PCR Device Research Articles

Development of a disposable and easy-to-fabricate microfluidic PCR device for DNA amplification

The production of microfluidic PCR devices within lab settings in a straightforward process and large numbers is limited due to complex methods of fabrication and high cost of development. In this study, we developed a unique disposable, low-cost, and easy-to-fabricate microfluidic PCR chip, comprised of a passive micromixer, reaction well, microheater, and a dynamic temperature control system. In addition to being compact, this device has the advantages of microfluidic PCR systems, such as low power and sample consumption, as well as being disposable and easy to fabricate. The disposable part of the chip is easy to fabricate and eliminates the run-to-run carryover contamination while its integration with a reusable heating system reduces the cost of producing a micro PCR device. The geometry of the micromixer and the pattern of microheater electrodes are analyzed through experiments to achieve comparable amplification to a conventional thermocycler. The temperature control system is designed to monitor the temperature within the reaction well with ±1 °C precision. Finally, the reliability and repeatability of the microfluidic device were verified by amplifying lambda (λ) DNA. The proposed device demonstrated high-quality amplification comparable to the conventional PCR and is an adaptable and low-cost on-chip PCR device for reliable molecular diagnostics.

Mini-thermal platform integrated with microfluidic device with on-site detection for real-time DNA amplification.

The recent cases of COVID-19 have brought the prospect of and requirement for point-of-care diagnostic devices into the limelight. Despite all the advances in point-of-care devices, there is still a huge requirement for a rapid, accurate, easy-to-use, low-cost, field-deployable and miniaturized PCR assay device to amplify and detect genetic material. This work aims to develop an Internet-of-Things automated, integrated, miniaturized and cost-effective microfluidic continuous flow-based PCR device capable of on-site detection. As a proof of application, the 594-bp GAPDH gene was successfully amplified and detected on a single system. The presented mini thermal platform with an integrated microfluidic device has the potential to be used for the detection of several infectious diseases.

Evaluation of a real-time mobile PCR device (PCR 1100) for the detection of the rabies gene in field samples

BackgroundThe Philippines is ranked among the top countries with 200–300 annual deaths due to rabies. Most human rabies cases have been reported in remote areas, where dog surveillance is inadequate. Therefore, a strategy to effectively improve surveillance in remote areas will increase the number of detections. Detecting pathogens using portable real-time reverse transcription-polymerase chain reaction (RT-PCR) has the potential to be accepted in these areas. Thus, we aimed to develop an assay to detect the rabies virus (RABV) genome by combining the robust primer system LN34 with the PicoGene PCR1100 portable rapid instrument targeting RABV RNA (PCR1100 assay).MethodsProcedures were optimised using an LN34 primer/probe set, KAPA3G Plant PCR Kit (KAPA Biosystems), FastGene Scriptase II (NIPPON Genetics), and an artificial positive control RNA.ResultsPositive control RNA showed an analytical limit of detection of 10 copies/µL without false positivity, generating results in approximately 32 min. Compared to dFAT or RT-qPCR using field samples, the sensitivity and specificity of the PCR1100 assay were 100%, and even lower copy numbers (approximately 10 copies/µL) were detected.ConclusionsThis study demonstrated that the developed assay can detect rabies RNA in field samples. Because dog-mediated rabies is endemic in remote areas, the rapidity, mobility, and practicality of the PCR1100 assay as well as the high sensitivity of the LN34 system make it an ideal tool for the confirmation of rabies in these areas.

Comparing artificial intelligence based diagnosis with expert results in SARS-COV-2 RT-qPCR

Objectives: Reverse transcription and real-time polymerase chain reaction (RT-qPCR) based on the SARS-CoV-2 viral RNA demonstration is the gold standard in diagnosis. Data files obtained from PCR devices should be analysed by a specialist physician and results should be transferred to Laboratory Information Management System (LIMS). CAtenA Smart PCR (Ventura, Ankara, Türkiye) program is a local bioinformatics software that assess PCR data files with artificial intelligence, submits to expert approval and transfers the approved results to LIMS. The aim of this study is to investigate its accuracy and matching success rate with expert analysis. Methods: A total of 9400 RT-qPCR test results studied in Ankara Provincial Health Directorate Public Health Molecular Diagnosis Laboratory were compared with respect to expert evaluation and CAtenA results. Results: It was determined that the preliminary evaluation results of the CAtenA matched 86% of the negative and 90% of the positive results provided by expert analysis. 987 tests which CAtenA determined as inconclusive and suggested repeating PCR were found either negative or positive by expert analysis. A significant difference between positive and negative matching success rates and artificial intelligence (AI) based software overall accuracy was found and associated with the missed tests of the AI. Conclusions: As a result, it was suggested there is a low risk of confirming false positive results without expert analysis and test repetitions would cause losing time along with extra test costs. It was agreed that the PCR analysis used in CAtenA should be improved particularly in terms of test repetitions.

Advanced Molecular-Genetic Methods and Prospects for Their Application for the Indication and Identification of <i>Yersinia pestis</i> Strains

The review provides an analysis of the literature data on the use of various modern molecular-genetic methods for the indication and identification of Yersinia pestis strains with different properties and degree of virulence, which is caused by the diverse natural conditions in which they circulate. The methods are also considered from the perspective of their promising application at three levels (territorial, regional and federal) of the system for laboratory diagnosis of infectious diseases at the premises of Rospotrebnadzor organizations to solve the problem of maintaining the sanitary and epidemiological well-being of the country’s population. The main groups of methods considered are as follows: based on the analysis of the lengths of restriction fragments (ribo- and IS-typing, pulse gel electrophoresis); based on the analysis of specific fragments (DFR typing, VNTR typing); based on sequencing (MLST, CRISPR analysis, SNP analysis); PCR methods (including IPCR, SPA); isothermal amplification methods (LAMP, HDA, RPA, SEA, PCA, SHERLOCK); DNA-microarray; methods using aptamer technology; bio- and nano-sensors; DNA origami; methods based on neural networks. We can conclude that the rapid development of molecular diagnostics and genetics is aimed at increasing efficiency, multi-factorial approaches and simplifying the application of techniques with no need for expensive equipment and highly qualified personnel for analysis. At all levels of the system for laboratory diagnosis of infectious diseases at the Rospotrebnadzor organizations, it is possible to use methods based on PCR, isothermal amplification, SHERLOCK, biosensors, and small-sized sequencing devices. At the territorial level, at plague control stations, the use of immuno-PCR and SPA for the indication of Y. pestis is viable. At the regional level, introduction of the technologies based on the use of aptamers and DNA chips looks promising. For the federal level, the use of DNA origami methods and new technologies of whole genome sequencing is a prospect within the framework of advanced identification, molecular typing and sequencing of the genomes of plague agent strains.

Rapid Diagnosis of Infectious Diseases through Miniature PCR Devices

Rapid Diagnosis of Infectious Diseases through Miniature PCR Devices

An amplification velocity-controlled PCR device for accurately detecting the initial content of target DNA templates

Taq DNA polymerase is the most widely used enzyme for PCR amplification and it contains 832 amino acids but no cysteines. Bovine serum albumin (BSA) is an 832-amino acid protein containing 35 cysteines, and at high temperature BSA can produce protein hydrogels through recombined disulfides and noncovalent bonds. When hydrogels are prepared using a mixture of Taq DNA polymerase and BSA, no disulfide bonds occur between them, and noncovalent bonds are the main forces holding together the Taq DNA polymerase with the hydrogels. During PCR amplification, temperature cycling can destroy the noncovalent bonds, thereby releasing Taq DNA polymerase from the hydrogels to synthesize DNA. The release rate, regulated through the variation of BSA concentration, allows for velocity-controlled amplification of DNA. As the amount of DNA products exceeds that of the Taq DNA polymerase released from hydrogels, a linear amplification replaces the exponential PCR amplification. When the amplification velocity begins to decrease, the time needed for reaching a plateau increase. This provides a broader detection window for the analysis of the end products, which correspond to the difference compared with the initial DNA concentration. The number of primers is continuously reduced with increasing cycles, which further causes a decline in the DNA amplification efficiency. The difference in the initial DNA template may cause a different consumption of primers, which can cause errors in the Ct values in the case of qPCR. Based on the slopes of the linear amplification curve, the Ct values can be calibrated to reduce this error, and a more accurate detection of the initial content of the target DNA can be implemented.

Artificial intelligence model with deep learning in nonalcoholic fatty liver disease diagnosis: genetic based artificial neural networks

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in the world. The NAFLD spectrum includes simple steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Genetic, nutritional factors, obesity, insulin resistance, gut microbiota are among the risk factors for NAFLD. The genetic variant Patatin-like phospholipase domain-containing protein 3 (PNPLA3) plays an important role in the development of a number of liver diseases ranging from steatosis, chronic hepatitis, cirrhosis and HCC. Due to the increase in the prevalence of NAFLD, new models are being developed with machine learning, deep learning, artificial neural network (ANN) algorithms in the field of artificial intelligence (AI) to determine low-cost, noninvasive diagnostic methods. Models developed with ANN from AI modules are important in order to examine biochemical and genomic information in detail in the diagnosis of NAFLD. The aim of this study is to develop a simple ANN model using biochemical and genotypic parameters in the diagnosis of NAFLD. A total of 300 patients followed up with the diagnosis of NAFLD and 100 controls were included in the study. The data set was divided into two as training and test set. Genotyping of PNPLA3 (CC, CG, GG) as genomic analysis was performed with real time PCR device. The algorithm used for the diagnosis of NAFLD was designed using age, body mass index (BMI), mean platelet volume (MPV), insulin resistance (IR), alanine aminotransferase (ALT), genotype PNPLA3 (CC, CG, GG) parameters. MLP Classifier algorithm from ANN was used in the development of the model. ANN algorithms are used in python programming language. Statistical analyzes were made in SPSS program. Percent accuracy, area under the ROC curve, confusion matrix, Positive (PPV) and Negative Predicted Value (NPV) values, precision, recall, and f1-score results were determined. The accuracy percentage was determined as 0.979 in the train set and 0.970 in the test set. The Log Loss value was set to 0.09. The developed neural network achieved an accuracy percentage of 97.0% during testing, with an area under the ROC curve value of 0.95. We think that the ANN model developed with genomic and biochemical parameters can be used as a cost-effective, noninvasive new predictive diagnostic model in clinical practice in the diagnosis of NAFLD.

Compact Camera Fluorescence Detector for Parallel-Light Lens-Based Real-Time PCR System.

The polymerase chain reaction is an important technique in biological research. However, it is time consuming and has a number of disadvantages. Therefore, real-time PCR technology that can be used in real-time monitoring has emerged, and many studies are being conducted regarding its use. Real-time PCR requires many optical components and imaging devices such as expensive, high-performance cameras. Therefore, its cost and assembly process are limitations to its use. Currently, due to the development of smart camera devices, small, inexpensive cameras and various lenses are being developed. In this paper, we present a Compact Camera Fluorescence Detector for use in parallel-light lens-based real-time PCR devices. The proposed system has a simple optical structure, the system cost can be reduced, and the size can be miniaturized. This system only incorporates Fresnel lenses without additional optics in order for the same field of view to be achieved for 25 tubes. In the center of the Fresnel lens, one LED and a complementary metal-oxide semiconductor camera were placed in directions that were as similar as possible. In addition, to achieve the accurate analysis of the results, image processing was used to correct them. As a result of an experiment using a reference fluorescent substance and double-distilled water, it was confirmed that stable fluorescence detection was possible.

Creation of a device for detecting fluorescence from microfluidic chips for studying biosystems

The article substantiates the need to develop devices using real-time PCR reaction on microfluidic chips for the study of biological systems (objects) to determine environmental pollution and the consequences of these pollution. In situations of radioactive or chemical contamination, it is necessary to detect and eliminate them as soon as possible. For these purposes, PCR is ideally suited, which is currently the leading tool for chemical and biological research. However, most of the currently available PCR devices use tubes or microtiter plates and have some serious disadvantages. Disadvantages: uneven heating/cooling of volumetric systems, analysis speed does not meet the requirements of modern medicine, biology, environmental services, etc., namely, the requirement for rapid analysis. The solution to this problem is microfluidic chips since they are planar systems. Using microfluidic chips, it is possible to analyze more samples in less time. This article presents a layout of a fluorescence detection device using real-time PCR reaction on microfluidic chips. The results of experimental studies on the created layout and their analysis are presented.

Fabrication of Pressure Conductive Silicone Rubber Socket Device by Shape-Controlled Nickel Powders Produced by High-Energy Ball Milling.

The pressure conductive silicone rubber socket (PCR) is one of the promising test socket devices in high-speed testing environments. In this study, we report highly dense PCR device channels comprised of high aspect-ratio flake-shaped Ni powders. The shape-controlled Ni powders are prepared by the high-energy milling process. The scanning electron microscopy (SEM) and particle size analyzer (PSA) results of the synthesized powder samples showed well-defined flake type Ni powder morphology, and the powder sizes are distributed in the range of ~24–49 μm. The cross-sectional SEM study of the fabricated PCR revealed that the channels consisting of flake Ni powder are uniformly, densely distributed, and connected as face-to-face contact. The resistance of the PCR channels comprised of flake-shaped Ni powders showed ~23% lower resistance values than the spherical-shaped Ni powders-based channels, which could be due to the face-to-face contact of the powders in the channels. The magnetic properties study for the flake-type Ni powder showed a high remanence (~2.2 emu/g) and coercivity (~5.24 mT), owing to the shape anisotropy factor. Finally, the fabricated highly dense and conductive channels of the silicone rubber socket device by shape-controlled Ni powder could be a potential test socket device.

Filtration extraction method using a microfluidic channel for measuring environmental DNA.

The environmental DNA (eDNA) method, which is widely applied in biomonitoring, is limited to laboratory analysis and processing. In this study, we developed a filtration/extraction component using a microfluidic channel, the Biryu-Chip (BC), and a filtration/extraction method, the BC method, to minimize the volume of the sample necessary for DNA extraction and subsequent PCR amplification. We tested the performance of the BC method and compared it with that of the Sterivex filtration/extraction method using aquarium and river water samples. We observed that using the BC method, the same concentration of extracted DNA was obtained with 1/20-1/40 of the filtration volume of the Sterivex method, suggesting that the BC method can be widely used for eDNA measurement. In addition, we performed on-site measurements of eDNA within 30 min using a mobile PCR device, demonstrating that filtration and extraction can be performed easily and quickly using the BC method. The PCR results obtained using the BC method were similar to those obtained using the Sterivex method. The BC method requires fewer steps; therefore, the risk of DNA contamination can be reduced. When combined with mobile PCR, the BC method can be applied to easily detect eDNA within 30 min from the collection of water sample, even on-site.

Outcomes of SARS-CoV-2 omicron infection in residents of long-term care facilities in England (VIVALDI): a prospective, cohort study

BackgroundThe SARS-CoV-2 omicron variant (B.1.1.529) is highly transmissible, but disease severity appears to be reduced compared with previous variants such as alpha and delta. We investigated the risk of severe outcomes following infection in residents of long-term care facilities.MethodsWe did a prospective cohort study in residents of long-term care facilities in England who were tested regularly for SARS-CoV-2 between Sept 1, 2021, and Feb 1, 2022, and who were participants of the VIVALDI study. Residents were eligible for inclusion if they had a positive PCR or lateral flow device test during the study period, which could be linked to a National Health Service (NHS) number, enabling linkage to hospital admissions and mortality datasets. PCR or lateral flow device test results were linked to national hospital admission and mortality records using the NHS-number-based pseudo-identifier. We compared the risk of hospital admission (within 14 days following a positive SARS-CoV-2 test) or death (within 28 days) in residents who had tested positive for SARS-CoV-2 in the period shortly before omicron emerged (delta-dominant) and in the omicron-dominant period, adjusting for age, sex, primary vaccine course, past infection, and booster vaccination. Variants were confirmed by sequencing or spike-gene status in a subset of samples.Results795 233 tests were done in 333 long-term care facilities, of which 159 084 (20·0%) could not be linked to a pseudo-identifier and 138 012 (17·4%) were done in residents. Eight residents had two episodes of infection (>28 days apart) and in these cases the second episode was excluded from the analysis. 2264 residents in 259 long-term care facilities (median age 84·5 years, IQR 77·9–90·0) were diagnosed with SARS-CoV-2, of whom 253 (11·2%) had a previous infection and 1468 (64·8%) had received a booster vaccination. About a third of participants were male. Risk of hospital admissions was markedly lower in the 1864 residents infected in the omicron-period (4·51%, 95% CI 3·65–5·55) than in the 400 residents infected in the pre-omicron period (10·50%, 7·87–13·94), as was risk of death (5·48% [4·52–6·64] vs 10·75% [8·09–14·22]). Adjusted hazard ratios (aHR) also indicated a reduction in hospital admissions (0·64, 95% CI 0·41–1·00; p=0·051) and mortality (aHR 0·68, 0·44–1·04; p=0·076) in the omicron versus the pre-omicron period. Findings were similar in residents with a confirmed variant.InterpretationObserved reduced severity of the omicron variant compared with previous variants suggests that the wave of omicron infections is unlikely to lead to a major surge in severe disease in long-term care facility populations with high levels of vaccine coverage or natural immunity. Continued surveillance in this vulnerable population is important to protect residents from infection and monitor the public health effect of emerging variants.FundingUK Department of Health and Social Care.

COVID-19 Real Time PCR Test Sonuçlarının PCR Cihazı ve CAtenA Smart PCR Bioinformatik Programı Üzerinden Değerlendirme Sürelerinin Karşılaştırılması

The COVID-19 pandemic, which was caused by the SARS-CoV-2 virus, emerged in Wuhan, China in December 2019, and has had a detrimental impact worldwide. The nucleic acid amplification tests are the recommended method for the diagnosis of COVID-19. CAtenA Smart PCR is an artificial intelligence-based bioinformatics tool that assists with PCR data interpretation and offers conclusion preferences before transaction to the web-based result systems. The aim of this study was to compare the turnaround times between the data analysis on a PCR instrument, including result submission, and the CAtenA Smart PCR-assisted analysis. The specialists assessed 139 PCR data sets, each with 94 samples and two internal controls, that were performed in the COVID-19 PCR Diagnostic Laboratory at Meram State Hospital in Konya between 1 September and 30 November 2021. The data analysis times for the PCR tool (Bio-Rad CFX96 Touch, Singapore) and the CAtenA Smart PCR Bioinformatics Program (Ventura, Ankara, Turkey) were recorded. The mean time duration of the 139 PCR data analyses for the PCR device was 14.05 ± 7.55 and 8.04 ± 3.93 minutes for the CAtenA. The Wilcoxon signed ranks test was used for the statistical analysis. The difference between the turnaround times for the PCR instrument and CAtenA Smart PCR was found to be statistically significant (p = 0.0001). We further divided the study period into two groups: the high-positivity phase and the low-positivity phase. We compared the two phases in order to assess the effect of the case positivity rates on the turnaround times. There was a significant difference between the turnaround times of the two groups (p = 0.0001). The findings showed that the positivity rate has affected the time duration of data analysis on both the PCR instrument and the CAtenA program. As a result, employing artificial intelligence-based CAtenA Smart PCR to interpret PCR data and send transactions to the web-based result systems reduces the time it takes to complete the task and gives the user more convenience.

Comparison of COVID-19 laboratory diagnosis by commercial kits: Effectivity of RT-PCR to the RT-LAMP.

Coronavirus disease 2019 or COVID‐19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of these novel approaches may also contribute to faster and cheaper field‐based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS‐CoV‐2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT‐LAMP as well as by conventional q‐RT‐PCR. The patient samples were tested with four different kits (SENSObiz COVID‐19 [SARS‐CoV‐2] LAMP Assay, the QIAseq DIRECT SARS‐CoV‐2 kit, Biospeedy SARS‐CoV‐2 Variant Plus kit, and CoVirion‐CV19‐2 SARS‐CoV‐2 OneStep RT‐PCR kit) and two different PCR devices (GDS Rotor‐Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor‐Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor‐Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS‐CoV‐2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.

Ultra-rapid real-time polymerase chain reaction assay for genotyping of the NT5E gene in Japanese black beef using a mobile PCR device, PCR 1100.

The concentration of inosine 5'-monophosphate (IMP) in beef is an important factor contributing to beef palatability. A previous study suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene strongly affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in beef. Genotyping of the NT5E gene is performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR assay. However, these conventional laboratory assays require large installed instruments. They also involve complicated procedures and are time-consuming. Here, the PCR primers and probes for the NT5E gene (rs42508588 SNP) were designed and synthesized, and we examined the rapid genotyping of the NT5E gene using a PicoGene PCR 1100 mobile PCR device. The results showed that this system enabled rapid amplification of each allele at approximately 19.4s per cycle, with a total run time of 13 min 36 s. This device is portable and does not require a power supply, which facilitates its use not only in specific laboratories but also in meat production farms and distribution stages of beef.

Evaluating sensitivity and specificity of the Biomeme Franklin™ three9 real-time PCR device and SARS-CoV-2 go-strips assay using clinical samples.

We evaluated the sensitivity and specificity of the Biomeme Franklin™ three9 Real-Time PCR Thermocycler and Biomeme SARS-CoV-2 Go-Strips in the detection of SARS-CoV-2. The Biomeme Franklin™ three9 platform is a portable, battery-operated system that could be used in remote settings. We assessed performance of the Biomeme SARS-CoV-2 detection system at a wide range of viral concentrations, examined cross-reactivity of the SARS-CoV-2 Go-Strips against several near-neighbor respiratory pathogens, and evaluated agreement against the BioFire® Respiratory Panel 2.1 in four clinical sample types. Our data indicate the Biomeme Go-Strips can reliably detect SARS-CoV-2 at a concentration of 4.2 × 103 copies/mL. No cross reactivity of the Go-Strips targets was detected against any of the tested near-neighbor respiratory pathogens. Cohen's kappa statistics ranged from 0.68 to 0.92 between results from the Biomeme SARS-CoV-2 Go-Strips and the BioFire® Respiratory Panel 2.1 in all the different sample types. Compared to the BioFire® Respiratory Panel 2.1, the Biomeme SARS-CoV-2 Go-Strips demonstrated statistically significantly lower sensitivity in 3 out of 5 sample types. Overall, our study demonstrates the Biomeme Franklin™ three9 used with the SARS-CoV-2 Go-Strips is an effective system for the detection of SARS-CoV-2 that could potentially be used in a remote or austere environment.

Management of a large outbreak of COVID-19 at a British Army training centre: lessons for the future

IntroductionThe COVID-19 pandemic has posed major challenges for infection control within training centres, both civilian and military. Here we present a narrative review of an outbreak that occurred at the...

POCT Detection of 14 Respiratory Viruses Using Multiplex RT-PCR

Over the past 6 years, acute respiratory infections have constituted an average of more than 70,000 cases in South Korea. It results in a high mortality rate in infants and the elderly with weak immunity. There are several types of respiratory viruses that invade the human respiratory tract and cause infectious disease. Reverse transcription PCR (RT-PCR) is mainly used for respiratory virus detection owing to its high sensitivity and reproducibility. In response, a multiplex real-time RT-PCR (rRT-PCR) assay was developed for the detection of influenza A and B viruses, parainfluenza viruses 1–4 (PIV1-4), human metapneumovirus, adenovirus, human rhinovirus, respiratory syncytial virus (RSV), and SARS-CoV-2. Detection ability of RT-PCR assay was confirmed by applying it to a portable device capable of point-of-care testing (POCT). Amplicons were synthesized using primer pairs and probe sets designed for each target virus, and a standard curve was constructed to confirm the limit of detection. An experiment using nasopharyngeal swab samples was conducted to understand the field applicability of the rRT-PCR assay. Detection was confirmed in most samples. This study demonstrated that rapid and accurate detection results can be obtained using the multiplex rRT-PCR based POC test, and that it is possible to detect 14 types of respiratory viruses that are generally difficult to distinguish at the same time, enabling timely treatment. Furthermore, we expect that the portable PCR device can significantly reduce the processing procedure of clinical samples before testing, which is the main disadvantage of common RT-PCR tests and can help reduce costs.

Multiple Compact Camera Fluorescence Detector for Real-Time PCR Devices.

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.