dPCR: A Technology Review Quan, Phenix-Lan; Sauzade, Martin; Brouzes, Eric
Sensors (Basel, Switzerland),
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.
Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox ...transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.
•A new mpox detection method based on mpox virus specific sequences.•The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction).•New multiplex real-time PCR system allows simultaneous and efficient detection of mpox and orthopoxvirus infections.
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.
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.
To date, four species of porcine circoviruses (PCVs), including PCV1‐4, have been reported to exist in the clinical cases. Fast and effective differential detection is critical to monitor the ...infection and co‐infection status of PCVs for adopting reliable control strategies. However, currently available methods cannot simultaneously differentiate the four species of PCV strains. In this study, a quadruplex real‐time PCR assay based on TaqMan probes was developed for differential detection of PCV1‐4. The new quadruplex real‐time PCR assay exhibited satisfied specificity, sensitivity, repeatability and reproducibility. In addition, the new assay was applied to the detection of 120 clinical samples collected from 2016 to 2020 in Jiangsu province of China and compared with previously reported PCV1‐4 singleplex conventional PCR assays. Based on the clinical performance, the results from the quadruplex real‐time PCR and conventional PCR assays showed 100% agreement. A total of 47 samples were detected as PCV positive by the quadruplex real‐time PCR assay, including 1, 2, 1 samples were co‐infected with PCV1 and PCV4, PCV2 and PCV3, PCV2 and PCV4, respectively. Full‐length ORF2 sequencing and phylogenetic analysis supported the real‐time PCR results that 5, 34, 8 and 4 of the 51 PCV sequences were PCV1, PCV2, PCV3 and PCV4, respectively. This study provides a promising alternative tool for rapid differential detection of PCVs and confirms the coexistence of all species of PCV1‐4 strains in Jiangsu province in recent years.
Insecticides are extensively exploited by humans to destroy the pests one such compound thiamethoxam is widely used over crops to offer control over wide-array of sucking insect pests. The present ...study unravels the detoxification potential of Pseudomonas putida in thiamethoxam exposed B. juncea seedlings. The thiamethoxam application curtailed the fresh weight, dry weight and seedling length by 106.22%, 80.29% and 116.78% while P. putida revived these growth parameters in thiamethoxam exposed B. juncea seedlings by 59.65%, 72.99% and 164.56% respectively. The exogenous supplementation of P. putida resuscitated the photosynthetic efficiency of B. juncea seedlings exposed to thiamethoxam as total chlorophyll, chlorophyll a, chlorophyll b, carotenoid, flavonoid and anthocyanin contents were enhanced by 169.42%, 62.90%, 72.89%, 78.53%, 47.36% and 515.15% respectively in contrast to TMX exposed seedlings. Further, P. putida pre-treatment reinvigorated the osmoprotectant content in B. juncea seedlings grown in thiamethoxam as trehalose, glycine betaine and proline contents were thrusted by 21.20%, 58.98% and 34.26% respectively. The thiamethoxam exposure exorbitated the superoxide anion, hydrogen peroxide and MDA levels by 223.03%, 130.18% and 74.63% while P. putida supplementation slackened these oxidative burst levels by 41.75%, 3.79% and 29.09% respectively in thiamethoxam treated seedlings. Notably, P. putida inoculation in thiamethoxam exposed seedlings upregulated the enzymatic antioxidant and non-enzymatic antioxidant activities as SOD, CAT and glutathione were enhanced by 163.76%, 99.29% and 114.91% respectively in contrast to thiamethoxam treated seedlings. The gene expression analysis exhibited the negative impact of thiamethoxam on B. juncea seedlings as conferred by upregulation of chlorophyllase by 443.86 folds whereas P. putida application in thiamethoxam exposed seedlings downregulated the chlorophyllase expression by 248.73 folds and upregulated CXE, GST, NADH and POD genes by 0.44, 4.07, 1.43 and 0.98 folds respectively suggesting the molecular-level thiamethoxam detoxification efficiency of P. putida.
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•Thiamethoxam treatment affected the growth of Brassica juncea seedling by reducing fresh weight, dry weight, and seedling length.•Thiamethoxam exposure impaired photosynthesis, induced oxidative stress and lipid peroxidation in Brassica juncea seedlings.•Pseudomonas putida application improved photosynthetic efficiency, enzymatic and non-enzymatic antioxidant levels of thiamethoxam stressed Brassica juncea seedlings.•Application of Pseudomonas putida in thiamethoxam stressed Brassica juncea seedlings upregulated the expression of genes involved in enzyme mediated pesticide detoxification.
•Optimized Mit1C real-time PCR improves C. cayetanensis detection specificity.•Study expands real-time PCR reagent repertoire for detection in produce and water.•A new dialysis filter was optimized ...for C. cayetanensis detection in water.•Optimized protocol addresses discontinued items in ag. water testing.•High Mit1C sensitivity for C. cayetanensis in produce and agricultural water.
Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal–oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.
To prevent the invasion of exotic species causing a decline in an endangered endemic species, it is important to determine the distribution of both species at an early stage, when the density of the ...exotic species is still low, and to manage the invasion immediately. However, distinguishing between closely related species is difficult because they share similar characteristics. The identification of DNA fragments sampled from a body of water (environmental DNA) has become a popular technique for rapidly determining the distribution of a target species. In this study, we analysed environmental DNA in water samples from 37 sites across the Katsura River basin in Japan. We used TaqMan real‐time PCR to distinguish the Japanese giant salamander Andrias japonicus from the closely related Chinese giant salamander Andrias davidianus, which is known to invade Japanese rivers and hybridize with the Japanese species. In environmental samples, we detected mtDNA of the endemic species at 25 sites and mtDNA of the exotic species at nine sites. The DNA detection sites were concentrated in the upstream region. The exotic species DNA was found beyond the limits of an earlier capturing survey. Synthesis and applications. Using environmental DNA to monitor the two salamander species requires less time and effort than traditional surveys, so a wide‐ranging survey can be conducted rapidly. Our results showed that performing three environmental DNA surveys for each site between autumn and winter is desirable for giant salamanders. Further collection of environmental DNA, in combination with conventional population surveys, will provide valuable information that can help protect rare endemic species in a variety of aquatic ecosystems and can help monitor the invasion of exotic species.
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