Convective Polymerase Chain Reaction Research Articles

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

Application of Artificial Neural Network to Nucleic Acid Analysis: Accurate Discrimination for Untypical Real-Time Fluorescence Curves With High Specificity and Sensitivity

Abstract As a division of polymerase chain reaction (PCR), convective PCR (CPCR) is able to achieve highly efficient thermal cycling based on free thermal convection with pseudo-isothermal heating, which could be beneficial to point-of-care (POC) nucleic acid analysis. Similar to traditional PCR or isothermal amplification, due to a couple of issues, e.g., reagent, primer design, reactor, reaction dynamics, amplification status, temperature and heating condition, and other reasons, in some cases of CPCR tests, untypical real-time fluorescence curves with positive or negative tests will show up. Especially, when parts of the characteristics between untypical low-positive and negative tests are mixed together, it is difficult to discriminate between them using traditional cycle threshold (Ct) value method. To handle this issue which may occur in CPCR, traditional PCR or isothermal amplification, as an example, instead of using complicated mathematical modeling and signal processing strategy, an artificial intelligence (AI) classification method with artificial neural network (ANN) modeling is developed to improve the accuracy of nucleic acid detection. It has been proven that both the detection specificity and sensitivity can be significantly improved even with a simple ANN model. It can be estimated that the developed method based on AI modeling can be adopted to solve similar problem with PCR or isothermal amplification methods.

(Digital Presentation) An Electrochemical Sensor in a Disc for Real-Time PCR Assisted with Thermal Convective Flow

Multiple microfluidic devices incorporating real-time polymerase chain reaction (PCR) for the quantification of DNA targets have been reported. They show features such as fast analysis time, reduced reagent consumption, and portability. Integration of real-time PCR in a centrifugal microfluidic disc has been also proposed due to the ease of automation and parallelization of microfluidic operations. Previously, it has been demonstrated the implementation of thermal convective PCR (TC-PCR) in a CD using a ring-structured microchannel. In this case, the microchannel is supplied with the temperature necessary to perform the complete thermal cycle: Denaturation, annealing, and extension. The flow generated by thermal convection, in combination with the centrifugal force moves the sample through the entire microchannel due to the difference in densities. In this work, we present the implementation in a CD of an electrochemical sensor combined with TC-PCR to quantify the amplification yield in real-time. We present advances related to the calibration of the electrochemical sensor, and the implementation of the TC-PCR in a CD as well as a preliminary simulation in COMSOL.

A hand-held, real-time, AI-assisted capillary convection PCR system for point-of-care diagnosis of African swine fever virus

A hand-held, low-cost and real-time convection polymerase chain reaction (CPCR) system is developed for point-of-care diagnosis of African swine fever (ASF) virus. In CPCR amplification, the reagent is transferred to denaturation, annealing and extension stages of the reaction repeatedly due to thermal convection. Two different modes of fiber-based optical detection are compared, and it shows that the detection sensitivity, repeatability, and consistency can be significantly improved with bottom collection comparing to sidewall collection. Each capillary tube is illuminated by its own LED from the top through a common optical filter, and meanwhile, a camera equipped with another optical filter is adopted to detect the fluorescence signal from optical fibers. To handle the untypical real-time fluorescence curves of negative tests due to non-specific amplification, AI-based classification methods, for example, artificial neural network (ANN) is adopted to improve the detection specificity up to 97.50% comparing to 47.5% or 75.0% which is achieved with Ct-based method. A smartphone is adopted to run the AI algorithm and custom software, as well as to report an ASF event with the geographical information. Experimental results show that with the hand-held CPCR system, within 30 min, ASF viruses can be successfully detected with acceptable sensitivity and specificity.

Convective polymerase chain reaction in standard microtubes

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 μl reaction volume, 2) the supply of external temperature 145°С for the denaturation zone and 0°С for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15–20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.

Automated optimization of double heater convective polymerase chain reaction devices based on CFD simulation database and artificial neural network model.

This paper presents a framework for automated optimization of double-heater convective PCR (DH-cPCR) devices by developing a computational fluid dynamics (CFD) simulation database and artificial neural network (ANN) model. The optimization parameter space that includes the capillary tube geometries and the heater sizes of DH-cPCR is established, and a database consisting of nearly 10,000 CFD simulations is constructed. The database is then used to train a two-stage ANN models that select practically relevant data for modeling and predict PCR device performance. The trained ANN model is then combined with the gradient-based and the heuristics optimization approaches to search for optimal device configuration that possesses the shortest DNA doubling time. The entire design process including model meshing and configuration, parallel CFD computation, database organization, and ANN training and utilization is fully automated. Case studies confirm that the proposed framework can successfully find the optimal device configuration with an error of less than 0.3 s, and hence, representing a cost-effective and rapid solution of DH-cPCR device design.

Deskilled and Rapid Drug-Resistant Gene Detection by Centrifugal Force-Assisted Thermal Convection PCR Device.

Here we report the improved Cyclo olefin polymer (COP) microfluidic chip and polymerase chain reaction (PCR) amplification system for point-of-care testing (POCT) in rapid detection of Carbapenem-resistant Enterobacteriaceae (CRE). The PCR solution and thermal cycling is controlled by the relative gravitational acceleration (7G) only and is expected to pose minimal problem in operation by non-expert users. Detection is based on identifying the presence of carbapenemase encoding gene through the corresponding fluorescence signal after amplification. For preliminary tests, the device has been demonstrated to detect blaIMP-6 from patients stool samples. From the prepared samples, 96.4 fg/µL was detected with good certainty within 15 min (~106 thermocycles,) which is significantly faster than the conventional culture plate method. Moreover, the device is expected to detect other target genes in parallel as determination of the presence of blaNDM-1 and blaOXA-23 from control samples has also been demonstrated. With the rising threat of drug-resistant bacteria in global healthcare, this technology can greatly aid the health sector by enabling the appropriate use of antibiotics, accelerating the treatment of carriers, and suppressing the spread.

Simulation and Optimization of a Single Heater Convective PCR Chip and Its Controller for Fast Salmonella Enteritidis Detection

The design and optimization of a Rayleigh- Bénard convection based polymerase chain reaction (PCR) microchip is presented. This PCR microchip simplifies the fabrication, implementation and control of microfluidic PCR while enhancing the detection of the bacterial pathogen Salmonella Enteritidis. The device's dimensions were optimized using multiphysics simulation then it was fabricated using high precision milling of Poly(methyl methacrylate) (PMMA) substrate. The device incorporated a single heater at the bottom end of its cylindrical chamber and two sensors at both ends. The heater was controlled using a fuzzy proportional, integral, derivative (PID) controller to maintain denaturation and annealing temperatures necessary for efficient PCR to take place. Salmonella Enteritidis DNA was successfully detected using the device while reducing the required assay time by 66%.

A novel point-of-care test of respiratory syncytial viral RNA based on cellulose-based purification and convective PCR

BackgroundRespiratory syncytial virus (RSV) infection is a global public-health problem. Timely diagnostics are needed for high-risk patients. Several methods have been used for RSV detection but not suitable for on-site detection due to the requirement of specialized laboratories and expensive equipment. MethodsWe developed a convenient, rapid and low-cost method of nucleic acids (NA) extraction based on cellulose paper, which could extract NA from nasopharyngeal swabs (NPSs) within 1 min. This extraction method was integrated with fluorescence convection polymerase chain reaction (CPCR), which easily affordable and easy-to-use NA detection of the RSV in 33 min. ResultsThe developed cellulose-based NA purification combine with CPCR (CP-CPCR) reliably detected as little as 0.01 TCID50/mL of RSV cultures. CP-CPCR performance was tested further using NPSs: it showed sensitivity of 100% and a specificity of 100% compared with traditional extraction and amplification methods. ConclusionsOur evaluation confirmed high specificity, sensitivity and efficient of the CP-CPCR, which can be used widely for RSV testing in resource-limited settings.

Surrogate- and possibility-based design optimization for convective polymerase chain reaction devices

This paper presents a surrogate- and possibility-based design optimization (SPBDO) methodology for robust design of convective PCR. Parametric computational fluid dynamics (CFD) models are built and simulated at sampled locations within the design space to capture the effect of design configurations on thermofluidic transport and convection–diffusion-reaction in the convective PCR. A support vector machine-based classifier model is trained to retain only practically relevant data for enhanced surrogate modeling accuracy. Surrogate models are constructed by Kriging interpolation and multivariate polynomial regression methods to establish the mapping between design configurations and DNA doubling time (indicative of reactor performance). Then a process to combine the sequential method of PBDO and the surrogate model is developed, and a trade study is carried out to evaluate the impact of possibility of failure ( $$\alpha$$ -value) and the balance between performance and design reliability. Our study demonstrates that the proposed SPBDO represents an effective method to consider robustness in PCR design for POC applications, especially when the uncertainty information or possibilistic characteristics of design variables is limited.

Computational Design of a Single Heater Convective Polymerase Chain Reaction for Point-of-Care

Abstract Recently, researchers have started working to develop polymerase chain reaction (PCR) devices as a means for point-of-care (POC) applications. Among the requirements are portability, affordability, and performing reliably and quickly. Proposed by the present study is a process to design a convective-PCR (CPCR) device with only a single heater. It is assumed that such a design developed using microfluidics and capillary tube should help make a CPCR to be portable and more economical for POC use. One of the challenges is to achieve steadily the prerequisite three temperature zones with a single heater. It is demonstrated that this can be done with the present methodology. The underlying physics of the convection driving the CPCR function is mathematically modeled, then verified by our experimental results. In search of better designs, the following parameters that affect the CPCR performance are considered: the heater's height, and the diameter, the height, and the wall thickness of the capillary tube. A large design space consisting of design candidates is defined by combining the values within the range of each of these parameters. The results of the corresponding design cases are obtained from our mathematical model, and the performance of each case is evaluated by their deoxyribonucleic acid (DNA) doubling time. The two best CPCR performing reactors are selected and discussed. It is, therefore, demonstrated that the present methodology is capable of enhancing the CPCR reactor performance with a single heater.

Development of fast and sensitive protocols for the detection of viral pathogens using a small portable convection PCR platform.

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.

PSO+: nonlinear fitting fluorescence data based on particle swarm optimizing combine with other iteration algorithm

The convective polymerase chain reaction (CCPCR) uses the principle of thermal convection to allow the reagent to flow in the test tube and achieve the purpose of amplification by the temperature difference between the upper and lower portions of the test tube. In order to detect the amplification effect in real time, we added a fluorophore to the reagent system to reflect the amplification in real time through the intensity of fluorescence. The experimental results show that the fluorescence curve conforms to the S-type trend of the amplification curve, but there is a certain jitter condition due to the instability of the thermal convection, which is not conducive to the calculation of the cycle threshold (CT value). In order to solve this problem, this paper uses the dynamic method, using the double S-type function model to fit the curve, so that the fluorescence curve is smooth and the initial concentration of the nucleic acid can be deduced better to achieve the quantitative purpose based on the curve. At the same time, the PSO+ algorithm is used to solve the double s-type function parameters, that is, particle swarm optimization (PSO) algorithm combined with Levenberg-Marquardt, Newton-CG and other algorithms for curve fitting. The proposed method effectively overcoms PSO randomness and the shortcoming of traditional algorithms such as Levenberg-Marquardt and Newton-CG which are easy to fall into the local optimal solution. The R 2 of the data fitting result can reach 0.999 8. This study is of guiding significance for the future quantitative detection of real-time fluorescent heat convection amplification.

Optimization of ultra-fast convection polymerase chain reaction conditions for pathogen detection with nucleic acid lateral flow immunoassay

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 μM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 μM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

Real-time capillary convective PCR based on horizontal thermal convection

A real-time, capillary, convective polymerase chain reaction (PCR) system based on horizontal convection is developed and analyzed. The capillary tube reactor is heated at one end in a pseudo-isothermal manner to achieve efficient thermal cycling based on horizontal thermal convection. Mathematical modeling and the in silico simulations indicate that, once consistent temperature gradient along the horizontal capillary tube has been established, a repeatable and continuous circulatory flow in the horizontal directions is created. The formed convection is able to transport PCR reagents through different temperature zones inside the horizontal capillary tube for different reaction stages, that is, DNA denaturing, annealing, and extension in a typical PCR cycle. Furthermore, the effectiveness and efficiency of the horizontal convection for PCR thermal cycling in a capillary tube is confirmed by experimentation. To evaluate the concept of horizontal convective PCR, a compact system, which is able to heat the capillary tube from one end and monitor the fluorescence in situ with a smartphone camera, is developed for real-time amplification. With horizontal thermal convection, influenza A (H1N1) virus nucleic acid targets with a limit of detection (LOD) of 1.0 TCID50/mL can be successfully amplified and detected in 30 min, which is promising for efficient nucleic acid analysis in point-of-care testing.

Performance of convective polymerase chain reaction by doubling time

We report in this article a novel computational methodology to estimate the doubling time, which is proposed as an effective performance evaluation tool for a convective polymerase chain reactor (cPCR). The method is a high-fidelity, in silico simulation for the development of a low-cost, robust, and portable cPCR intended for point-of-care applications. Our in-house computer code, based on the well-established OpenFOAM solver, outputs the number of generated double-stranded DNA copies and the doubling times by solving for the concentration species. For this purpose, a three-dimensional, conjugate heat and momentum transport model is developed. In the first step, a simulation of a steady, circulating flow, due to convection, is computed by solving the coupled Navier-Stokes and heat transfer equations. Flow and temperature fields are then used to obtain the concentrations of the double-stranded, the primal-annealed, and the single-stranded DNAs while solving the convection-diffusion-reaction equations. The methodology is demonstrated through multiple design cases and validated by experimental results. The results strongly suggest that the performance of a cPCR reactor can be measured by computing the doubling time, and such an approach reduces the otherwise needed extensive experimental efforts to a minimum.

Effect of tris(2-carboxyethyl)phosphine and tertiary butyl alcohol on the performance of convection polymerase chain reaction.

Rapid and on-site DNA-based molecular detection has become increasingly important for sensitive, specific, and timely detection and treatment of various diseases. To prepare and store biomolecule-containing reagents stably, reducing agents are used during protein preparation, and freeze-drying technology has been applied to the protein reagents. Some of the additives used during these processes may affect subsequent processes such as polymerase chain reaction (PCR). In this study, we evaluated the impact of TCEP, a reducing agent, and TBA, a freeze-drying medium, on the performance of convection PCR (cPCR) using a battery-operable PCR device. Singleplex cPCR detection of a 249bp amplicon from human genomic DNA suggested that approximately 82% of performance was achieved in the presence of 0.1mM TCEP and 1% TBA. The limit of detection and the minimum number of cycles at which amplicons began to appear was a little lower (~ 82% efficiency) or higher (20 vs 15 cycles), respectively, in the chemical-treated group than in the control group. With larger amplicons of 500bp, the chemical-treated group revealed approximately 78% of performance and amplicons started to appear at 20 cycles of cPCR in both groups. Similar results were obtained with multiplex cPCR amplification.

Ultra-Fast On-Site Molecular Detection of Foodborne Pathogens Using a Combination of Convection Polymerase Chain Reaction and Nucleic Acid Lateral Flow Immunoassay.

Rapid detection and timely treatment of diseases caused by foodborne pathogens is important for improving the curative efficiency and preventing the spread of disease. In this study, we developed an assay utilizing a recently introduced ultra-fast convection polymerase chain reaction (cPCR) in conjunction with a simple nucleic acid lateral flow (NALF) immunoassay for ultra-fast on-site molecular detection of foodborne pathogens. Two Salmonella enterica serovars, Salmonella Enteritidis and Salmonella Typhimurium, and Escherichia coli O157:H7 were used as the target pathogens. We confirmed the specific amplification of the target species with specifically designed modified primer sets for cPCR in singleplex and duplex modes. After cPCR amplification, we compared the detection specificity and sensitivity using agarose gel electrophoresis and NALF assays with one or two test lines. The cPCR amplicons were readily and sensitively detected using the NALF assay, and the sensitivity was comparable with that of agarose gel electrophoresis. To confirm the application of the assay in real-life samples, the assay was used to test artificially contaminated milk. Without sample pre-enrichment, the limit of detection (LOD) was 4.5 × 103 colony-forming units (CFU)/mL for Salmonella species; 4.5 × 104 CFU/mL to differentiate Salmonella Enteritidis and Salmonella Typhimurium; and 2.3 × 103 CFU/mL for E. coli O157:H7, in a duplex assay. With a 6 h pre-enrichment, the LOD was 4.5 CFU/mL for Salmonella Enteritidis and Salmonella Typhimurium, and 2.3 CFU/mL for E. coli O157:H7. The cPCR amplification took only 14 min, and the NALF assay took ca. 5 min. The total analysis time was less than 20 min. Based on these observations, we propose that the developed assay is simple, ultra-fast, and applicable for on-site detection of foodborne pathogens.

A Rapid On-Site Assay for the Detection of Influenza A by Capillary Convective PCR

Morbidity and mortality from influenza A (Flu A) have increased in recent years. Timely diagnosis and management are critical for disease control. Therefore, the development of a rapid, accurate, and portable analytical method for on-site analysis is imperative. The aim of this work was to develop a rapid, on-site, automated assay for the detection of Flu A and to evaluate the assay. A handheld instrument (TD-01) based on capillary convective polymerase chain reaction (PCR) was developed for rapid on-site detection of Flu A. Since a previous version of the instrument, an automated motion mechanism has been introduced to TD-01 to achieve RNA automated testing. The primers and probe used for Flu A detection were designed according to the Flu A gene sequence of matrix proteins. Finally, we evaluated the detection spectra, sensitivity, specificity, and diagnostic performance of the assay. The TD-01 was able to successfully automatically detect Flu A RNA within 30min. Results for serially diluted viruses indicated that the lower limit of detection for Flu A was 0.1 TCID50/ml (50% tissue culture infective dose). After evaluating known virus stocks, including 15 strains of Flu A, four strains of Flu B, and two strains of respiratory syncytial virus (RSV), the assay had a favorable detection spectrum and no obvious cross-reactivity. Method verification based on 554 clinical samples indicated that the sensitivity and specificity of TD-01 were 98.30% (231/235) and 98.75% (315/319), respectively. The results indicate that Flu A detection by TD-01 is particularly suitable for on-site testing and has the potential for application in point-of-care testing.

Development of a Novel, Rapid Multiplex Polymerase Chain Reaction Assay for the Detection and Differentiation of Salmonella enterica Serovars Enteritidis and Typhimurium Using Ultra-Fast Convection Polymerase Chain Reaction.

Salmonella enterica serovars Enteritidis and Typhimurium are the most common causative agents of human nontyphoidal salmonellosis. The rapid detection and timely treatment of salmonellosis are important to increase the curative ratio and prevent spreading of the disease. In this study, we developed a rapid multiplex convection polymerase chain reaction (PCR) method to detect Salmonella spp. and differentiate Salmonella Enteritidis and Salmonella Typhimurium. We used the invA gene for Salmonella spp. detection. Salmonella Enteritidis-specific primers and Salmonella Typhimurium-specific primers were designed using the insertion element (IE) and spy genes, respectively. The primer set for Salmonella spp. detection clearly detected both Salmonella Enteritidis and Salmonella Typhimurium after a 21-min amplification reaction. Serovar-specific primer sets for Salmonella Enteritidis and Salmonella Typhimurium specifically detected each target species in a 21-min amplification reaction. We were able to detect Salmonella spp. at a single copy level in the singleplex mode. The limits of detection for Salmonella Enteritidis and Salmonella Typhimurium were 30 copies in both the singleplex and multiplex modes. The PCR run time could be reduced to 10.5 min/15 cycles. The multiplex convection PCR method developed in this study could detect the Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium in artificially contaminated milk with as few as 100 colony-forming unit/mL after 4-h enrichment. The PCR assay developed in this study provides a rapid, specific, and sensitive method for the detection of Salmonella spp. and the differentiation of Salmonella Enteritidis and Salmonella Typhimurium.