Polymerase Chain Reaction System Research Articles

Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of intraductal papillary mucinous neoplasm in Japan.

This study aimed to clarify whether several single-nucleotide polymorphisms (SNPs)-related chronic pancreatitis such as carboxypeptidase A1 (CPA1), carboxypeptidase B1 (CPB1), Gamma-glutamyltransferase 1 (GGT1), G-protein-coupled receptor Class C Group 6 Member A (GPRC6A), and serine protease inhibitor, Kazal type 1 (SPINK-1) genotypes were associated with clinical characteristics of patients with intraductal papillary mucinous neoplasm (IPMN) and worrisome features of IPMN. We enrolled 100 patients with IPMN and 116 patients as a control. Serum p-amylase, lipase, trypsin, phospholipase A2 (PLA2), and elastase-1 levels were measured. An Olympus EUS (GF-UCT 260) was used to perform endosonography in 100 patients with IPMN. Total EUS score was evaluated using endosonography. DNA was isolated from the duodenal tissue using a commercial system and polymerase chain reaction (PCR) was performed on 7500 Fast PCR System. There were no associations between glucose tolerances, lipid levels and genotypes of CPA1, GGT1, GPRC6A, and SPINK-1 in patients with IPMN. CPA1 genotype was significantly associated with the pathophysiology of IPMN. Then, GGT1 genotype was also significantly associated with EUS total score and the size of cyst more than 20 mm and more than 30 mm as one of worrisome features of IPMN. Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of IPMN.

EGFR T790M mutation detection in NSCLC patients resistant to tyrosine kinase inhibitor therapy.

The finding of mutations that activate epidermal growth factor receptor (EGFR) in people with lung adenocarcinoma resulted in the creation of a new class of biological treatments called tyrosine kinase inhibitors (TKI). These medications have changed how patients with EGFR mutations are clinically managed, nearly doubling their survival rate compared to standard chemotherapy. Though 1st and 2nd generation EGFR TKIs are initially highly effective, typically within 9-14 months all tumors with the mutation progress due to secondary resistance mutations involving alternative molecular pathways. In most cases (up to 60%), this is due to the T790M mutation emerging in the EGFR gene. The study included 85 patients with NSCLC with progression of the disease after treatment with TKI 1st and 2nd generation. The T790M mutation was determined by digital polymerase chain reaction (PCR) on the QIAcuity One 5plex digital PCR system and traditional real-time PCR. Real-time PCR analysis of the presence of the T790M mutation was performed using the Therascreen EGFR Plasma RGQ PCR Kit (Qiagen). Using a digital PCR system in QIAcuity One (Qiagen) nanoplanets, the T790M mutation was analysed by digital PCR. The age of the patients ranged from 37 to 85 years. Of 85 patients with NSCLC with disease progression after TKI treatment, T790M mutations were detected during digital PCR in 30 of 85 patients, which is 35.2% of the sample, and with traditional real-time PCR, positive mutations came out only in 3 out of 85 patients.

The potential relationship between EasyNAT system Tt values and Cobas z480 Ct values in the detection of SARS-Cov-2

ObjectivesThis study aimed to evaluate the potential relationship between the time threshold (Tt) values of a commercial EasyNAT system, which is based on cross priming amplification (CPA) technology, and the cycle threshold (Ct) values of the Cobas z480 analyzer, which is based on a real-time fluorescence polymerase chain reaction (PCR) method, in the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from oropharyngeal swabs. Design and Methods: Data were retrospectively collected from a clinical laboratory between December 4, 2022 and July 1, 2024. ResultsA total of 277 EasyNAT-positive samples (Tt values from 3.83 to 29.5) were simultaneously investigated using the Cobas z480 analyzer (Ct values from 10.74 to 38.78). The concordance rate between the two systems was 100 %. Among the positive samples, the mean and maximum PCR Ct values of O and N genes increased in line with increasing Tt values of the left and right amplification areas of the EasyNAT system. The maximum Ct values of the O or N gene determined by the Cobas z480 analyzer were no more than 29.52 when the Tt values of the left or right amplification areas of the UC0116 analyzer were no more than 6. ConclusionsThe safe, simple, fast, accurate, and automatic EasyNAT system used in conjunction with a PCR system might be a better choice for the detection of SARS-CoV-2 in hospitals, especially in settings without sophisticated PCR facilities. The Tt value (≤6) of the EasyNAT system can be a reference index for estimating the maximum Ct value (29.52) in SARS-CoV-2-positive samples.

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

The risk-based operational safety strategy for Perkin-Elmer/Cetus DNA Thermal Cycler PCR systems

Introduction: this article was conducted in the field of polymerase chain reaction (PCR) test safety, where it is essential to understand the contribution and impact of risk analysis methods to improve test safety medical diagnosis at the institutional level. In this context, the current study focuses on failure modes and effects analysis (FMEA) applied to the Perkin-Elmer/Cetus deoxyribonucleic acid (DNA) thermocycler.Objective: to describe the application of FMEA to improve the operational safety of Perkin-Elmer/Cetus DNA thermal cyclers used in medical diagnostics in ataxic patients. Methods: to carry out this study, a methodology based on an exhaustive review of the scientific literature available in academic databases and relevant sources was used. Previous studies on FMEA analysis of DNA thermocyclers in common and expanded forms were collected and analyzed.Results: the results obtained demonstrated that FMEA is an effective tool to identify and evaluate possible failure modes of Perkin-Elmer/Cetus DNA thermal cyclers. Several defects were identified and their consequences, causes and classification based on their severity and frequency were determined. Likewise, corrective actions are proposed to reduce the identified risks.Conclusions: early identification of possible failure modes and implementation of corrective actions can reduce risk and guarantee the quality of the diagnoses carried out and therefore improve operational safety

Characterization of genetic diversity in Curcuma longa L. cultivars using Start Codon Targeted (SCoT) markers

Curcuma longa L. (Family- Zingiberaceae), commonly called turmeric, is one of the most important commercial spices. Being a center of turmeric's diversity, India boasts of more than fifty cultivars with variable agronomic characters and curcumin content. However, the quest for calibrating their genetic variability remains debatable as popular molecular marker-based studies have yielded inconsistent results. A promising PCR-based approach, known as the Start Codon Targeted (SCoT)-PCR system has been established to be more efficient than random DNA marker systems because this technique can generate more information on genetic polymorphism patterns. Hence, the present study proposes to use Start Codon Targeted (SCoT) markers to assess inter-cultivar genetic diversity of turmeric. We report an orthogonal L16 (45) design-based approach to investigate the influence of five crucial factors (dNTPs, Taq DNA polymerase, primer, template DNA and annealing temperature of primer) on the efficiency of the polymerase chain reaction (PCR) for development of an optimized Start Codon Targeted (SCoT) amplification protocol using twenty-seven different cultivars of C. longa. The optimized PCR protocol yields best results with a 20 uL reaction mixture, containing 10×PCR Buffer (with 15 mM MgCl2), 2.5 mM dNTPs, 10 mM SCoT primer, Taq polymerase (2 U), template DNA (50 ng), which was verified by using twenty-five SCoT primers. This standardized rapid protocol of SCoT-PCR reported here was employed to examine genetic diversity among twenty-seven Indian cultivars of turmeric, revealing high range of polymorphism ranging from 81.25 % to 100 %. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data segregated the twenty-seven cultivars into two main clusters. The first cluster comprised of nine cultivars (36 % of the cultivars) which was further divided into three sub-clusters and the second one consisted of eighteen cultivars (64 %) which further divided into four sub-clusters. From the results of this study, it can be inferred that the employment of SCoT markers can be an effective tool for analysing genetic variability, phylogenetic relationships and develop a rich genetic resource management system for C. longa cultivars.

A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

Poliomyelitis and COVID-19: a repeated history.

The measurement of the enterovirus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage water is relevant in the early detection of the introduction or disappearance of these viruses in the ecosystem. We evaluated the co-circulation of the enteroviruses and SARS-CoV-2 in 81 sewage water samples collected between September 2021 and April 2023 from different regions of north and southeast Romania, at the border with Ukraine. We used, for the molecular detection of the pathogens, the multiplex real-time polymerase chain reaction (PCR) assay produced for respiratory samples and the Respiratory 2.1 Plus panel Biofire Film array. The isolation of enteroviruses was performed on cell culture lines, in accordance with the World Health Organization (WHO) recommendations. By molecular investigations, we detected the SARS-CoV-2 in 22 (27%) samples, and the human rhinovirus/enterovirus in 64 (79%) samples. By isolation on cell culture lines, 27 samples (33,33%) were positive for non-polio enteroviruses, and no poliovirus strains were isolated, proving the maintenance of the polio-free status in Romania. In an emergency situation, the molecular detection of the pathogens in sewage water using a PCR system integrating sample preparation, amplification, detection, and analysis in 1 h could be implemented.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Development of a Rapid Epstein-Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay.

The quality of cellular products used in biological research can directly impact the ability to obtain accurate results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. We developed three EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 103 copy numbers for the RPA-based and RPA-LFA systems and 1 × 104 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those obtained by the EBV assay methods specified in the pharmaceutical industry standards of the People's Republic of China. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Immunochromatographic Detection of SARS CoV-2 Antigen In Comparison With the Polymerase Chain Reaction for Laboratory Diagnosis of Covid-19 in Karachi, Pakistan

Introduction: The Covid-19 pandemic raised the question of which laboratory test was relevant for a rapid and accurate diagnosis of the disease. The current two methods for the detection of SARS CoV-2 are the ICT (Immunochromatographic Technique) and the PCR (Polymerase Chain Reaction) tests; therefore comparative analysis of test performance has to be done. Objective: To perform a comparative analysis of two different techniques (Immunochromatographic technique for Antigen and Polymerase Chain Reaction for RNA) which are implicated for the detection of SARS CoV-2. Materials & Methods: The Rapid Antigen Test (RAT) and Polymerase Chain Reaction (PCR) were performed for the detection of Covid-19 on individuals having the signs and symptoms of SARS Covid-19. Immunochromatographic technique (ICT) was performed by SD Biosensor kits, while for Polymerase Chain Reaction RNA extraction was performed by Liferiver® auto extraction analyzer, and amplification was performed by SLAN®-48P Real-Time PCR System. Results: Among of 100 samples tested, 62 (23.56%) were positive on ICT while PCR had 66 (22.44%) and 38 were negative on ICT while PCR had 34. Thus the sensitivity of ICT was 90.77% (95% CI, 80.98% - 96.54%) and specificity was 91.43% (95% CI 76.94%-98.20%). The Positive Predictive Value was 95.16% while the Negative Predictive Value was 84.21%. However the Cohen’s Kappa Index Value was recorded as 0.806. Conclusion: The clinical performance of SD Biosensor Roche kits was excellent. On the basis of sensitivity and specificity it was concluded that RAT have comparable results with RT PCR. Keywords: Antigen, COVID-19, diagnosis, immunoassay, PCR, SARS-CoV-2.

Gene-Environment Interaction of Interleukin 10 Gene Polymorphism, rs1800896, with Lifestyle on Cardiovascular Risk in Type 2 Diabetes

Abstract Background: Gene–environment interactions play a major role in the phenotypic expression of complex disease traits such as those for cardiovascular diseases. Aims: This study aimed to determine the gene–environment interactions that underpin the relationship between interleukin-10 (IL-10) single-nucleotide polymorphism (1082 G/A [rs1800896]) with lifestyle on cardiovascular disease risk in adult Nigerians with type 2 diabetes mellitus (DM). Setting and Design: This case–control study involved patients with type 2 DM with high cardiovascular risk, determined by the Framingham’s classification, and age, sex, and diabetes-duration matched subjects with low and intermediate cardiovascular risks. Subjects and Methods: The genotypes were detected by polymerase chain reaction (PCR) followed by allelic discrimination using the Applied Biosystems 7900HT Fast Real-Time PCR System. One-way analysis of variance and logistic regression were employed for analysis. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated to estimate the risk caused by the polymorphism. P <0.05 was considered significant. Results: The odds for cardiovascular risk decreased progressively in individuals with the GG, GA, and AA genotypes (OR = 0.80, 95% CI = 0.49–1.28, P = 0.345 and OR = 0.46, 95% CI = 0.24–0.88, P = 0.018 for GA and AA genotypes, respectively. A significantly higher proportion of homozygous (AA) individuals were in the low cardiovascular risk group (54.2%, P = 0.018). Compared with the whole study population, individuals with the AA genotype had consistently lower odds for cardiovascular risk in subpopulations like alcohol users (OR = 0.25 [0.11–0.55], P = 0.001), but the odds were higher among smokers (OR = 1.80 [1.14–2.90], P = 0.017) and those with sedentary lifestyles (OR = 2.46, 95% CI = 1.14–5.33, P = 0.024). Conclusions: The homozygous mutant genotype AA of the IL-10 gene 1082 G/A had a protective effect on cardiovascular risk in type 2 DM. However, this protection was absent in those leading a sedentary lifestyle.

(Invited) COVID-19 As a Technology Accelerator for High-Speed Polymerase Chain Reaction (PCR)

Limit of detection (LOD), speed and cost for some of the most important diagnostics tools for COVID-19 i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) all benefitted from both the financial and regulatory support brought about by the pandemic. Before COVID-19, LFA was the fastest and least expensive but came with the poorest limit of detection (LOD). The slowest, most expensive but with the best (lowest) LOD was PCR. In between those two extremes, in terms of cost, LOD and speed, was an ELISA . Today, through the “COVID-19 accelerator effect” there are point of care (POC) reverse transcriptase quantitative PCR (ie., RT-qPCR) COVID-19 tests on the market that provide sample to answer in under 20 minutes, while retaining the same very low LOD but at a lower cost [1]. There are even POC PCR tests under development that come in at less than 10 minutes: beating both LFA and ELISAs in terms of time and LOD [2]. Although RT-qPCR has gained the most in overall performance, signal-amplified POC LFAs are possible now with an LOD that rivals that of an ELISA.However, implementing the next generation PCR equipment in point-of-care settings, remains very challenging because of difficulties in reaching a 10 minute or below sample to answer time , instrument complexity and cost. In this work, we discuss the engineering challenges involved in the development of high-speed microfluidic PCR equipment. To guide this review/comparison of PCR advances we introduce three parameters. The first one is the overall sample to answer time (t), the second is called lambda (λ) and is a measure of the degree to which stochastic effects interfere with obtaining analytical results and thereby setting the minimal number of copies required per volume. The third parameter gamma (γ), introduced first by Dong et al [3], is the ratio of thermal cycling time (in min) to the heat cycled sample volume (in mL). It describes the trade-off between the speed of the PCR system and the desired level of sensitivity. It can be interpreted as a system efficiency: the most efficient system heat cycles the largest amount of sample in the smallest amount of time.With these three criteria in mind we set out to suggest designs for an optimized POC reverse transcriptase (RT) quantitative qPCR or RT-qPCR system. Applications of RT-qPCR include quantifying gene expression levels, validating RNA interference (RNAi), and detecting pathogens such as viruses. The aim of this specific POC RT-qPCR system is to provide sample to answer in ten minutes or less for a respiratory pathogens panel, with an LOD of 500 copies/mL and the capability to multiplex (8 chambers) while remaining portable and low cost.Finally we introduce two of the most daunting, remaining, microfluidics challenges that both need to be tackled to implement an ideal microfluidic platform like the POC RT-qPCR system described above. One is liquid storage and the other is proportional valves on a disposable microfluidic platform. Liquid storage is essential when the assay requires repeated washes, if these washes can come from one and the same reservoir via a proportional valve, real estate is gained and system complexity is reduced. The storage issue is further complicated when the design also calls for storing lyophilized or dried reagents on the same disposable. We compare some of the most recent approaches to liquid storage and proportional valving that we believe will enable the fast POC diagnostic platforms of the future. 1. Tsang, Hin Fung, Wai Ming Stanley Leung, Lawrence Wing Chi Chan, William Chi Shing Cho, and Sze Chuen Cesar Wong. “Performance Comparison of the Cobas® Liat® and Cepheid® GeneXpert® Systems on SARS-CoV-2 Detection in Nasopharyngeal Swab and Posterior Oropharyngeal Saliva.” Expert Review of Molecular Diagnostics 21, no. 5 (May 4, 2021): 515–18. 2. BUCKLAND, Justin, Tom JELLICOE, Alex STOKOE, and Amaru ARAYA-WILLIAMS. “VARIABLE TEMPERATURE REACTOR, HEATER AND CONTROL CIRCUIT FOR THE SAME.” Patent Application, January 30, 2020. 3. Dong, Xiaobin, Luyao Liu, Yunping Tu, Jing Zhang, Guijun Miao, Lulu Zhang, Shengxiang Ge, Ningshao Xia, Duli Yu, and Xianbo Qiu. “Rapid PCR Powered by Microfluidics: A Quick Review under the Background of COVID-19 Pandemic.” Trends in Analytical Chemistry 143 (October 2021): 116377.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Development of a Thermal Cycler for a Low-Cost Real-Time PCR Application

With the COVID 19 pandemic and the rise of polymerase chain reaction (PCR) testing in its wake, affordable large-scale testing became necessary. A thermal cycler for PCR has been developed that is affordable, fast, and accurate. The thermocycler must follow a specific temperature profile consisting of three different temperature levels. To ensure the success of any PCR, the tempering elements must accurately follow the tempering profile and be stable heat sources to maintain any given temperature. A control mechanism is required to approximate this prescribed temperature behavior. The basis for this is a Proportional-Integral-Differential (PID) controller, whose functionality is ensured by a feedback mechanism consisting of a temperature sensor. The PID controller provides precise attainment and maintenance of the temperature levels. A high performance Peltier element is used to heat and cool the PCR system. This is controlled by a programmable power supply and, in combination with a heat dissipation system, achieves heating and cooling powers in excess of 200 W. In addition, different design variants of the thermal cycler were created and their temperature behavior was simulated. These were then implemented, tested and the PID controller tuned accordingly. These tests help to find the best design for the thermal cycler with the optimal heat distribution.

Multiplexed detection of single-point mutations in unprocessed human serum with SERS-integrated PCR

Early detection and monitoring of tumor mutations is an important area of research in diagnostics. Polymerase chain reaction (PCR) is considered the gold standard for monitoring tumor mutations. Recently, integration of PCR with SERS has led to enhanced sensitivity and multiplexity of methods for monitoring pathogens and mutations. However, limitations on increasing the number of targets that can be detected using a single set of reaction still exist. The number of mutations that can be analyzed in a single reaction is influenced by the interaction among different fluorophores. For simultaneous detection of more than three mutations with SERS, complicated extraction, and subsequent statistical analysis is required. Here, we present a SERS Hairpin-blocked Integrated PCR (SHIP) system, which integrates PCR with SERS using hairpin-shaped blocking DNA to prevent false positive enhancement of SERS intensity. SHIP enables simultaneously detection of up to four different KRAS single-point mutations in unprocessed serum. The SHIP system holds a great potential for multiplexed detection of mutations and pathogens for diagnostic purposes.

A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa.

The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.

Development of rapid on-site detection of Salmonella Enteritidis, S. Typhimurium, and S. Thompson in food samples using an ultrafast PCR system

The conventional Salmonella serotyping methods are labor intensive and time-consuming; therefore, rapid identification method is needed to detect outbreaks early and prevent their spread. In this study, we developed for the first time a detection method for the rapid and accurate on-site serotyping of Salmonella enterica subsp. enterica serovar Enteritidis, Thompson, and Typhimurium, all of which have epidemiological importance worldwide. This method, which is based on specific marker genes, was constructed with portable equipment and a simple DNA extraction step. For the specific detection of the three serovars, genetic markers obtained via a bioinformatics analysis were selected and showed high specificity for 97 strains. The ultrafast polymerase chain reaction (PCR) showed high linearity (≥0.997) in both pure culture and a spiked food sample, with a detection specificity of 102 colony forming unit (CFU)/ml; these results are consistent with real-time PCR. Moreover, ultrafast PCR was further improved for developing direct ultrafast PCR method for field detection, the entire procedure of which could be completed within 25 min. The test results of an artificially contaminated food sample showed that the three serovars could be detected using the direct ultrafast PCR with a very short (3 h) enrichment step when inoculated at 1 CFU/g. This method exhibited various advantages, such as its ultrafast and labor-saving characteristics, which can be applied to the diagnosis of Salmonella serovar in the field.

A-297 Usefulness of Non-Skin Samples in the PCR Diagnosis of Monkeypox

Abstract Background During 2022, human mpox cases were reported in several European countries. Until November 2022, in Spain, 7404 cases were detected, most of them linked to sexual intercourse. In that context, fast identification of cases and sources of infection were essential for an optimal management. Polymerase chain reaction (PCR) was the preferred laboratory test. Our objective was to evaluate the usefulness of sera and nasopharyngeal samples for MPXV detection, and its application in contact studies without skin or mucosal injuries. Methods In the Hospital Clínico Universitario of Zaragoza (Spain), 106 samples from 50 patients, 38 male and 12 female, aged between 11 to 62 years, with skin lesions suspected MPXV infection were analysed. Samples were collected between May and September 2022 in inactivating universal viral transport medium (Biocomma). Samples were obtained between 1 and 19 days of evolution. To perform the real-time PCR, the VIASURE Monkeypox Virus Real Time PCR Detection Kit and the VIASURE 48—Real Time PCR System were used. Nucleic acid extraction was performed with the automated nucleic acid extraction system MagLEAD12gC using the MagDEA®Dx SV reagents. Results Table 1 shows the results expressed in Ct values. Skin, anogenital and serum samples were the most efficient for MPXV diagnosis. Nevertheless, these data are influenced by the sample collection date. Conclusion Real-time PCR was positive in samples from all locations in 71,4% of the infected patients. Respiratory samples (pharyngeal and nasopharyngeal) were the ones that showed the highest number of false negative results, whereas there was only one skin sample and one serum sample with a negative result. This study shows that sera samples can be used in the diagnosis of MPXV infection because of ease of collection, applicability to contacts without lesions and the option of serology testing for other STI agents (HBV, HCV, HIV, Treponema pallidum…).

Simple confirmation methods for rare but impaired variants of human flavin-containing monooxygenase 3 (FMO3) found in an updated genome resource databank

Forty-seven new nonsense or missense human flavin-containing monooxygenase 3 (FMO3) variants were recently identified in an updated Japanese population reference panel. Of these, 20 rare single-nucleotide substitutions resulted in moderately or severely impaired FMO3 activity. To easily identify these 20 FMO3 variants (2 stop codon mutations, 2 frameshifts, and 16 amino-acid substitutions) in the clinical setting, simple confirmation methods for impaired FMO3 variants are proposed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) or allele-specific PCR methods. Using PCR-RFLP, FMO3 variants p.Arg51Gly, p.Met66Lys, p.Asn80Lys, p.Val151Glu, p.Val187fsTer25, p.Gly193Arg, p.Val283Ala, p.Asp286His, p.Val382Ala, and p.Phe451Leu were digested by the designated restriction enzymes and confirmed using reference cDNAs. In contrast, the FMO3 variants p.Gly39Val, p.Arg238Ter, p.Arg387Cys, p.Arg387His, p.Leu457Trp, and p.Met497Arg were not digested, whereas the wild type was digested. FMO3 variants p.Gly11Asp, p.Lys416fsTer72, p.Gln427Ter, and p.Thr453Pro were confirmed using allele-specific PCR systems. The previously identified FMO3 p.Arg500Ter variant has a relatively high frequency and was differentiated from p.Arg500Gln in two steps, i.e., enzyme restriction followed by allele-specific PCR, similar to the method for p.Arg387Cys and p.Arg387His. These systems should facilitate easy detection in the clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearance possibly caused by impaired FMO3 function.