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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.

Three molecular assays were used to detect and quantify white spot syndrome virus (WSSV) in DNA extracted from seston size-fractioned (0.02, 0.2, 1.2, and 20 μm) samples collected from a coastal lagoon and an adjacent shrimp farm. From 107 DNA extracts, only two from one sample tested positive for WSSV with nested PCR in the 1.2 and 20 μm fractions. These results were confirmed by a semi-quantitative (IQ2000TM WSSV Detection and Prevention System) and a quantitative (IQREALTM WSSV Quantitative System) detection system based, based, respectively, on nested PCR and real-time PCR. A first viral load reference value (6.54 × 104 WSSV copies/mL) was established in a seston size fraction (1.2−20 μm). The results suggest that WSSV could be associated with both resuspension of fine clays and silts, and nanoplankton and organic colloids during infectious events.

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.

Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size‐fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small‐sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 µm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future. This study showed that the Japanese Jack Mackerel eDNA shedding rate increased at higher water temperatures and larger fish biomass, and the most supported model for the eDNA decay curves included both temperature and fish density as explanatory variables. In addition, eDNA size distribution varied depending on temperature, fish biomass, and time passage.

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.

Ornithobacterium rhinotracheale (ORT) has been associated with poultry respiratory disease worldwide. The organism is fastidious and isolation is challenging. One TaqMan real-time PCR (qPCR) assay has been developed for the detection of ORT. However, during validating the ORT qPCR, the assay performance was suboptimal. During the in silico evaluation, deviations from the basic parameters for primers and probes designs (e.g., presence of stable undesirable primer-dimers) were observed. The suboptimal design led to low efficiency and low sensitivity of the assay. Initially, modification on the probe was carried out to improve the performance of the assay. However, the assay’s performance (efficiency and sensitivity) was still suboptimal. In this manuscript, we describe the development of a new qPCR assay and the comparison of its performance with the currently available assay. A highly efficient, sensitive, and specific qPCR assay was developed with approximately 1000-folds reduction in the limit of detection (from 3 × 106 plasmid DNA copies/mL to 1 × 103 plasmid DNA copies/mL). Additionally, the efficiency of the new assay (E = 98.70%) was significantly better than the current assay (E = 73.18%). The newly developed assay is an improved diagnostic tool for the sensitive and efficient diagnosis of ORT from clinical samples.

The efficiency of three diagnostic methods, i.e. PCR, real-time PCR and LAMP, for detection of Xylella fastidiosa (Xf) genomic DNA from Philaenus spumarius (Ps) and Neophilaenus campestris (Nc) insect vectors was evaluated using three total nucleic acids (TNA) extraction methods (EM). In addition, a new real-time LAMP technology, Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOSLAMP), originally developed for human virus diagnoses, was optimized and assessed for detection of Xf in insect vectors. EM1 consisted of entire insects heated in an extraction buffer (EB) containing Tris-EDTA and TRITON-X100. In EM2, TNAs were extracted only from excised heads of insects, and heated again in the EB of EM1. EM3 consisted of grinding entire insects, heads and bodies recuperated from EM2, with a CTAB buffer. The molecular analyses conducted on 100 specimens of Ps and 50 of Nc, collected from a Xf-infected olive orchard (Lecce province, Italy), showed that 29% of specimens (40 Ps and four Nc) were positive to the presence of Xf. The comparison between the three methods revealed that EM3 is the most efficient for extracting Xf-genomic DNA from insect vectors, of which 44 specimens were positive for Xf in each of the diagnostic methods used, including the newly optimized FLOS-LAMP assay. In general, the real-time PCR and LAMP assays were more competent than the conventional PCR for detection of Xf in insect vectors, independently from the EM used. The newly optimized FLOS-LAMP technique had a detection limit of 1 fg [micro][L.sup.-1] of Xf-genomic DNA, compared to the 10 fg [micro][L.sup.-1] for conventional LAMP. The high sensitivity of the FLOS-LAMP was evident through the greater number of overall Xf-infected insect vectors detected (60%), compared to those for LAMP (45%,), real-time PCR (28%) and PCR (10%). FLOS-LAMP, being a more sensitive and specific assay, together with EM3, were the most appropriate approaches for an accurate detection of Xf in insect vectors. Keywords. Philaenus spumarius, Neophilaenus campestris, PCR, real-time PCR, LAMP, FLOS-LAMP

Forty nine sputum specimens were collected from patients with aspergillosis attended to four hospitals in Baghdad. The frequent species of Aspergillus identified included Aspergillus fumigatus 23(46.9%), Aspergillus niger 14 (28.6%), followed by Aspergillus flavus 12 (24.5%). According to age group factor, the age group (50-59) years appeared to be more susceptible to infected by aspergillosis with percentage at (24.5%). The results was revealed that no significant differences between male and female with aspergillosis infection. To detect A.fumigatus isolates by molecular methods, the genomic DNA were extracted and amplification to detect the aspHS gene by the singleplex PCR method using species-specific primers for these A.fumigatus, to sum up 17 of isolates from 23 isolates of A.fumigatus which identified the previous by morphological and microscopic methods, by observing the singleplex PCR product of aspHS gene with ~108 bp. The total RNA of A.fumigatus was extracted by using TRIzol purification kit and convert to cDNA and submit for further amplification to detect the Heat Shock protein 70 genes (Hsp70 genes) expression as virulence factor in variable temperature activation include 28 ºC, 37 ºC and 45 ºC by real time PCR. The results of HSP70 gene expression showed the level increased at 37 ˚C but decreased when the temperature increases to 45 ˚C. 

Aqueous environmental DNA (eDNA) analysis has been applied to the monitoring of various ecosystems and taxa, and the characteristics of aqueous eDNA have been previously studied. In contrast, although sedimentary eDNA has been used to restore past information, the characteristics of sedimentary eDNA are not well understood. In this study, we compared the properties of sedimentary and aqueous eDNA of macro‐organisms. First, to clarify the preservation ability of sediments, we compared the difference in decay rates between aqueous and sedimentary eDNA using samples collected from a biotope (an artificial pond prepared with concrete). Next, to clarify the biological information retained in sedimentary eDNA both qualitatively and quantitatively, we compared eDNA concentrations between sediment and water samples collected simultaneously from a lake, and the fish species detected by eDNA metabarcoding were also compared. The results demonstrated the following: (a) the decay rate (decreased eDNA copy number divided by the initial eDNA copy number per unit time) of sedimentary eDNA (0.00033 ± 0.000049 [mean ± SE]/hr) was lower than that of aqueous eDNA (0.01863 ± 0.0011/hr); (b) sedimentary eDNA concentration of the mitochondrial marker of three fish species was higher than aqueous eDNA concentration for the same sample weight (12.5–1,456.9 times); and (c) the species composition obtained by metabarcoding was not significantly different between sediment and water; however, considering the lower decay rate of sedimentary eDNA, using both sample types may provide more comprehensive information of species distribution. Thus, sedimentary eDNA analysis will expand future biomonitoring and ecological studies by providing a difference in timescale. We found that the degradation rate of sedimentary eDNA was lower than that of aqueous eDNA. Additionally, sedimentary eDNA concentration was higher than aqueous eDNA concentration for the same sample weight. Further, different fish species were detected in sediment and water samples by eDNA metabarcoding.