was verified by PCR amplification
of a β-globulin gene using the genomic DNA extracted from WBCs. Consequently, we demonstrated that the proposed automatic
micro-SPE device can extract nucleic acids from biological samples, thereby facilitating its integration with downstream genetic
analyses in a micro format. 相似文献
5.
Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL–5 μL), amplification speed
(100–400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic
separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or
calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely
hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate
and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of
PCR in 1 μL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min
to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering
in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from
λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple,
relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering. 相似文献
6.
The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle
eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing
DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time
PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold
cycle (C
T) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of
QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C
T values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first
6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle
such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification
of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition
of a standard: exogenous DNA target.
This work was supported in parts by Pfizer Animal Health, South Dakota State University Research Support Grant, the SDSU Functional
Genomics Core Facility, and the South Dakota State University Agricultural Experiment Station. 相似文献
7.
Sheau Wei Tan Aini Ideris Abdul Rahman Omar Khatijah Yusoff Mohd Hair-Bejo 《Journal of virological methods》2009,160(1-2):149-156
SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (Ct) 18.19 ± 3.63 and a melting temperature (Tm) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the Ct value 14.70 ± 2.32 and Tm 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log10 concentration range, 109–105 copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV. 相似文献
8.
Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C
T
difference” formula 总被引:2,自引:0,他引:2
Schefe JH Lehmann KE Buschmann IR Unger T Funke-Kaiser H 《Journal of molecular medicine (Berlin, Germany)》2006,84(11):901-910
For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative C
T
(ΔΔC
T
) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression’s C
T
Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data. 相似文献
9.
This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic
gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by
using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached
onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature
gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the
temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions
on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the
MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced
by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed
in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification
times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology
can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination
measurements, and fluorescent melting curve analysis. 相似文献
10.
In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex
PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput
and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets
associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through
microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow
multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of
cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with
a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is
about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model,
the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10−2 ng/μl (278-bp, S. enterica), 11.2 × 10−2 ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10−2 ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 104 copies/μl, 3.58 × 104 copies/μl, and 1.79 × 104 copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow
PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA
samples from different food sources in parallel, thereby achieving another level of multiplexing. 相似文献
11.
Parin Chaivisuthangkura Chutima Srisuk Sombat Rukpratanporn Siwaporn Longyant Pattarin Sridulyakul Paisarn Sithigorngul 《Journal of virological methods》2009,162(1-2):188-193
Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 °C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng−1 genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV). 相似文献
12.
B.?Leticia?Fernández-Carballo Ian?McGuiness Christine?McBeth Maxim?Kalashnikov Salvador?Borrós Andre?Sharon Alexis?F.?Sauer-Budge
In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm?×?30.9 mm?×?0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device. 相似文献
13.
14.
Yong Chen Wei‐Ching Lo Jesse I. Hamilton Kestutis Barkauskas Haris Saybasili Katherine L. Wright Joshua Batesole Mark A. Griswold Vikas Gulani Nicole Seiberlich 《NMR in biomedicine》2018,31(6)
The quantification of cardiac T1 relaxation time holds great potential for the detection of various cardiac diseases. However, as a result of both cardiac and respiratory motion, only one two‐dimensional T1 map can be acquired in one breath‐hold with most current techniques, which limits its application for whole heart evaluation in routine clinical practice. In this study, an electrocardiogram (ECG)‐triggered three‐dimensional Look–Locker method was developed for cardiac T1 measurement. Fast three‐dimensional data acquisition was achieved with a spoiled gradient‐echo sequence in combination with a stack‐of‐spirals trajectory and through‐time non‐Cartesian generalized autocalibrating partially parallel acquisition (GRAPPA) acceleration. The effects of different magnetic resonance parameters on T1 quantification with the proposed technique were first examined by simulating data acquisition and T1 map reconstruction using Bloch equation simulations. Accuracy was evaluated in studies with both phantoms and healthy subjects. These results showed that there was close agreement between the proposed technique and the reference method for a large range of T1 values in phantom experiments. In vivo studies further demonstrated that rapid cardiac T1 mapping for 12 three‐dimensional partitions (spatial resolution, 2 × 2 × 8 mm3) could be achieved in a single breath‐hold of ~12 s. The mean T1 values of myocardial tissue and blood obtained from normal volunteers at 3 T were 1311 ± 66 and 1890 ± 159 ms, respectively. In conclusion, a three‐dimensional T1 mapping technique was developed using a non‐Cartesian parallel imaging method, which enables fast and accurate T1 mapping of cardiac tissues in a single short breath‐hold. 相似文献
15.
Kausik Das M. Rohan Fernando Sara Basiaga Stephanie M. Wigginton Tom Williams 《Acta histochemica》2014
Stabilization of nucleated blood cells by cell stabilizing reagent (BCT reagent) present in the Cell-Free DNA BCT™ blood collection device and consequent prevention of cell-free DNA contamination by cellular DNA during sample storage and shipping have previously been reported. This study was conducted to investigate the effect of this novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional cell stabilizing reagents, formaldehyde and glutaraldehyde. A 787 bp long DNA fragment from human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified by PCR and used as model system. DNA samples and blood samples were treated with BCT reagent, 0.1% formaldehyde or 0.1% glutaraldehyde at room temperature. DNA amplification was studied using conventional and real-time quantitative PCR. Results indicate that exposure of DNA to the BCT reagent for up to 14 days had no effect on DNA amplification by PCR as compared to the untreated control DNA. However, there was statistically significant decrease in DNA amplification in the DNA samples treated with formaldehyde and glutaraldehyde. We conclude that the BCT reagent used in Cell-Free DNA BCT blood collection device to prevent cell-free DNA contamination by cellular DNA had no effect on DNA amplification by PCR. 相似文献
16.
We report on a novel, polymer-based, multi-channel device for polymerase chain reaction that combines, for the first time,
rapid sample processing in less than 5 min with high throughput at low costs. This is achieved by sample shuttling, during
which submicroliter sample plugs (∼100 nl) are oscillated rapidly over three constant-temperature zones by pneumatic actuation
with integrated system. The accuracy and the speed of the liquid handling have been significantly increased, while the design
of the device can be kept very simple and allows for mass production using conventional low-cost polymer fabrication processes.
Massive parallelization can lead to a throughput up to 100 samples in 10 min including the preparation time. The amplification
can be optically monitored by means of online fluorescence detection. Successful real-time PCR and the determination of the
threshold cycle, C
t, using the developed device were demonstrated with plasmid DNA in a fluorescent real-time format. 相似文献
17.
On-line pH detection of cell culture environment is necessary in a bioprocess or tissue engineering. Devices by means of electrochemical
mechanisms for this purpose have been reported to be less suitable compared with optical-based sensing principles. More recently,
some non-invasive optical sensing systems have been proposed for online pH monitoring of cell culture environment. However,
these devices are not for multi-target pH monitoring purpose, and are large in scale and thus not appropriate for the pH monitoring
at a micro scale such as in microbioreactor or microfluidic-based cell culture platform. To tackle these issues, an optical
fiber sensor array for on-line pH monitoring was proposed using microfluidic technology. The working principle is based on
the optical absorption of phenol red normally contained in culture medium. Different from other device of the similar working
principle, the proposed device requires less liquid volume (less than 0.8 μl), is non-invasive, and particularly can be configured
as an array for high throughput pH monitoring. The present device has been optimized for the shape of detection chamber in
a microfluidic chip with the aid of computational fluid dynamics (CFD) simulation, to avoid flow dead zone and thus to reduce
the response time of detection. Both simulation and experimental results revealed that the design of oval detection chamber
(axis, 1.5 and 2.0 mm) can considerably reduce the response time. Preliminary test has proved that the optical pH detection
device is able to detect pH with average detection sensitivity of 0.83 V/pH in the pH range of 6.8–7.8, which is normally
experienced in mammalian cell culture. 相似文献
18.
Miniature real time PCR on chip with multi-channel fiber optical fluorescence detection module 总被引:1,自引:0,他引:1
This paper presents the design and implementation of a miniature real time PCR system consisting of a disposable reactor chip,
a miniature thermal cycler, and a multi-channel fiber optical fluorescence excitation/detection module. The disposable PCR
chip is fabricated by using soft photolithography by PDMS (Polydimethylsiloxane) and glass. The miniature thermal cycler has
a thin film heater for heating and a fan for rapid cooling. The fiber optical detection module consists of laser, filter cube,
photo-detector and 1×4 fiber optical switch. It is capable of four-well real time PCR analysis. Real-time PCR detection of
E. coli stx1 has been demonstrated successfully with this system. 相似文献
19.
A. Müller R. Bialek G. Fätkenheuer B. Salzberger V. Diehl C. Franzen 《European journal of clinical microbiology & infectious diseases》2000,19(8):631-634
The aim of the present study was to use small-subunit (SSU)-rRNA sequences of Isospora belli to design specific primer pairs and a hybridization probe for the detection of Isospora belli in human samples by PCR and Southern blot hybridization. PCR amplification with the primer pairs produced correct DNA fragments
with target DNA from samples of Isospora belli-infected patients and from cloned SSU-rRNA of Isospora belli. The nature of the PCR products was confirmed by Southern blot hybridization. No amplification was seen with template DNA
extracted from other parasites. Although Isospora belli infections can be easily diagnosed using light microscopy, molecular-based techniques may prove useful as an additional diagnostic
tool. 相似文献
20.
Cedric Hurth Jianing Yang Matthew Barrett Carla Brooks Alan Nordquist Stanley Smith Frederic Zenhausern 《Biomedical microdevices》2014,16(6):905-914
We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated. 相似文献