PCR Device Research Articles

Evaluation of a triplex assay for detecting equine influenza virus and equine herpesvirus types 1 and 4 by using a microfluidic‐chip‐based mobile real‐time PCR device for point‐of‐care testing

Evaluation of a triplex assay for detecting equine influenza virus and equine herpesvirus types 1 and 4 by using a microfluidic‐chip‐based mobile real‐time PCR device for point‐of‐care testing

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing

This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 μm to 5 μm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.

On-site detection of methicillin-resistant Staphylococcus aureus (MRSA) utilizing G-quadruplex based isothermal exponential amplification reaction (GQ-EXPAR)

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.

Field-based molecular detection of Batrachochytrium dendrobatidis in critically endangered Atelopus toads and aquatic habitats in Ecuador.

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus.

An Inexpensive System for Rapid and Accurate On-site Detection of Garlic-Infected Viruses by Agarose Gel Electrophoresis Followed by Array Assay.

Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.

The role of circulating miRNAs in the diagnosis of osteoporosis miRNAs in osteoporosis.

Osteoporosis, defined as a systemic skeletal disease, is characterized by increased bone fragility and fracture risk. Studies have shown that dysregulation of the functions of miRNAs or the mechanisms they mediate may be an important pathological factor in bone degeneration. Therefore, the aim of the study was to determine the role of miRNAs, which are thought to play a role in bone metabolism, in osteoporosis. The study included 48 patients who were diagnosed with osteoporosis according to the results of a bone mineral density assessment by quantitative computed tomography and 36 healthy individuals. MiRNAs from plasma samples obtained from blood samples taken into ethylenediaminetetraacetic acid (EDTA) tubes were isolated with the miRNA isolation kit and converted to cDNA. Expression analysis of miR-21-5p, miR-34a-5p, miR-210, miR-122-5p, miR-125b-5p, miR-133a, miR-143-3p, miR-146a, miR-155-5p, and miR-223 was performed on the real-time PCR (RT-PCR) device. When miRNA expression levels in the patient group were compared with the control group, all miRNAs were found to be downregulated in the patients. When fold changes in expression levels in the patient group were examined, significant differences were found in miR-21-5p, miR-133a, mir143-3p, miR-210, and miR-223. In the receiver operating curve analysis, area under the curve=0.882 for the combination of miR-34, miR-125, miR-133, and miR-210. In this study, it was determined that the combined effects of miRNAs, as well as their single effects, were effective in the development of osteoporosis. Therefore, a miRNA panel to be created can make a significant contribution to the development of novel diagnostic and treatment approaches for this disease.

1691. Use of multiplex PCR in pleural effusion: a case series study in a pediatric reference center in Colombia

Abstract Background The microbiological diagnosis of pleural effusion (PE) is largely based on methods of classical microbiology. Culture-based methods allow for testing of bacterial susceptibility tests, but these methods take time and frequently have false negative results. We describe the utility of multiplex PCR in pleural fluid for the diagnosis of complicated pneumonia in children using a commercial platform for respiratory samples. Methods We conducted a case series of children with complicated pneumonia by PE, empyema or necrotizing pneumonia. Pleural fluid was sent for laboratory to culture, cytochemical and molecular biology testing analysis between 2021 and 2022 in a reference children hospital in Bogotá, Colombia. We used BIOFIRE® FILMARRAY® Pneumonia Panel plus (BioFire Diagnostics, Salt Lake City, Utah). The results were strictly analyzed by a multidisciplinary team. Any change in patient’s therapeutics was decided by this group. Results We identified 17 samples that were processed for multiplex-PCR from 16 patients (Table 1). Median age was 3 years (IQR 2-4 years). Pleural empyema was the most common presentation (7/17, 41%), followed by free pleural effusion (6/17, 35%) and necrotizing pneumonia with empyema (4/17, 23,5%). Pathogens were documented in 16 out of 17 (94%) molecular panels performed. Streptococcus pneumoniae was detected in 11/17 (64.7%) multiplex PCR tests, adenovirus was detected in 7/17 (41.1%), and H. influenzae in 2/17 (11.7%) tests. Pneumococcus was isolated in classical cultures in 4 patients (23.5%), of which only one was in pleural fluid. Changes in antibiotic management in 9/17 (52.9%) of the patients. In 5 patients, there was an increase in the antibiotic spectrum, and in 4 patients, there was a decrease in it. Conclusion Although the use of this multiplex PCR device is not approved by the FDA for use in pleural fluid samples (off-label), this case series described its usefulness and importance in contexts where there is no other approved PCR technology for these samples. Disclosures All Authors: No reported disclosures

Plasmonic digital PCR for discriminative detection of SARS-CoV-2 variants

We developed a novel strategy for discriminative detection of SARS-CoV-2 variants based on the plasmonic photothermal effect of gold nanofilms and digital polymerase chain reaction (dPCR) technology. This method consists of the gold nanofilm-based dPCR chip fabrication for ultrafast heating and cooling cycles by the plasmonic photothermal effect, the LED quencher immobilization through the interfacing compound on the surface of the gold nanofilm to prevent photoquenching of PCR signaling dye, and the discriminative detection of the variant viruses from the COVID-19 clinical samples by photothermal cycles with fabricated dPCR chips and a portable plasmonic PCR device. Compared to conventional sequencing or RT-qPCR-based variant detection methods, this technology can be effectively applied to point-of-care testing by enabling ultrafast quantitative analysis with a small device. With this method, we successfully detected the delta variant and the omicron variant with a high sensitivity of 10 copies from COVID-19 patients’ clinical samples within 25 min, including reverse transcription. This method can be applied universally to rapid and accurate point-of-care testing for various pandemic viruses as well as the coronavirus.

PATH-44. INTRA-OPERATIVE ANALYSES OF GENETIC ALTERATIONS IN THE CENTRAL NERVOUS SYSTEM TUMORS USING RAPID QUANTITATIVE PCR DEVICE

Abstract Recent comprehensive molecular analyses of central nervous system (CNS) tumors identified several diagnostic or prognostic markers. IDH mutation, TERT promoter (pTERT) mutation and CDKN2A homozygous deletion in adult-type diffuse gliomas, BRAF p.V600E mutation in circumscribed astrocytic gliomas, histone H3 gene mutations in pediatric-type diffuse high-grade gliomas or MYD88 mutation in primary CNS lymphoma (PCNSL) are quite important markers. Intra-operative identification of these molecular alterations could be effective for decision-making of tumor removal strategy. In this study, we established intra-operative gene analysis method using rapid quantitative PCR device, GeneSoC, by which we could obtain genotype of these important genes for 20 minutes in an operating room. Using GeneSoC, we could perform 50 cycles of quantitative PCR for approximately 12 minutes. We established rapid gene analyses of mutations of IDH1 p.R132H, pTERT C225T, pTERT C250T, H3 K27M, BRAF p.V600E and homozygous deletion of CDKN2A for diffuse gliomas and circumscribed astrocytic tumors and MYD88 p.L265P for PCNSL. For elution of DNA, we incubated at least 1mg of tumor tissue at 95°for 5 minutes. Combined this boiling method with GeneSoC, we could obtain those genetic alterations for 20 minutes after collection of tumor tissue. For 42 glioma cases and 10 PCNSL cases, we could obtain accurate genetic alterations (IDH1 p.R132H, pTERT C225T, pTERT C250T for gliomas or MYD88 p.L265P for PCNSL) in all cases, using this method. For detection of CDKN2A homozygous deletion, sensitivity and specificity are 100% and 83% in analyzed 50 cases, respectively. In this study, we could obtain the genotype of important molecular markers intra-operatively, not only during tumor removal but even during tumor biopsy, using GeneSoC device. This rapid genetic analysis might be effective not only for intra-operative diagnosis but also for detection of boundary between tumor and normal tissue.

The Correlation of Two Different Real-Time PCR Devices for the Analysis of CYP2C19 Pharmacogenetic Results

CYP2C19 is a highly polymorphic gene responsible for the metabolism of commonly used drugs. CYP2C19*1, the wild-type allele, is associated with normal enzyme activity, whereas CYP2C19*2 and CYP2C19*17 lead to null and increased enzyme activity, respectively. The use of different instruments to perform the same pharmacogenetic tests should not affect the reliability of the results reported to clinicians, as required by the ISO 15189 standard. Genotyping assays allowed for the identification of gene variants corresponding to the CYP2C19*2 and CYP2C19*17 haplotypes in 44 selected samples. Each sample was analyzed in duplicate using the Thermo Fisher Taqman Drug Metabolism probes CYP2C19*2: c_25986767_70 (rs4244285) and CYP2C19*17: c_469857_10 (rs12248560). The experiments were performed on two widely used types of real-time PCR analyzers: ABI PRSIM™7500 and QuantStudioTM12KFlex (both from Applied Biosystems, Thermofisher). The data were analyzed in a Thermo Fisher Cloud facility. The analysis was performed independently by two qualified professionals. Both different instruments and analysts’ interpretations were consistent in identifying the native homozygous, heterozygous, and mutant homozygous variants for CYP2C19*2 and CYP2C19*17. The results provided by both the primary and backup analyzers showed a perfect correlation. This would allow for the use of the backup analyzer in case the main one is not available.

Numerical Simulation and Application of a Channel Heat Sink with Diamond Ribs

This paper presents a channel radiator with ribbed ribs and primarily investigates the fluid flow and heat-transfer characteristics of the channel radiator. A three-dimensional numerical simulation of the radiator’s pressure-drop and heat-transfer process was conducted using the finite volume method. A comparison between the experimental data and the simulation results demonstrates that the simulation in this paper is accurate, with a maximum error not exceeding 5%. Furthermore, the radiator was further subjected to geometric parameter studies, principally including the height ratio between the fins and the channel, the fin angle, and the spacing between the fins. The thermal resistance, Nusselt number, friction factor, and heat-transfer enhancement factor were calculated. The results indicate that if the geometric parameters are selected appropriately, the heat sink will enhance heat-transfer performance within an acceptable pressure drop. When the Reynolds number is greater than 507.5, the height ratio of 25%, the rib angle of 135°, and the rib spacing of 2.5 mm can be given priority. This heat sink is used in PCR devices, and experimental results show that the novel channel heat sink can meet the heat dissipation requirements of the TEC during the PCR process.

B-247 Rapid and Field-deployable Microchip-based Real-time PCR for Hepatitis B Virus DNA Detection

Abstract Background Hepatitis B is one of the most common infectious diseases with global significance, seriously causing chronic liver complications and a heavy health burden in developing countries. There is no doubt that real-time polymerase chain reaction (RT-PCR) has been a golden standard for monitoring the Hepatitis B virus (HBV) level in clinical health management. However, the conventional PCR protocol cannot fulfill the field test requirement in some resource-poor areas due to its drawbacks, such as huge size, long turnaround time, and demand for professional laboratory facilities. For solving these problems, a portable microfluidic chip-based PCR detection system was developed to enable the nucleic acid test in a flexible, rapid, and economical way. With the efficient temperature control module and advanced optical path design, this novel panel can achieve fast thermal circulation with a 30°C/s heating rate and multiple fluorescent detection with a throughput of 6 samples at a time. Methods In this study, we assessed the detection performance of the system using a commercial HBV nucleic acid detection kit. The reproducibility, limit-of-detection (LoD), and linearity were validated by reference samples with gradient concentrations. We evaluated the clinical sensitivity and specificity of the microdevice by a total of 200 extracted serum samples (100 positive and 100 negative cases). The analytical results were compared with a traditional PCR device with FDA certification. Results The variation coefficients of the microdevice for reference samples with different concentrations are 1.78%, 1.98%, and 1.50% (n = 10), respectively. The field-deployable PCR system can provide the HBV DNA assay with a detection limit of 300 IU/ml, whose reaction time was approximately 67 mins rather than the 108 mins performed by the comparator. During the establishment of the standard curve, this fast PCR test shows a strong linear correlation (R2 = 0.9919) between targeted gene concentration and dilution factor. Additionally, among the 200 clinical samples, we used the microchip-based system to rapidly complete quantitative detection for each sample with high sensitivity of 100% (100/100) and specificity of 100% (100/100), which is critically consistent with the traditional PCR method. Conclusion Hence, the point-of-care microfluidic PCR system exhibits great feasibility in offering timely, accurate, and reliable molecular diagnosis for early epidemic disease screening and therapeutic guidance of chronic infections.

Simultaneous detection using a portable multiplex PCR-dual lateral flow immunoassay for P. carotovorum subsp. brasiliense and E. coli O157:H7

A portable multiplex polymerase chain reaction (mPCR)-dual LFA system was developed for the simultaneous detection of P. carotovorum subsp. brasiliense and E. coli O157:H7 in fresh agricultural products. The field applicability of the system was improved using a portable PCR device and dual LFA strip, enabling rapid and sufficiently sensitive results to be obtained within 60 min. The mPCR-LFA method was validated for each bacterial pure culture and six agricultural products, with successful detection of both bacteria up to 102 CFU/g, a sufficiently sensitive level for ensuring the safety of all fresh agricultural products. The results demonstrate that the mPCR-LFA method developed in this study can identify pathogens much more conveniently in a point-of-care detection setting.

CD4+ and CD8+ T cells and antibodies are associated with protection against Delta vaccine breakthrough infection: a nested case-control study within the PITCH study.

Defining correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine breakthrough infection informs vaccine policy for booster doses and future vaccine designs. Existing studies demonstrate humoral correlates of protection, but the role of T cells in protection is still unclear. In this study, we explore antibody and T cell immune responses associated with protection against Delta variant vaccine breakthrough infection in a well-characterized cohort of UK Healthcare Workers (HCWs). We demonstrate evidence to support a role for CD4+ and CD8+ T cells as well as antibodies against Delta vaccine breakthrough infection. In addition, our results suggest a potential role for cross-reactive T cells in vaccine breakthrough.

Kruppel benzeri faktör 2 polimorfizmlerinin koroner arter hastalığı riski ile ilişkisi

Purpose: Coronary artery disease is defined as complications that develop in proportion to the prevalence of ischemia due to occlusion of the coronary arteries and the resulting cell death. The development of atherosclerosis is significantly influenced by endothelial cell dysfunction. Kruppel-like factor 2, a transcription factor, has been shown to regulate critical biological events in endothelial biology, such as vascular tone, migration, proliferation, vasoreactivity, and angiogenesis. In our study, it was aimed to clarify the relationship between coronary artery disease and Kruppel-like factor 2 protein levels and C1239A polymorphisms.
 Materials and Methods: 191 individuals who underwent coronary angiography at Mersin University Faculty of Medicine, Department of Cardiology, Health, Research and Application Center were included in the study. Measurements of serum fasting blood glucose, HDL cholesterol, total cholesterol, and triglyceride levels were performed on the AU5800 (Beckman Coulter, United States) autoanalyzer. Serum LDL levels were calculated using the Friedwald equation. Serum Kruppel-like factor 2 protein levels were measured by sandwich enzyme-linked immunoassay method on the Multiskan GO (Thermo Scientific, Finland) device. Kruppel-like factor 2 C1239A variations were detected on the Applied Biosystem VIIA™ 7 Real-Time PCR (Life Technologies Co., United States) device by TaqMan® single nucleotide polymorphism (SNP) genotyping method.
 Results: Men had a 3.8-fold higher risk of CAD than women. (Odd’s ratio 3.83, 95% Confidence interval 1.98-7.39; p

Microdroplet PCR in Microfluidic Chip Based on Constant Pressure Regulation.

A device and method for the constant pressure regulation of microdroplet PCR in microfluidic chips are developed to optimize for the microdroplet movement, fragmentation, and bubble generation in microfluidic chips. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that microdroplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20 μL will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87 μm, and the microdroplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect human genes. As indicated by the experimental results, a good linear relationship exists between the detection signal and DNA concentration ranging from 101 to 105 copies/μL (R2 = 0.999). The microdroplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, microdroplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, microdroplet PCR devices based on constant pressure regulation chips have promising applications for nucleic acid quantification.

A portable, high-throughput real-time quantitative PCR device for point-of-care testing

Nucleic acids detection has become essential in the identification of many infectious diseases and tumors. Conventional qPCR instruments are not suitable for point-of-care Moreover, current miniaturized nucleic acid detection equipment has limited throughput and multiplex detection capabilities, typically allowing the detection of a limited number of samples. Here, we present an affordable, portable, and high-throughput nucleic acid detection device for point-of-care detection. This portable device is approximately 220 × 165 × 140 mm in size and about 3 kg in weight. It can provide stable and accurate temperature control and analyze two fluorescent signals (FAM and VIC) and run 16 samples simultaneously. As a proof of concept, we used the two purified DNA samples from Bordetella pertussis and Canine parvovirus and the results showed good linearity and coefficient of variation. Moreover, this portable device can detect as low as 10 copies and has good specificity. Therefore, our device can provide advantages in real-time diagnosis of high-throughput nucleic acid detection in the field, especially for resource-limited conditions.

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.