dPCR: A Technology Review Quan, Phenix-Lan; Sauzade, Martin; Brouzes, Eric
Sensors,
04/2018, Letnik:
18, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules ...in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.
•We evaluated a real-time PCR assay for diagnosing five Plasmodium and three Babesia species.•Plasmodium detection limit: 30 to 0.0003 copies/µL; mixed infections: 0.3 to 3 copies/µL.•Babesia ...detection limit: 0.2 copies/µL; no cross-reactivity in 64 microorganism DNA samples.•Assay sensitivity: 100 % for most species; P. falciparum: 97.7 %, B. microti: 12.5 %.
Malaria and babesiosis are global health threats affecting humans, wildlife, and domestic animals, particularly in Africa, the Americas, and Europe. Malaria can lead to severe outcomes, while babesiosis usually resembles a mild illness but can be severe and fatal in individuals with weakened immune systems. Swift, accurate detection of these parasites is crucial for treatment and control. We evaluated a real-time PCR assay for diagnosing five Plasmodium and three Babesia species from blood samples, assessing its sensitivity, specificity, and analytical performance by analyzing 46 malaria-positive and 32 Babesia spp-positive samples diagnosed through microscopy. The limit of detection for Plasmodium species ranged from 30 to 0.0003 copies/µL. For mixed infections, it was 0.3 copies/µL for P. falciparum/P. vivax and 3 copies/µL for P. malariae/P. knowlesi. Babesia species had a detection limit of 0.2 copies/µL. No cross-reactivity was observed among 64 DNA samples from various microorganisms. The assay showed good sensitivity, detecting Plasmodium and Babesia species with 100 % accuracy overall, except for P. falciparum (97.7 %) and B. microti (12.5 %). The low sensitivity of detecting B. microti was attributed to limitations in microscopy for species identification. This technique heavily relies on the proficiency of the examiner, as species within the genus cannot be distinguished under a microscope. Additionally, Babesia can be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. The findings support multiplex qPCR's diagnostic superiority over the gold standard, despite higher costs. It offers enhanced sensitivity, specificity, and detects mixed infections, crucial for effective monitoring and diagnosis of malaria and babesiosis in endemic regions with significant public health challenges.
Background
The use of environmental DNA analysis has revolutionized biodiversity monitoring. Initially, eDNA monitoring surveys in aquatic environments utilized a targeted approach, but there has ...been a steady shift toward whole community assessments (eDNA metabarcoding). Both approaches can increase the detection sensitivity for rare and elusive species, compared to more conventional methods. However, it is important to understand the benefits and limitations of targeted and whole community eDNA monitoring to tailor surveys to research questions and management objectives.
Aims
Here, we aimed to test the relative merits of targeted eDNA analysis versus eDNA metabarcoding in an intermittent river system.
Methods
First, samples collected during different seasons were used to assess the influence of seasonality on the detection probabilities of both methods. Second, detection probabilities from the two monitoring approaches for one focal species were compared to evaluate the sensitivity of both methods. Finally, the data from an eDNA metabarcoding survey conducted across the outer distribution limits of an invasive species were used to evaluate whether species interactions can be inferred by this method.
Results
Analyses showed that sampling intermittent river systems during low flow events increases the performance of the targeted eDNA surveys, while sampling season does not influence the performance of eDNA metabarcoding surveys. Environmental DNA metabarcoding was found to be less sensitive than a targeted monitoring approach, thus making the latter more suitable for generating detailed distribution data. Nevertheless, eDNA metabarcoding survey data can be interpreted in a semiquantitative manner and can provide insights into biological interactions.
A comparison of targeted eDNA monitoring and eDNA metabarcoding showed a higher detection sensitivity for targeted surveys. Environment DNA metabarcoding surveys can, however, be used to monitor species interaction.
MicroRNAs (miRNAs) have emerged as important regulators in the post‐transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, ...including blood, urine and cerebro‐spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma‐ and serum‐derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.
qPCR primer design revisited Bustin, Stephen; Huggett, Jim
Biomolecular detection and quantification,
12/2017, Letnik:
14
Journal Article
Recenzirano
Odprti dostop
Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, ...poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications.
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•Detection of Yersinia enterocolitica in bivalve mollusks by a reference test (ISO 10273:2017).•Validation “in house” of the molecular method employed to detect pathogenic Yersinia ...enterocolitica in bivalve mollusks.•Detection of Yersinia enterocolitica in real samples of bivalve mollusks.
Yersinia enterocolitica (Ye) is a foodborne pathogen isolated from humans, food, animals, and the environment. Yersiniosis is the third most frequently reported foodborne zoonosis in the European Union. Ye species are divided into six biotypes 1A, 1B, 2, 3, 4, and 5, based on biochemical reactions and about 70 serotypes. Biotype 1A is non-pathogenic, 1B is highly pathogenic, and biotypes 2–5 have moderate or low pathogenicity. The reference analysis method for detecting pathogenic Ye species underestimates the presence of the pathogen due to similarities between Yersinia enterocolitica-like species and other Yersiniaceae and/or Enterobacteriaceae, low concentrations of distribution pathogenic strains and the heterogeneity of Yersinia enterocolitica species.
In this study, the real-time PCR method ISO/TS 18867 to identify pathogenic biovars of Ye in bivalve molluscs was validated. The sensitivity, specificity and accuracy of the molecular method were evaluated using molluscs experimentally contaminated. The results fully agree with those obtained with the ISO 10273 method. Finally, we evaluated the presence of Ye in seventy commercial samples of bivalve molluscs collected in the Gulf of Naples using ISO/TS 18867. Only one sample tested resulted positive for the ail gene, which is considered the target gene for detection of pathogenic Ye according to ISO/TS 18867. Additionally, the presence of the ystB gene, used as target for Ye biotype 1A, was assessed in all samples using a real-time PCR SYBR Green platform. The results showed amplification ystB gene aim two samples.
Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In ...Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into “Hi” as phase 1 and “H1 or H2” as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.
•a real-time PCR was developed to analyze flagellin phase for S. typhimurium.•the real-time PCR is based on the inversion of hin gene.•the real-time PCR provides an accurate measurement for flagellin expression status.•the real-time PCR is useful for field strains as well as for laboratory strains.
There are more than 350 real‐time polymerase chain reaction (RT‐PCR) coronavirus disease‐2019 (COVID‐19) testing kits commercially available but these kits have not been evaluated for pooled sample ...testing. Thus, this study was planned to compare and evaluate seven commercially available kits for pooled samples testing. Diagnostic accuracy of (1) TRUPCR SARS‐CoV‐2 Kit (Black Bio), (2) TaqPath RT‐PCR COVID‐19 Kit (Thermo Fisher Scientific), (3) Allplex 2019‐nCOV Assay (Seegene), (4) Patho detect COVID‐19 PCR kit (My Lab), (5) LabGun COVID‐19 RT‐PCR Kit (Lab Genomics, Korea), (6) Fosun COVID‐19 RT‐PCR detection kit (Fosun Ltd.), (7) Real‐time Fluorescent RT‐PCR kit for SARS CoV‐2 (BGI) was evaluated on precharacterised 40 positive and 10 negative COVID‐19 sample pools. All seven kits detected all sample pools with low Ct values (<30); while testing weak positive pooled samples with high Ct value (>30); the TRUPCR Kit, TaqPath Kit, Allplex Assay, and BGI RT‐PCR kit showed 100% sensitivity, specificity, and accuracy. However, the Fosun kit, LabGun Kit, and Patho detect kit could detect only 90%, 85%, and 75% of weakly positive samples, respectively. We conclude that all seven commercially available RT‐PCR kits included in this study can be used for routine molecular diagnosis of COVID‐19. However, regarding performing pooled sample testing, it might be advisable to use those kits that performed best regarding positive identification in samples' pool, that is TRUPCR SARS‐CoV‐2 Kit, TaqPath RT‐PCR COVID‐19 Kit, Allplex 2019‐nCOV Assay, and BGI Real‐time RT‐PCR kit for detecting SARS CoV‐2.
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