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Developing a low-cost thermal cycler for a polymerase chain reaction (PCR) is becoming interested in the pandemic era caused by viruses. PCR is the standard gold for the diagnostic. However, in a low-income country, the availability of the device is limited. In this work, the development of a thermal cycler uses electronic modules available in the market. The central part is thermoelectric for heating and cooling, an embedded system to control, and a low-profile cooling fan. The system temperature control used a combination of feedforward, bang-bang, and proportional-integral-derivative (PID) control. The control parameter of the PID was successfully obtained by using Chien servo tuning. The feedforward and bang-bang control are used to optimize the cooling cycle and minimize the rise time. The system shows a well-suited temperature accuracy at the denaturation, annealing, and extension temperature with a temperature deviation of less than 0.5 °C. System performance is maintained even though the system has been running non-stop for 24 hours. The low-profile cooling fan, which is usually used for CPU cooling, shows good results in maintaining temperature stability.

The article covers molecular genetic analysis of intermicrosatellite DNA sequences of dairy cattle productivity. Molecular markers based on DNA polymorphism were used for this monitoring. Such markers make it possible to assess quickly the genetic polymorphism of taprin in the herd. Іnsofar as a large number of intermicrosatellite repeats is in the genome of cattle, that increases the probability of detecting polymorphic loci. The ISSR markers selected for the study are based on multiclocus synthesis in polymerase chain reaction (PCR) and allow an objective study of the breed and interbreed diversity. And it makes possible to assess quickly and accurately genetic diversity for the presence of genes associated with economically useful traits. The purpose of this work is the selection and evaluation of ISSR-markers for the analysis and study of genetic diversity of Ukrainian and imported breeds of dairy cattle. Samples of biological material from representatives of three herds of cattle (Ukrainian Red-and-White spotted dairy, Montbeliard breed and their crossbreeds) were selected for the study by the method of groups-analogues. For the analysis of this material the generally accepted zootechnical methods of studying of a selection material and methods of an estimation of animals on molecular – genetic markers are included. According to standard methods, DNA was isolated from peripheral blood lymphocytes using a set of reagents "DNA Sorb B". Amplification of total DNA with ISSR primers was performed on a thermal cycler "Tertsyk" ("DNA technology" of the Russian Federation). Electrophoretic separation of DNA fragments was performed in an agarose gel according to conventional methods. The size of the obtained PCR products was detected using a molecular weight marker SM1343. As a result of the study of the biological material of these animals, the obtained ISSR-PCR products were quite heterogeneous. The vast majority of polylocus spectra had clear discrete bands, but there were amplicons without clear discrete bands. Analyzing the results of the study of the genetic structure of animals of the Ukrainian Red-and-White dairy breed, using primers ISSR-1, ISSR-2, ISSR-3 and ISSR-4, the range of obtained PCR products ranges from 250 bp. up to 3000 bp. The range of amplification products in Montbeliard animals has a smaller range and ranges from 250 bp, respectively. up to 1500 bp.The obtained amplicons for the use of primers ISSR-1 and ISSR-2, ISSR-3 and ISSR-4 in the turf of Ukrainian Red-and-White dairy and Montbeliard breeds have sizes from 350 bp to 2000 bp. Having analyzed the information you can determine the distribution of the number and length of DNA fragments using 4 ISSR-markers. The total number of amplified DNA fragments varies depending on the primer from 21 to 106, and their size ranges from 250 BP up to 3000 bp. Based on the analysis of DNA plymorphism, it is possible to assess the heterogeneity of selected populations of cattle. Thus, as a result of studying the genetic structure of animals of two breeds of dairy cattle and their crossbreeds by intermicrosatellite DNA loci, their individual polymorphism was revealed. The amplification products have significant variations depending on the primer used. Primers ISSR-1 and ISSR-2 were the most informative for the analysis of cattle DNA polymorphism.

Nucleic acid amplification using polymerase chain reaction (PCR) method has been widely used in different fields such as agricultural science, medicine, pathogen identification, and forensics to name a few. Today, it seems inevitable to have a robust, simple PCR system for diagnostics at the point-of-care (POC) level. Many photonic PCR systems have been proposed in the literature that benefit from plasmonic photothermal heating to achieve the common PCR thermal cycling. However, non-homogeneous temperature distribution is a challenge in some of them. In the present work, to achieve more efficient gene amplification, the effect of adding TiO2 nanoparticles has been investigated in a photonic PCR benefiting from plasmonic photothermal heating. The system enjoys higher heating and cooling rates than those of conventional PCR systems while having much lower energy consumption. The average heating and cooling rates are 4.44 °C/s and 2.65 °C/s, respectively. The system demonstrates acceptable temperature stability and accuracy with negligible deviation from the set points. Furthermore, the system is capable of amplifying eight gene samples simultaneously which makes it a viable choice for POC diagnostics. A part of the Alcohol Oxidase gene has been amplified using the plasmonic thermal cycler. It is demonstrated that adding TiO2 nanoparticles with a proper concentration to the sample provides a more specific band that can be ascribed to more homogeneous temperature distribution owing to enhanced thermal conductivity. The best amplification of the target gene has been achieved with a concentration of 0.4 nM of nanoparticles.

We have tested 16 primer mixes for RT‐LAMP targeting SARS‐CoV‐2 RNA from the available literature in three rounds of sensitivity assays. The selected RT‐LAMP primer mix has a limit of detection of six copies of viral RNA per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes interfered with the RT‐LAMP analysis, but this effect was prevented with the ribonuclease inhibitor, which was confirmed on a small set of correctly diagnosed clinical samples., Summary In the fight against the recent COVID‐19 pandemics, testing is crucial. Nasopharyngeal swabs and real‐time RT‐PCR are used for the detection of the viral RNA. The collection of saliva is non‐invasive, pain‐free and does not require trained personnel. An alternative to RT‐PCR is loop‐mediated isothermal amplification coupled with reverse transcription (RT‐LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS‐CoV‐2 RNA can be detected directly in saliva using RT‐LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT‐LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT‐PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT‐LAMP analysis. Neither Chelex‐100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT‐LAMP assay was 12 copies per reaction of RNA in Salivette® saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT‐LAMP for SARS‐CoV‐2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.

This research study utilized a Thermal Cycler to simulate the field aging conditions and compared with standard laboratory aging methods. The Thermal Cycler intends to simulate asphalt binder aging...

A B S T R A C TIntroduction. Along with the COVID-19 pandemic, the need for a thermal cyclerdevice for examining COVID-19 is getting bigger. Many laboratories have overusedthe use of thermal cyclers due to the limited availability of this tool. This study aimsto test the effectiveness of the Sriwijaya thermal cycler based on a smart controllerin conducting PCR DNA sequences. Methods. The research design was anexperimental study with a posttest control group approach, in order to see anoverview of the results of the polymerase chain reaction (PCR) in the form ofintermediate DNA bands using Sriwijaya Thermal Cycler compared to groups usingfactory-made Thermal cyclers. Results. The PCR results showed precise results fromthe image and band separation, and there was a clear separation of the bands onthe marker. The PCR results from the ACE I / D gene showed quite good and optimalresults in the separation of the PCR results band. Conclusion. Sriwijaya thermalcycler is effective in DNA polymerase chain reaction (PCR) sequences comparable tothat of the manufacturer's Thermal Cycler.

This study aimed at investigating the tilapia fish (Oreochromis Niloticus) DNA. In analyzing Tilapia DNA, DNA extraction was carried out and PCR mixture was used for 20 μL of reaction, making Master Mix such as 1 μL Forward Primer, 1 μL Reverse Primer, 10 μL HotStar Taq Master Mix, 1 μL DNA Sample, 7 μL Nuslease Free Water. Mixing is done by adding the enzyme at the last stage. Then turn on the thermal cycler and set it according to the desired PCR conditions. Electrophoresis is carried out through stages such as Pre-Denaturation, Denaturation, Annealing, Extention, Final extension. The results showed that the virus did not infect tilapia (Oreochromis Niloticus). Tilapia sprayed on the gills with various doses showed the same results, namely no detection of KHV in mucus, kidneys, and liver.

Food product authentication is important at every level of the food manufacturing process, starting from raw materials until finished products. Authentication also plays an important role in assuring accurate food labelling, which is required to help consumers select suitable types of food products. Food adulteration is one of the vital issues addressed by halal authentication, especially for food products that contain pig traces or porcine ingredients. Various methods that aim to guarantee the authenticity of foods have been developed over the past years. In this article, a short review of recent food analytical methods related to authenticity studies, with special regard to pork identification, is provided. The focus of this review is DNA-based methods, which have gained the interest of the scientific community. The specificity, sensitivity and fast and high throughput of the methods are highlighted. In the present case, methods that are capable of detecting pork by using DNA barcode, polymerase chain reaction (PCR)-restriction fragment length polymorphism, conventional PCR, real-time PCR and isothermal amplification are discussed. Although PCR is the most popular method, recent studies have shown that isothermal amplification is a potential alternative because it is rapid, simple and does not require the use of any complicated instruments, such as a thermal cycler and sequencer.

Gene amplification technology is essential in the fields of diagnostic medicine. The polymerase chain reaction (PCR) is central in the molecular studies and provides ways for diagnostic advancement in the areas. However, the requirement for thermal cycler in a dedicated facility for amplification of target genes in the PCR technique has been a bottleneck to many researchers. The limitations associated with PCR include cost implication, strict expertise necessity and relatively higher turn-around time. The emergence of loop-mediated isothermal amplification (LAMP) in the last two decades assists in bridging the undesirable gaps. This review aims to highlight the natural advantages of the LAMP technique over the existing conventional PCR and other isothermal molecular techniques. Available published articles on LAMP techniques reviewed, listed many outstanding advances of the method in comparison to traditional PCR technique. The mentioned advantages include simplicity, affordability, naked-eye result detection and many more. That made LAMP becomes a rapidly accepted method in the field of molecular diagnosis. Other essential features of LAMP in comparison with other emerging nucleic acid amplification techniques were adequately explained and presented in tabular form for research and quick reference purposes. Though LAMP has some few limitations, its advantages outweigh its flaws by filling the gap in the field of molecular biology diagnostics.

The generation of growth curves is a common and established method for determining the properties of bacterial strains. Such properties can be growth rates in enrichment broths or resistance to antibiotics. Enrichment methods are the gold standard for enumerating bacteria and determining growth properties. Newer methods can use agents that are metabolized during bacterial growth, which leads to a shift in fluorescence. This principle is used, for example, to measure viable bacterial cells. In this work, a real-time thermal cycler was used to cultivate bacteria and measure the change in fluorescence during bacterial growth to determine the generation time of different bacterial strains. The ability of the real-time diagram enables a significant simplification and increase in cost efficiency in the generation of bacterial growth curves. It was also possible to monitor the susceptibility of bacteria to antibiotic resistance, which opened another important application of this technique.

Sclerotinia sclerotiorum is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of S. sclerotiorum can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of S. sclerotiorum (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of S. sclerotiorum and assist in pathogen management and control.

The efficiency of selection and breeding work in animal husbandry depends largely on the accuracy of the evaluation of the breeding value of animals. The integration of molecular genetics into applied science made it possible to assess the genetic potential of animal productivity not only on the basis of phenotype data but also directly at the DNA level. The study was conducted on the basis of Tatar Scientific Research Institute of Agriculture, FRC Kazan Scientific Center, Russian Academy of Sciences. The object of the study was DNA samples obtained from the blood of Holstein cows belonging to the stud farm “named after Lenin” of Atninsky district of the Republic of Tatarstan. Animal blood was taken from the tail vein using vacutainer tubes with 100mM EDTA. DNA was isolated from the blood in the amount of 100 µl using a set of reagents for DNA extraction from the “Ampliprime DNA-sorb-B” clinical material (NextBio, Russia), according to the method presented by the producer. DNA fragment amplification was performed in T100 Thermal Cycler (Bio-Rad, USA) and on MyCycler programmable Thermal Cycler (Bio-Rad, USA). After amplification, each DNA fragment obtained by us in the study of TG5, CSN3, LGB genes, was subjected to cleavage by a restriction endonuclease. Hydrolysis was conducted at 37°C for 12 hours. The analysis of polymorphism of candidate genes of dairy productivity of leptin, thyroglobulin, kappa casein and beta-lactoglobulin with the use of PCR-RFLP was carried out. The most common were homozygous TG5CC, CSN3AA and heterozygous LEPCT, LGBAB genotypes. The study of the effect of polymorphism of these genes on milk production showed that animal carriers of LEPCT, TG5TT, CSN3AB, LGBBB genotypes have the best indicators of dairy productivity.

From simple 2-dimensional (2D) structures to highly complex 3-dimensional (3D) structures, DNA is widely used as a building block material. Despite this advancement, mass production of DNA nanostructures was a challenge due to the high cost that accompanies the preparation of diverse DNA strands. Rolling circle amplification (RCA) is one of the cost-effective methods for the mass production of repetitive DNA structures without using an expensive laboratory thermal cycler. Here, we report a DNA hydrogel containing various single-stranded DNA nanostructures (Y, X, Tetrahedron) in a one-pot system, prepared by RCA, and their selective separation with various restriction enzymes. Each DNA nanostructure in the DNA hydrogel contained distinctive restriction sites that were responsive to different kinds of restriction enzymes, achieving selective separation of Y-, X- and Tetrahedron-shaped DNA structures. This study has the potential to contribute towards DNA-based biomedical applications by employing mass production and precisely controlled release of DNA nanostructures.

Astronauts face serious health threats during spaceflight. The two major factors that may lead to an astronauts' physiological dysfunction are space radiation and microgravity. Heavy ion radiation, one of the important components of charged particles in orbit, can cause deoxyribonucleic acid (DNA) damage and gene mutations. Microgravity can also affect a series of cell physiology functions, including cytoskeleton remodeling, DNA modification, interactions between molecules, etc. Several ionizing radiation experiments performed in our previous study suggested the variation in the mutation across different selected immune genes. Here, we performed a further experiment (IGM-BIT-1) on board the International Space Station (ISS) to explore the molecule evolution rules of the selected DNA. To conduct the on-orbit amplification of the DNA fragments from the antibody encoding genes in the ISS, a self-developed portable and programmable PCR device was designed and produced. We developed a novel PCR chip that consists of a multi-channel optical adhesive reaction chamber and a miniature thermal cycler. The reaction chamber was cost effective and disposable. The thermal cycler was used to achieve both rapid heating and cooling. As the DNA amplification yield of IGM-BIT-1 PCR device was much similar or even higher than the commercial devices, the IGM-BIT-1 payload has been proven to be suitable for space life science research.

In this paper, we consider the creation and testing of a prototype for recording fluorescence from microfluidic chips during the polymerase chain reaction (PCR). The paper presents the characteristics of the main elements used to create the layout of the device for fluorescence detection. The results of experiments in testing the performance of mock-up elements and microfluidic chips are presented. The operability of the assembled layout was demonstrated during the real-time PCR reaction., В данной работе рассматривается создание и проверка на работоспособность макета для регистрации флуоресценции от микрофлюидных чипов при проведении полимеразной цепной реакции (ПЦР). В работе приведены характеристики основных элементов, использованных при создании макета устройства для детектирования флуоресценции. Представлены результаты экспериментов при проверке работоспособности элементов макета и микрофлюидных чипов. Продемонстрирована работоспособность собранного макета при проведении ПЦР реакции в реальном времени.

Herein, a pipette-tip-enabled digital nucleic acid analyzer for high-performance COVID-19 testing is demonstrated. This is achieved by digital loop-mediated isothermal amplification (digital LAMP or dLAMP) using common laboratory equipment and materials. It is shown that simply fixing a glass capillary inside conventional pipette tips enables the generation of monodisperse, water-in-oil microdroplets with benchtop centrifugation. It is shown that using LAMP, the ORF1a/b gene, a standard test region for COVID-19 screening, can be amplified without a thermal cycler. The amplification allows counting of fluorescent microdroplets so that Poisson analysis can be performed to allow quantification with a limit of detection that is 1 order of magnitude better than those of nondigital techniques and comparable to those of commercial dLAMP platforms. It is envisioned that this work will inspire studies on ultrasensitive digital nucleic acid analyzers demanding both sensitivity and accessibility, which is pivotal to their large-scale applications.

Quantifying pathogenic genes with q-PCR in complex samples to determine the pathogen loads is influenced by a wide range of factors, including choice of extraction method, standard curve, and the decision to use relative versus absolute quantification of the genes. The aim was to investigate the standardisation of q-PCR methods to determine enumerated E. coli gene ratios grown with the IDEXX Colilert® Quanti-Trays® using enteropathogenic E. coli as the model pathogen. q-PCR targeting the eaeA and gadAB genes was used to calculate the eaeA: gadAB ratios for clinical strains collected between [2005–2006 (n = 55)] and [2008–2009 (n = 19)] using the LinRegPCR software and Corbett Research Thermal cycler software. Both programs grouped the isolates into two distinct groups based on the gene ratios although the Corbett Research Thermal cycler software gave results one log higher than the LinRegPCR program. Although the eaeA: gadAB ratio range was determined using extracted E. coli DNA, the impact of free DNA and other bacteria present in the sample needed to be understood. Standard curve variations using serially diluted extracted E. coli DNA, serially diluted pure E. coli culture followed by DNA extraction from each dilution with or without other bacteria was tested using the eaeA q-PCR to quantify the genes. Comparison of the standard curves showed no significant difference between standard curves prepared with diluted DNA or with cells diluted before the DNA is extracted (P = 0.435). Significant differences were observed when background DNA was included in the diluent or Coliform cells added to the diluent to dilute cells before the DNA is extracted (P < 0.001). The “carrier” DNA and Coliform cells enhanced the DNA extraction results resulting in better PCR efficiency. This will have an influence on the quantification of gene ratios and pathogen load in samples containing lower numbers of E. coli.

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.

Covid-19 is pandemic to which the world is fighting. Various precautionary measures are being imposed all over the world which is affecting the routine life of an individual and also the economy worldwide. Although, a definite vaccine is still not known to medical science but they are able to distinguish Covid-19 from the other types of flu. Presently this is being done by detecting the SARS-CoV-2 virus using RT-PCR technique as recommended by the World Health Organization (WHO) (WHO, Geneva, 2020). Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a nucleic acid amplification test that converts the RNA into DNA and subsequently amplifies the specific DNA targets. This method was already being employed to detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (Emery et al. in Emerg Infect Dis 10(2):311, 2004). The entire process of DNA amplification takes place in three steps: denaturation, annealing, and extension for which the sample is required to be maintained at constant temperatures of 95, 55 and 72 °C, respectively. This article introduces the technology to achieve a constant temperature which can be tweaked to develop on-chip RT-PCR.

Background Porcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples. Results This assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 102 copies/μL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97). Conclusions These results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

We report improvements on the heat transfer and on the control strategy of a thermal cycler implemented on a novel device for performing a fast polymerase chain reaction (PCR). The reduction of the thermal mass of the sample holder and the direct contact with the sample allow for a significant reduction of the transition times, while the hybrid feedforward/feedback controller can rely on inexpensive components (actuators, sensors, processor) and still achieve minimal over/undershooting and good temperature stability. The design of the device has been improved by performing transient heat conduction analysis on the highly heat-conductive sample holder and on the solid metal body of the device which rapidly dissipates the excess heat produced during the thermal cycling. In the current setup, the sample holder hosts nine 1 μL samples covered with mineral oil, which can be simultaneously read in real time by the detection module designed in-house and installed on the device. An increase in speed of the PCR amplification was achieved with a reduction of the transition time of 63.8% when compared against a commercial real-time PCR machine. Our work shows that a complete, stand-alone, and ready-to-use quantitative PCR instrument can be fabricated from inexpensive and easily available components and it can achieve fast thermal cycling thanks to a hybrid control strategy.

Analysis of frequency and structure of paternity exclusions in the material collected by the Department of Forensic Medicine, Medical University of Bialystok in 2008-2017.The paper is based on paternity test reports involving alleged father-child-mother trios. In a total of reviewed 958 cases, 187 exclusions were identified. The analysis was carried out on the basis of the results of DNA tests. DNA extraction was performed using QIAamp DNA Mini Kit (Qiagen) and DNA quantitation using Quantifiler Human DNA Quantification Kit and 7500 Real-Time PCR System (Applied Biosystems). AmpFLSTR Identifiler PCR Amplification Kit and a PCR System 9700 thermal cycler (Applied Biosystems) were used for DNA amplification.Over the analyzed period, the number of paternity tests was nearly halved, whereas the percentage of exclusions in individual years varied significantly (33.9-13.3%), with the average of 26.3%. The highest efficiency of exclusions was observed for D18S51 (0.7166) and FGA (0.7059), and the least effective system was TPOX (0.3048).The applied set of markers has been demonstrated to be an efficient tool in genetic paternity tests in the context of the recommended rules of exclusion.Celem pracy była analiza częstości i struktury wykluczeń ojcostwa na podstawie materiału zgromadzonego w Zakładzie Medycyny Sądowej Uniwersytetu Medycznego w Białymstoku w latach 2008–2017.Dokonano analizy dokumentacji obejmującej wyniki testów ojcostwa w trójkach: domniemany ojciec–dziecko–matka. Ocenie poddano 958 spraw, wśród których 187 stanowiły wykluczenia. Analiza opierała się na wynikach badań, w czasie których dokonano ekstrakcji DNA przy użyciu zestawów QIAamp DNA Mini Kit (Qiagen) i oznaczenia ilościowego DNA za pomocą zestawu Quantifiler Human DNA Quantification Kit w aparacie 7500 Real-Time PCR System (Applied Biosystems). Próbki DNA amplifikowano w zakresie markerów zestawu AmpFLSTR Identifiler PCR Amplification Kit w termocyklerze PCR System 9700 (Applied Biosystems).W analizowanym okresie zanotowano blisko dwukrotny spadek liczby testów ojcostwa, natomiast odsetek wykluczeń w poszczególnych latach ulegał znacznym fluktuacjom (33,9–13,3%), osiągając średnią 26,3%. Najwyższą obserwowaną skuteczność wykluczania wykazały układy D18S51 (0,7166) i FGA (0,7059), najniższą – TPOX (0,3048).Zastosowany zestaw markerów okazał się skutecznym narzędziem w genetycznych testach ojcostwa w kontekście przyjętych reguł wykluczania.

Highlights • NxTAG chemistry offers an easy workflow, low hands-on-time and scalable throughput. • All post-extraction assay steps take place in a single, closed tube. • Clinical performance of NxTAG RPP is comparable to that of the xTAG RVP assay. • Analytical performance is also similar or better than xTAG and xTAG FAST assays., The Luminex® NxTAG® Respiratory Pathogen Panel (NxTAG RPP) is an IVD-cleared assay for the simultaneous detection and identification of nucleic acids from 18 respiratory viruses and 2 (or 3 outside of the U.S.) atypical bacterial pathogens in nasopharyngeal swabs. Its scalability allows concurrent testing of up to 96 samples in a single batch. Nucleic acid extracted from 200 µL of raw specimen using the easyMAG® extractor is added directly to pre-plated, lyophilized bead reagents (LBRs), where multiplexed RT-PCR and hybridization to MagPlex-TAG™ microspheres occurs within a sealed reaction well using a single cycling program. Data acquisition is done on the MAGPIX® instrument which reads and sorts the reaction products directly from the sealed well following transfer of the assay plate from the thermal cycler. NxTAG is the newest innovation in bead-based nucleic acid chemistry developed by Luminex. Here we provide the detailed assay protocol and present data which describe the clinical and analytical performance characteristics of NxTAG RPP.

Microfluidic polymerase chain reaction (PCR) has been of great interest owing to its ability to perform rapid and specific nucleic acid amplification and analysis on small volumes of samples. One of the major drawbacks of microfluidic PCR is bubble generation and reagent evaporation, which can cause malfunctions. Here, through theoretical modeling and characterization of bubble behavior, we propose a bubble-free microfluidic PCR device via controlled fluid transfer. Our approach exploits a thin impermeable polyethylene (PE) top layer that minimizes the generation of bubbles by inhibiting mass transport along a vertical direction. Simulation results demonstrate that a calculated mass flow difference of approximately 370% can be obtained by utilizing an impermeable membrane as the vertical barrier layer. To demonstrate proof-of-concept, two nanoporous polymeric materials, poly(dimethylsiloxane) (PDMS) and PE, were used for stand-alone self-powered sample loading (approximately 70 s) and for use as a vertical barrier layer, respectively. Consequently, we demonstrate successful amplification of the cMET gene, a nucleic acid (NA) biomarker for lung cancer, and complete an ultrafast PCR test in less than 3 min using a high powered Peltier-based thermal cycler under bubble-free conditions. This approach will result in a new paradigm for ultrafast molecular diagnosis and can facilitate NA-based nearly instantaneous diagnostics for point-of-care testing and for personalized and preventive medicine.

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring.

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (

Introduction. The evolving SARS-CoV-2 coronavirus pandemic presents a series of challenges to clinical diagnostic services. Many proprietary PCR platforms deployed outside centralised laboratories have limited capacity to upscale when public health demands increase. We set out to develop and validate an open-platform mobile laboratory for remote area COVID-19 diagnosis, with a subsequent field trial. Gap Statement. In regional Western Australia, molecular diagnostic support is limited to near point-of-care devices. We therefore aimed to demonstrate open-platform capability in a rapidly deployable format within the context of the COVID-19 pandemic. Methodology. We compared, selected and validated components of a SARS-CoV-2 RT-PCR assay in order to establish a portable molecular diagnostics laboratory. The optimal combination of PCR assay equipment, reagents and consumables required for operation to national standards in regional laboratories was identified. This comprised RNA extraction and purification (QuickGene-480, Kurabo, Japan), a duplex RT-PCR assay (Logix Smart COVID-19, Co-Diagnostics, USA), a Myra liquid handling robot (Biomolecular Systems, Australia) and a magnetic induction thermal cycler (MIC, Biomolecular Systems). Results The 95 and 99% limits of detection were 1.01 copies μl−1 (5.05 copies per reaction) and 2.80 copies μl−1 (14.00 copies per reaction) respectively. The Co-Diagnostics assay amplified both SARS-CoV-1 and −2 RNA but showed no other cross reactivity. Qualitative results aligned with the reference laboratory SARS-CoV-2 assay (sensitivity 100% [95 % CI 96.48–100%], specificity 100% [95% CI 96.52–100%]). In field trials, the laboratory was operational within an hour of arrival on-site, can process up to 36 samples simultaneously, produces results in two and a half hours from specimen reception, and performed well during six consecutive runs during a 1 week deployment. Conclusion. Our mobile laboratory enables an adaptive response to increased test demand, and unlike many proprietary point-of-care PCR systems, rapid substitution with an alternative assay if gene targets change or reagent supply chains fail. We envisage operation of this RT-PCR assay as a standby capability to meet varying regional test demands under public health emergency operations guidance.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies&rsquo, MinIONTM sequencer and miniPCR bio&rsquo, s miniPCR&trade, thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods, however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

The gold standard for the diagnosis of SARS-CoV-2, the causative agent of COVID-19, is real-time polymerase chain reaction (PCR), which is labor-intensive, expensive, and not widely available in resource-poor settings. Therefore, it is imperative to develop novel, accurate, affordable, and easily accessible assays/sensors to diagnose and isolate COVID-19 cases. To address this unmet need, we utilized the catalytic potential of peroxidase-like DNAzyme and developed a simple visual detection assay for SARS-CoV-2 RNA using a conventional thermal cycler by the PCR-induced generation of DNAzyme sensor. The performance of RT-PCR DNAzyme-based sensor was comparable to that of real-time PCR. The pilot scale validation of RT-PCR DNAzyme-based sensor has shown ~100% sensitivity and specificity in clinical specimens (nasopharyngeal swab, n = 34), with a good correlation (Spearman r = 0.799) with the Ct-value of fluorescence probe-based real-time PCR. These findings clearly indicate the potential of this inexpensive, sensitive, and specific molecular diagnostic test to extend our testing capabilities for the detection of SARS-CoV-2 to curtail COVID-19 transmission.

Acute hepatopancreatic necrosis disease (AHPND) is currently the most important bacterial disease of shrimp that has caused enormous losses to the shrimp industry worldwide. The causative agent of AHPND are Vibrio spp. Carrying plasmids containing the pirA and pirB genes which encode binary toxins, PirAB. Currently, AHPND is mostly diagnosed by PCR-based platforms which require the use of sophisticated laboratory instrumentation and are not suitable for a point-of-care diagnostics. Therefore, the availability of an alternative method based on isothermal amplification would be suitable for AHPND detection outside a laboratory setting and extremely useful at a pond side location. Isothermal amplification is based on the nucleic acid amplification at a single temperature and does not require the use of a thermal cycler. In this study, we developed an isothermal Recombinase Polymerase Amplification (RPA) assay for AHPND detection targeting both pirA and pirB genes, simultaneously and evaluated the specificity and sensitivity of the assay. The assay could detect AHPND without any cross-reaction with other microbial pathogens and Specific Pathogen Free (SPF) shrimp. The limit of detection of the assay was 5 copies of pirAB genes. To evaluate the reliability of the assay in detecting AHPND, DNA from Penaeus vannamei shrimp displaying acute and chronic infection were analyzed by the RPA assay and the results were compared with SYBR Green real-time PCR assay. While there was a 100% conformity between the two assay while detecting acute phase infection, RPA appeared to be more sensitive in detecting chronic phase infection. The data suggest that RPA assay described here would be a reliable method in detecting AHPND outside a standard laboratory setting.

Background Pathogenic bacteria cause significant economic damages in agriculture. The detection of such bacteria is considered as a continual interest for plant pathologists to prevent disease dissemination. Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting yield and quality of stone fruits throughout the world. Biochemical assays such as a LOPAT and GATTa are common methods to detect this pathogen. Serological tests and culturing on King’s B selective medium also used to isolate this bacterium. Selective media is composed of specific and effective ingredients to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow. These are used for the growth of only selected microorganisms. King’s B medium can be used as a general medium for the non-selective isolation cultivation and pigment production of Pseudomonas species from foods, cosmetic samples, plants, etc. Nevertheless, the mentioned methods are not enough accurate to differentiate the strains. On the other hand, PCR-based techniques are sensitive and efficient in detecting plant diseases. However, these techniques are not practicable for those researchers who do not have access to a thermal cycler. We have used loop-mediated isothermal amplification to couple with a target. The amplification of syrD gene using loop and bumper primers can be used to prevent disease dissemination. Results The outcome of this investigation indicated more sensitivity of LAMP in comparison to PCR. The direct addition of SYBR Gold in microtube is more sensitive than gel in both LAMP and PCR byproducts so we can eliminate gel electrophoresis, while the LAMP showed high sensitivity and high specificity in comparison to results obtained by cultivation. The described molecular test could detect Pseudomonas syringae pv. syringae type in nearly 1 h, and this is the first time that Lamp molecular detection of Pseudomonas syringae pv. syringae particularly on stone fruits is described and introduced. Conclusions The obtained data confirmed that LAMP is a fast, cheap, and high specific method for the rapid detection of Pseudomonas syringae pv. syringae to the comparison of PCR and culture.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

The rapid growth of in vitro nucleic acid replication has offered a powerful tool for clinical diagnosis, food safety detection and environmental monitorning. Successful implementation of various isothermal nucleic acid amplification methods enables rapid replication of target sequences without the participant of a thermal cycler. Point-of-need analysis possesses great superiorities in user-friendly, instant results analysis, low manufacturing, and consumable costs. To meet the great challenge of point-of-need analysis, developing simple and rapid visual methods becomes crucial. Submicro- and nanomaterials possess unique surface properties, which enables their rapid response to DNA amplicons. Their unique optical, magnetic, catalytic, and other physical/chemical properties have been frequently employed for the visual detection of in vitro nucleic acid replications. Herein, we aim to review the submicro- and nanomaterials-based visual methods for detection of nucleic acid amplification. The visual methods are classified according to the designing strategies (e.g. LSPR, bridging flocculation, luminescence, catalytic reaction, separation, etc.). The basic principles, merits and drawbacks of each strategy are described. The application in analysis of nucleic acid targets and non-nucleic acid targets are discussed. The main challenges and future research directions are also highlighted in this rapidly emerging field.

Background Alpha-1-antitrypsin deficiency (AATD) is one of the most common hereditary disorders occurring in populations of European origin and is due to variants in SERPINA1, which encodes a protease inhibitor of neutrophil elastase, limiting lung damage from this enzyme. The World Health Organization has recommended that individuals with chronic obstructive pulmonary disease and asthma be tested for AATD. The development of inexpensive and simple genetic testing will help to meet this goal. Methods Primers and synthetic SERPINA1 gene fragments (gBlocks) were designed for 5 AATD-associated variants. PCR was run on a CFX96 Thermal Cycler with High Resolution Melting (HRM) capacity and data analyzed using the supplied HRM-analysis software. Genomic DNA from individuals (n = 86) genotyped for the S and Z variants were used for validation. HRM-analysis was performed on 3 additional samples with low alpha-1-antitrypsin levels inconsistent with the genotype determined in our clinical laboratory. Results Unique normalized melt curve and difference curve patterns were identified for the AAT variants Z, S, I, F, and MMalton using gBlocks. Similar curve shapes were seen when these primers were used to analyze the gDNA samples. HRM identified the genotypes of the gDNA correctly with 100% concordance. The curve shapes of some samples did not match the melting patterns of the targeted variant. Sequencing was used to identify the variants, including rare AATD variants c.1108_1115delinsAAAAACA (p.Glu370Lysfs*31) and c.1130dup (p.Leu377fs). Conclusion We developed a rapid and inexpensive HRM-analysis method for genotyping of Z, S, MMalton, I, and F variants that was also capable of detecting other variants.

A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed to detect the Potato virus S (PVS) in potato. Two sets of six novel primers that recognize the coat protein gene sequence of the PVS were designed and RT-LAMP assay was optimized for the parameters such as different concentrations of primers, MgSO(4), betaine, dNTPs, Bst DNA polymerase, temperature and duration. The RT-LAMP was carried out under isothermal conditions without the thermal cycler using PVS infected leaf and tuber samples, LAMP specific primers with amplification at 65 °C for 60 min, and 80 °C for 5 min. The results were assessed by gel electrophoresis and visual observation of colour change using SYBR Green I dye. The detection limit of the developed RT-LAMP assay was determined and compared with a conventional reverse transcription-polymerase chain reaction (RT-PCR). RT-LAMP was found 100 times more sensitive than RT-PCR. The optimized RT-LAMP assay is robust, reliable, sensitive and convenient for the detection of the PVS in infected potato tubers including asymptomatic plants. No cross-reactions were observed with healthy plants and other potato viruses. The assay is economical and can be employed in large scale testing of potato plants against PVS under healthy seed potato production programme.