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.
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.
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.
Display omitted
•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.
Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with ...no clear epidemiological links to travel or animal exposure and with ever‐increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real‐time PCR‐assay for MPXV diagnosis in humans and compare the performance of this novel assay against a Food & Drug Administration‐cleared pan‐Orthopox RT‐PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT‐PCR assay targeting the viral F3L‐gene. In addition, we further evaluated MPXV‐PCR‐positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT‐PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.
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.
PDF
