Coronavirus disease 2019 or COVID‐19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is an ongoing pandemic that has emerging global effects ...and requires rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of these novel approaches may also contribute to faster and cheaper field‐based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS‐CoV‐2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT‐LAMP as well as by conventional q‐RT‐PCR. The patient samples were tested with four different kits (SENSObiz COVID‐19 SARS‐CoV‐2 LAMP Assay, the QIAseq DIRECT SARS‐CoV‐2 kit, Biospeedy SARS‐CoV‐2 Variant Plus kit, and CoVirion‐CV19‐2 SARS‐CoV‐2 OneStep RT‐PCR kit) and two different PCR devices (GDS Rotor‐Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor‐Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor‐Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS‐CoV‐2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.
Keypoints
Novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 or 2019‐nCoV) is a pandemic that has emerging globallyand needs rapid and reliable diagnostic testing. Quantitative reverse transcription‐polymerase chain reaction (q‐RT‐PCR) is the gold standard method for SARS‐CoV‐ 2 detection. On the other hand, reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) as one of thenovel approaches that contribute to the faster and cheaper field‐based testing on the field of COVID‐19 disease diagnosis. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30–45 min and to compare the effectiveness of LAMP to the q‐RT‐PCR.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Immuno-polymerase chain reaction (I-PCR) assay with advantages of both enzyme-linked immunosorbent assay (ELISA) and PCR exhibits several-fold enhanced sensitivity in comparison to respective ELISA, ...which has wide applications for ultralow detection of several molecules, i.e. cytokines, protooncogenes and biomarkers associated with several diseases. Conjugation of reporter DNA to the detection antibodies is the most crucial step of I-PCR assay that usually employs streptavidin-protein A, streptavidin-biotin conjugate or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) system by a covalent binding. However, coupling of antibodies and oligonucleotides to nanoparticles (NPs) is relatively easier in the NP-based I-PCR (NP–I-PCR) that also displays better accuracy. This article is mainly focused on the detection of important biomarkers associated with several outrageous infectious and non-infectious diseases by NP-I-PCR assays, which would expedite an early initiation of therapy thus human health would be improved. Similarly, ultralow detection of environmental pollutants by these assays and their elimination would certainly improve human health.
•Immuno-PCR (I-PCR) has advantages of both ELISA and PCR assays.•Coupling of detection antibodies to reporter DNA is crucial step of I-PCR assay.•Coupling of antibodies and DNA to nanoparticles (NPs) is relatively easier.•We described ultralow detection of biomarkers related with diseases by NP-I-PCR.•We also described ultralow detection of environmental pollutants by NP-I-PCR.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Quantification of DNA methylation in neoplastic cells is crucial both from mechanistic and diagnostic perspectives. However, such measurements are prone to different experimental biases. Polymerase ...chain reaction (PCR) bias results in an unequal recovery of methylated and unmethylated alleles at the sample preparation step. Post‐PCR biases get introduced additionally by the readout processes. Correcting the biases is more practicable than optimising experimental conditions, as demonstrated previously. However, utilisation of our earlier developed algorithm strongly necessitates automation. Here, we present two R packages: rBiasCorrection, the core algorithms to correct biases; and BiasCorrector, its web‐based graphical user interface frontend. The software detects and analyses experimental biases in calibration DNA samples at a single base resolution by using cubic polynomial and hyperbolic regression. The correction coefficients from the best regression type are employed to compensate for the bias. Three common technologies—bisulphite pyrosequencing, next‐generation sequencing and oligonucleotide microarrays—were used to comprehensively test BiasCorrector. We demonstrate the accuracy of BiasCorrector's performance and reveal technology‐specific PCR‐ and post‐PCR biases. BiasCorrector effectively eliminates biases regardless of their nature, locus, the number of interrogated methylation sites and the detection method, thus representing a user‐friendly tool for producing accurate epigenetic results.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Uveitis is a sight-threatening intraocular inflammatory disorder which may occur from both infectious and non-infectious or autoimmune causes. The frequency of infectious uveitis and autoimmune ...uveitis varies depending on countries and regions. According to a nationwide survey conducted by the Japanese Ocular Inflammation Society, infectious and non-infectious uveitis accounted for 16.4 and 50.1% of new patients, respectively while the remaining 33.5% of new uveitis cases were not classified or were idiopathic uveitis. Infectious uveitis is particularly important because it causes tissue damage to the eye and may result in blindness unless treated. However, it can be treated if the pathogenic microorganisms are identified promptly and accurately. Remarkable advancements in molecular and immunological technologies have been made in the last decade, and the diagnosis of infectious uveitis has been greatly improved by the application of molecular and immunological investigations, particularly polymerase chain reaction (PCR). PCR performed on a small amount of ocular samples provides a prompt, sensitive, and specific molecular diagnosis of pathogenic microorganisms in the eye. This technology has opened a new era in the diagnosis and treatment of uveitis, enabling physicians to establish new clinical entities of uveitis caused by infectious microorganisms, identify pathogens in the eyes of many patients with uveitis, and determine prompt diagnosis and appropriate therapy. Here we review the PCR process, new PCR tests specialized for ocular diseases, microorganisms detected by the PCR tests, diseases in the eye caused by these microorganisms, and the clinical characteristics, diagnosis, and therapy of uveitis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive ...(e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations.
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•Milk protein polymorphism and “A1/A2 hypothesis” (β-caseins) are highlighted.•Phenotyping of genetic variants of milk proteins in Simmental cows (n = 568) by IEF.•Genotyping of ...β-caseins variants by AS-PCR, ACRS-PCR, and PCR-RFLP techniques.•Detection of β-casein A1 in retail “A2 milk” is a deception/misleading of consumers.
Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial “A2 milk” in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled “A2 milk”) to protect consumers from this unexpected fraud.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, ...and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.
Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk ...in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC‐PCR), propidium monoazide PCR (PMA‐PCR), droplet digital PCR (ddPCR) and loop‐mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long‐term rotation study at Normandin, Québec, with replicated plots representing 0‐, 1‐, 2‐, 3‐, 5‐ and 6‐year breaks following susceptible canola infested with clubroot. CPIC‐PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC‐PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA‐PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
30.
Three-color crystal digital PCR Madic, J.; Zocevic, A.; Senlis, V. ...
Biomolecular detection and quantification,
12/2016, Volume:
10
Journal Article
Peer reviewed
Open access
Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in ...complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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