Gotowa bibliografia na temat „Droplet Recombinase Polymerase Amplification”

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was &lt;20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.

Abstract BACKGROUND First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years. The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5–20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms. CONTENT This review summarizes the current knowledge on RPA. The molecular diagnostics of various RNA/DNA pathogens is discussed while highlighting recent applications in clinical settings with focus on point-of-care (POC) bioassays and on automated fluidic platforms. The strengths and limitations of this isothermal method are also addressed. SUMMARY RPA is becoming a molecular tool of choice for the rapid, specific, and cost-effective identification of pathogens. Owing to minimal sample-preparation requirements, low operation temperature (25–42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices. RPA is undoubtedly a promising isothermal molecular technique for clinical microbiology laboratories and emergence response in clinical settings.

Cette thèse de doctorat porte dans l’ensemble une étude approfondie sur une technologie émergente pour l’amplification isotherme des acides nucléiques appelée recombinase polymerase amplification (RPA). L’introduction porte une description détaillée sur la RPA. Cette revue de littérature documente et discute les diverses applications de la RPA en soulignant les connaissances actuelles concernant les applications diagnostiques. Malgré la composition complexe de la RPA (6 à 7 protéines dans le même mélange réactionnel), cette dernière s’avère une technologie rapide (générant des résultats < 20 min), spécifique et sensible (détection de l’ordre de quelques copies de génome), et largement appliquée dans différentes disciplines. Ces avantages nous permettent de croire que la RPA possède la flexibilité nécessaire pour être utilisée comme outil de diagnostic rapide des maladies infectieuses en réduisant le temps d’obtention des résultats à moins d’une heure au lieu de 2 à 3 jours avec les tests de cultures standards. En conséquence, il sera possible d’intégrer la RPA dans des plateformes microfluidiques ou laboratoire sur puce qui permettent la préparation d’échantillons, l’amplification et la détection des acides nucléiques des microbes causant des infections. En premier lieu, les travaux de cette thèse ont généré des lignes directrices additionnelles pour la conception des amorces/sondes RPA. En second lieu, nos travaux ont permis de développer un essai diagnostic RPA pour la détection des streptocoques du groupe B, responsables de la septicémie et la méningite chez les nouveau-nés. Cet essai fut le premier à évaluer la performance de la RPA avec des échantillons cliniques humains. Ce test diagnostic RPA a été comparé à une méthode de référence, la réaction en chaîne par polymérase (PCR). Cette démonstration sur des échantillons cliniques nous à inciter à pousser notre étude pour réaliser le dernier objectif de ce projet qui consistait à automatiser la RPA par intégration dans un système microfluidique miniaturisé centripète. Une collaboration avec des experts en génies et en matériaux a permis de générer un dispositif microfluidique appelé blade ainsi de l’instrument impliqué dans l’opération des différentes tâches mécanistiques. Ces résultats préliminaires suggèrent qu’il sera important d’offrir un système automatisé complet applicable au chevet du patient. Par conséquence, il sera possible d’exécuter une analyse complète des agents infectieux en moins d’une heure sans le besoin des procédures complexes de préparation et de transport des échantillons cliniques ni le recours à du personnel qualifié.
This dissertation consists of an exhaustive study on an emerging technology for isothermal amplification of nucleic acids called recombinase polymerase amplification (RPA). The introduction of this thesis is a detailed description of the RPA. This review documents and discusses the various applications of this technology by pointing to the current knowledge about RPA for diagnostic applications. Despite the complex composition of RPA (6 to 7 proteins in the same reaction mixture), the latter was shown to be rapid (generating results in < 20 min), specific and sensitive (detecting few target genome copies), and applied widely in different fields. Based on these advantages, we assume that RPA has a flexibility allowing it to be used for the rapid diagnosis of infectious diseases thus reducing time-to-result to less than an hour. Consequently, it will be possible to integrate RPA in microfluidic platforms providing a lab-on chip system. The first part of this doctoral project generated additional guidelines for RPA primers/probes design to develop specific RPA diagnostic assays. Second, we developed an RPA diagnostic test for the detection of group B streptococci, responsible for sepsis and meningitis in newborns. This assay was the first to evaluate RPA with human clinical samples. This diagnostic test was compared to a reference method, the polymerase chain reaction (PCR). This demonstration with clinical samples served to carry out the final objective of this project that was to automate RPA in a miniaturized microfluidic centripetal system. Collaboration with engineers and experts in materials has generated the microfluidic device called "blade" and the instrument involved in the operation of various mechanistic tasks. These preliminary results suggested that it will be important to provide an automated system applicable at bedside. Consequently, it will be possible to perform a complete analysis of infectious agents in less than an hour without the need for complex procedures for the preparation and transport of clinical specimens or the assistance of qualified personnel.

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine.
Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.

Nucleic acid quantification (NAQ) is extensively employed for gene expression analysis, monitoring viral loads, detecting rare or dysfunctional cells, and assessing treatment regimes. The gold standard, quantitative polymerase chain reaction (qPCR), and the recent alternative, droplet digital PCR (ddPCR), provide accurate quantification of nucleic acids (NA). Albeit the requirement of thermal cycling and separate platforms for droplet generation, NA amplification, and signal detection, in the case of ddPCR increases the assay complexity and time, limiting its broad applicability. In this work, we have developed an integrated droplet isothermal amplification-based NAQ (idNAQ) platform that enables facile and fast NAQ with a large dynamic range. First, we adapted the isothermal amplification method, Recombinase Polymerase Amplification (RPA), for NAQ. We demonstrate a fast (• 40 minutes) semi-quantitative RPA (qRPA) assay with the endpoint intensity ratio (EIR) for DNA quantification with a 6-log order range. Since the EIR model estimates the amplicon levels at the end of the reaction, real-time monitoring of the amplification reaction (unlike in the case of qPCR) is no longer required. With qRPA, we demonstrate viral load detection from the serum of dengue-infected patients with comparable performance to qPCR. The later section discusses the translation of the qRPA NAQ to a microfluidic droplet format. Droplet RPA (dRPA) displays similar kinetics to the bulk reaction suggesting successful optimization of droplet conditions for RPA. dRPA in the low concentration regime follows Poisson distribution that enables digital quantification as in the case of ddPCR. On the other hand, at a higher starting concentration of DNA (non-digital regime, >10 DNA per droplet), the RPA amplification in droplets exhibits heterogeneous intensity puncta due to rapid amplification and incomplete mixing leading to the formation of DNA ‘amplification globules’. We use a supervised machine, learning-based regression model with these intensity features as inputs to accurately predict the target concentration of up to 10^5 molecules per droplet. Combining these two modalities of dRPA yields a dynamic range of >7 log orders of concentration that are comparable to qPCR. Finally, we demonstrate the successful integration of all unit operations onto a single microfluidic device for droplet RPA and quantification. Different microfluidic designs were optimized for monodisperse droplet generation and image acquisition from a large incubation area that allowed the successful implementation of quantitative dRPA in a single device.

碩士
國立交通大學
生物資訊及系統生物研究所
106
At present, most of the deoxyribonucleic acid (DNA) amplification techniques such as polymerase chain reaction (PCR). PCR relies on the thermal cycle machine, through denaturation, annealing, extension, the process requires precise control the temperature. Recombinase polymerase amplification (RPA) technology is developed by TwistDx in 2006. First, it makes the primer sequence and protein form a complex, the complex will find the location of the homologous sequence on the DNA template and open double-stranded DNA helix structure. Next, amplification was performed by recombinase polymerase. The temperature of the whole process is maintained at about 37 to 42°C, which will allow the DNA amplification technology get a new breakthrough. DNA amplification technology at constant temperature will no longer need to rely on the thermal cycle machine, enhances this DNA amplification technology’s portability and convenience. However, the most important part of the technology is how to design primers to make the RPA correctly amplify the target gene or sequence. In addition, design primers for multiplex PCR or RPA, it needs to avoid the two primers because the sequence with excessive similarity leads to form primer dimers so that reduce the amplification efficiency. So far, it is still not found that someone provides a primer design for multiplex RPA platform. In this study, we collect RPA primers from literature, and statistics out RPA primer features and integrate the recommendations of primer design from literature. Next, according to as above, use a series of bioinformatics methods like we use Primer3 to generate candidate primer groups, and then we use Bowtie to confirm the specificity of each primer pairs. Finally, the genetic algorithm was used to find out optimized primer group that the temperature between the two primers will not be too high to form primer dimers. In this study, we respectively designed primer sets for multiplex PCR and multiplex RPA to provide future experimental verification, such as gel electrophoresis, next-generation sequencing or Nanopore MinION sequencing platform. In summary, this study develops a web platform and a standalone tool allows users to design multiplex PCR or RPA primer sets that meet their own experimental needs.

The article demonstrates the prospects for using a new experimental approach for the detection of the fungus Cercospora Sojina Hara. The main principle underlying the presented method is a combination of two technologies: RPA (Recombinase polymerase amplification) and CRISPR/Cas12a. RPA - is an alternative to classical PCR, with features in the form of a faster reaction rate and its passage under isothermal conditions. Using RPA technology will reduce amplification to 15-30 minutes. Amplified genomic DNA can be detected fluorescently labeled with CRISPR/Cas12a. The difficulties of this method lie in the selection of specific primers and the selection of spacers for the guide RNA within the amplicon. As a result of the work carried out, using the primer3 plugin on the Unipro Ugene platform, it was possible to select a specific pair of primers that would make it possible to identify this particular phytopathogen. The genome spacer was identified in the ChopChop web toolkit. The resulting primer pair and spacer having complementarity exclusively to the CP036215 locus in Cercospora Sojina H.

The polymerase chain reaction (PCR) has revolutionised molecular biology, and is at the forefront of many current efforts to document and understand human genetic diversity. Recent years has seen a move towards incorporating the PCR technique into a micro Total Analysis System (μTAS) thus exploiting its full potential. Micro scale PCR design offers the opportunity to integrate all functional steps of DNA expression analysis into a miniaturised device allowing for high throughput and reduced analysis times, reduced sample volume requirements and cost efficiency. Consequently, it is desirable to replace the traditional stationary or well based thermal cyclers with continuous flow designs. A continuous flow polymerase chain reaction device consisting of a cylindrical heating core, which is segmented axially into three symmetric heating zones providing the denaturating, annealing and extension phases of the polymerase chain reaction, and a flow through capillary tube which is wound helically around the core has been fabricated and shown to consistently amplify target plasmid DNA samples. At the inlet to the device, PCR samples are segmented into droplets and entrained in an immiscible carrier fluid to eliminate cross contamination between PCR samples. This approach also prevents degradation of the micro-reactor droplets from inhibitory effects posed by the high surface to volume ratios associated with the device. The droplet train is then cycled through the capillary tube with each complete revolution constituting one cycle of the PCR reaction. The results reported in this paper include, initial validation of the spiral cycler design in comparison to an existing commercial PCR platform, and subsequent optimisation of the reaction time and its effects on the devices performance. The spiral thermal cycler has demonstrated successful PCR amplification at the nano scale with stable trains of 30–35nl droplet volumes being processed in an amplification time of 32 minutes. At this level the device offers the potential to process approximately 3500 such droplets in of order one hour.

Polymerase Chain Reaction (PCR) is an enzymatic process that has dramatically advanced many fields of life sciences, where it is an indispensable tool in a burgeoning range of applications, including diagnostic medicine, molecular biology, forensics and food testing. Recent increased demand for extremely high throughput PCR systems has led to the development of miniaturised continuous flow microfluidic PCR devices, which may have extremely high throughput compared to standard commercial PCR thermal cyclers. A novel continuous flow microfluidic PCR device has been designed and fabricated, consisting of two thermal zones maintained on aluminium thermal blocks providing the precise temperatures required for denaturation and annealing/extension. Polycarbonate sideplates retain the denaturation thermal block vertically above the annealing/extension thermal block while allowing for a variable air gap to be maintained between them. Heating of the denaturation thermal block is achieved using a Labview controlled Thermofoil heater, while the annealing/extension thermal block is maintained at temperature by optimised heat transfer from the denaturation block. Flow-through capillary tubing is positioned into a grooved serpentine channel machined into these thermal blocks. This serpentine channel passes through each thermal block fifty times, providing fifty PCR thermal cycles. Contamination free high throughput continuous flow PCR necessitates that the samples be encapsulated in an immiscible carrier fluid to eradicate cross contamination between samples and suppress the likelihood of the sample contacting the capillary leading to sample degradation. Encapsulation of the PCR reaction mixture is achieved upstream of the thermal cycler through segmentation of the sample into droplets entrained within an immiscible carrier fluid, which are then cycled through the thermal cycler. High throughput DNA amplification of two genes, GAPDH and LEF1, from the REH cell line has been successfully demonstrated on this microfluidic platform without any detectable contamination between samples. The PCR droplet reactors were approximately 250nl which is two orders of magnitude less than the standard sample size for most commercial PCR thermal cyclers.

In this paper are presented the results of a thermal analysis on a high throughput droplet based, nucleic acid amplifier. Initial data of successful amplification of DNA is also presented. The advent of microfluidics offers many opportunities to integrate all functional steps of DNA analysis onto a single platform allowing for reduced analysis times, reduced sample volume requirements and higher throughput. There are many technical challenges facing this goal, thermal control being pivotal. This paper involves fundamental theoretical and experimental characterization of the core element of such a platform; a polymerise chain reaction (PCR) thermal cycler. The PCR process can be either a two-step or three-step temperature cycling procedure, depending on the chemistry involved. Presented in this paper is a thermal analysis of four, two-step thermocyclers arrayed in parallel. Two step PCR requires samples to be cycled through temperature ranges of 92–95°C and 60°C, the preciseness of these temperatures again depending on the chemistry involved. For optimum efficiency, fast heating and cooling between steps, and uniformity within each step is crucial. Our thermocycler design comprises a flow conduit in a serpentine pattern, machined in two segmented aluminium blocks, in which the conduit extends through the denaturation (92–95°C) and the annealing and extension (60°C) zones. Circular tubing, in which the samples are passed through to ensure biocompatibility for the reaction, is embedded in the machined channel which results in high heat transfer from the block to the sample. The device is then positioned in the vertical plane and an array of thermocyclers are formed by combining multiple planar systems. Thermofoil heaters are attached to the underside of the upper blocks creating the denaturation temperature, and by adjusting the air gap, passive thermal control is used to create the temperature required for the annealing and extension zone, thereby reducing the number of heaters and thus the power required. Theoretical thermal analysis of the device is performed, in conjunction with CFD simulations, experimental testing, and thermal imaging. Finally successful PCR of the gene ABL1 was performed.