Grapevine is a well-known and popular cultural plant that is grown on thousands of hectares worldwide. It possesses a lot of nutritional benefits but, on the other side, some of the people may suffer ...an allergic reaction when eating grapes or even in the case of consumption of the processed forms. Although grape allergy is not a common allergic reaction, the prevalence of this type of allergy is increasing mainly in southern European countries. One of the main proteins that has the potential to cause grapes allergy is chitinase. Here, a real-time PCR protocol was optimized to analyse the expression of chitinase in the mature grape berries. Different parameter such as analytic kit, primer annealing temperatures or cDNA dilution effects were tested. The final optimized protocol was proved for the chitinase unique isoform amplification by post melting analysis and testing of the protocol was performed for selected grape varieties.
The Luminex Gastrointestinal Pathogen Panel (xTAG® GPP) detects in one assay the most common gastroenteritis-causing pathogens and toxins, namely adenovirus 40/41, norovirus genogroup (NG) I/II, ...rotavirus A, Clostridium difficile toxin A/B, Campylobacter sp., Escherichia coli O157, Enterotoxigenic E. coli heat-labile enterotoxin/heat-stable enterotoxin, Salmonella sp., Shiga-toxin producing E. coli, Shiga-like toxin (Stx) 1/2, Shigella sp., Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium sp., Entamoeba histolytica and Giardia sp. In this study, we compared the results that were obtained by testing 393 faecal samples, collected during November and December 2011 at our laboratory, using the xTAG® GPP assay with the results of the routine diagnostic procedure. This procedure includes culture for bacteria and real-time PCR for viruses and parasites, but only if the test was requested by the clinician. If the clinician did not request the test for an xTAG® GPP-positive target, real-time PCR assays were used to confirm xTAG® GPP positivity. Discrepant results were also tested with real-time PCR assays. A total of 83 targets were detected in 76 samples using xTAG® GPP. The xTAG® GPP assay detected 43 additional positives compared with the routine diagnostic procedure, of which 11 targets could not be confirmed by real-time PCR. The non-confirmed targets were Campylobacter (one sample), Salmonella (four samples), Shigella (one sample) and E. histolytica (five samples). The xTAG® GPP was shown to be a convenient and sensitive assay for detection of 15 major gastrointestinal pathogens in a single molecular test, but for detection of E. histolytica and Salmonella, a confirmatory assay is indicated.
Background: Understanding the profile of antibody responses following acute COVID-19 infection is required. Aim: to describe the pattern of IgG anti-COVID-19 antibody production in patients with ...acute infection using the LABScreen COVID Plus assay. Results: The overall seropositivity was 69/73(94.5%). Anti-Spike, Spike 1 and spike S2 subunits were positive in 78.1%, while anti spike receptor binding domain (RBD) was detected in 68.4% and anti nucleocapsid protein in 61.6%. The overall positivity of the assay reached 100.0% during the second week post symptoms. The mean fluorescent intensities (MFI) of anti-Spike S1 was higher in the second week than the first week, p < /em>=0.03. MFI of anti-Spike S2 was significantly higher in PCR positive patients in comparison with the negative ones, p < /em>=0.006. When compared to the RT-PCR results; the overall antibodies positivity, anti-Spike, and anti-Spike2 antibodies had sensitivities (100% and 84.7%) and specificities (28.6% and 50.0%) and accuracies (86.3% and 78.1%). Patients' outcome correlated significantly with the time of hospital admission, p < /em>=0.001. Conclusion: COVID-19 IgG antibodies are detectable with considerable frequencies during the first two weeks post infection. Anti S2 antibodies correlates well with the RT-PCR results. The LABScreen COVID Plus is a sensitive assay for the detection of post-acute COVID-19 infection antibody responses. Background: Understanding the profile of antibody responses following acute COVID-19 infection is required. Aim: to describe the pattern of IgG anti-COVID-19 antibody production in patients with acute infection using the LABScreen COVID Plus assay. Results: The overall seropositivity was 69/73(94.5%). Anti-Spike, Spike 1 and spike S2 subunits were positive in 78.1%, while anti spike receptor binding domain (RBD) was detected in 68.4% and anti nucleocapsid protein in 61.6%. The overall positivity of the assay reached 100.0% during the second week post symptoms. The mean fluorescent intensities (MFI) of anti-Spike S1 was higher in the second week than the first week, p < /em>=0.03. MFI of anti-Spike S2 was significantly higher in PCR positive patients in comparison with the negative ones, p < /em>=0.006. When compared to the RT-PCR results; the overall antibodies positivity, anti-Spike, and anti-Spike2 antibodies had sensitivities (100% and 84.7%) and specificities (28.6% and 50.0%) and accuracies (86.3% and 78.1%). Patients' outcome correlated significantly with the time of hospital admission, p < /em>=0.001. Conclusion: COVID-19 IgG antibodies are detectable with considerable frequencies during the first two weeks post infection. Anti S2 antibodies correlates well with the RT-PCR results. The LABScreen COVID Plus is a sensitive assay for the detection of post-acute COVID-19 infection antibody responses.
•The current goat genome harbors 50 β-defensin genes.•Some duplicated β-defensin genes in goat chromosome 27 may be derived from gene copy number variation.•The annotation of sheep and cattle ...β-defensins appears to be incomplete in the genome.•Goat β-defensin 1 is the highest expressed β-defensins in the small intestine.
Defensins represent a family of cysteine-rich peptides that have broad-spectrum antimicrobial activities and serve as a typical kind of effector molecule in the immunity. Ruminant species have a large number of β-defensins in the absence of α- and θ-defensins. It is well-known that the genomes of sheep and cattle harbor at least 43 and 57 β-defensin genes, respectively. However, the repertoire of the goat β-defensin gene family has not been fully elucidated. In this study, we identified a total of 50 β-defensins from the goat genome, including 48 functional genes and 2 pseudogenes. Cross-species genomic and evolutionary analyses showed that all of the β-defensins in goat chromosomes 8, 13 and 23 present one-to-one orthologous relationships to their sheep and cattle counterparts, whereas some β-defensin genes in goat chromosome 27 are goat-specific. Moreover, we observed that some duplicated genes in goat chromosome 27 may be derived from gene copy number variation, and the annotation of sheep and cattle β-defensins appears to be incomplete in the genome. Importantly, real-time PCR analysis showed that 17 β-defensins are expressed in the small intestine with abundant cBD1s expression. These findings significant increased our knowledge of ruminant β-defensin and provided useful information for genetic studies, as well as providing a foundation for future research exploring the role of defensins in the immune response.
In the age of a pandemic, such as the ongoing one caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the world faces a limited supply of tests, personal protective equipment, and ...factories and supply chains are struggling to meet the growing demands. This study aimed to evaluate the efficacy of specimen pooling for testing of SARS‐CoV‐2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten previously tested nasopharyngeal and throat swab specimens by real‐time polymerase chain reaction (PCR), were pooled for testing, containing either one or two known positive specimens of varying viral concentrations. Specimen pooling did not affect the sensitivity of detecting SARS‐CoV‐2 when the PCR cycle threshold (Ct) of original specimen was lower than 35. In specimens with low viral load (Ct > 35), 2 of 15 pools (13.3%) were false negative. Pooling specimens to test for Coronavirus Disease 2019 infection in low prevalence (≤1%) areas or in low risk populations can dramatically decrease the resource burden on laboratory operations by up to 80%. This paves the way for large‐scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with a low incidence of infection, or with lower‐risk populations.
Highlights
Specimen pooling did not affect the sensitivity of detecting SARS‐CoV.
Pooling specimens to test for COVID‐19 infection can dramatically decrease the resource burden on laboratory operations.
Specimen pooling in samples whose cycle threshold (Ct) value is greater than 35 may yield false‐negative results.
Pooling specimens is especially useful for large‐scale population screening.
The advent of environmental DNA (eDNA) analysis methods has enabled rapid and wide‐range ecological monitoring in aquatic ecosystems, but there is a dearth of information on eDNA degradation. The ...results of previous studies suggest that the decay rate of eDNA varies depending on the length of DNA fragments. To examine this hypothesis, we compared temporal change in copy number of long eDNA fragments (719 bp) with that of short eDNA fragments (127 bp). First, we isolated rearing water from a target fish species, Japanese Jack Mackerel (Trachurus japonicus), and then quantified the copy number of the long and short eDNA fragments in 1 L water samples after isolating the water from the fish. Long DNA fragments showed a higher decay rate than short fragments. Next, we measured the eDNA copy numbers of long and short DNA fragments using field samples, and compared them with fish biomass as measured by echo intensity. Although a previous study suggested that short eDNA fragments could be overestimated because of nontarget eDNA from a nearby fish market and carcasses, the eDNA concentrations of long fragments were correlated with echo intensity. This suggests that the concentration of longer eDNA fragments reflects fish biomass more accurately than the previous study by removing the effects of the fish market and carcasses. The length‐related differences in eDNA have a substantial potential to improve estimation of species biomass.
Background
Theileria equi and Babesia caballi cause equine piroplasmosis (EP), one of the most important tick‐borne diseases of horses due to its high negative impact to the equine industry. Although ...infections with these parasites have been reported for decades in Spain, epidemiological studies have only been carried out in certain regions.
Objectives
To determine the (sero)prevalence of these parasites in asymptomatic horses nationwide in Spain and to identify potential individual and environmental factors associated with seropositivity to EP.
Study design
Sample size was calculated according to the horses registered in Spain in 2013 and by autonomous community using a random stratified sampling. A questionnaire was used to collect data on factors associated with EP seropositivity.
Methods
Serological (cELISA and complement fixation test) and molecular (real‐time PCR) analyses were carried out in 740 horses. Risk factors were identified computing two independent logistic regression models with the collated data.
Results
Antibodies against EP were detected in 42.9% (95% CI 39.4‐46.5) of horses, whereas 30.3% (95% CI 27.0‐33.6) were EP positive by PCR. A substantial strength of agreement (k = 0.721) was estimated between serological tests. Exposure to T. equi was significantly higher than to B. caballi and the highest (sero)prevalence was detected in the northern communities. Increasing horse age, presence of ticks and contact with cows were factors related to EP seropositivity in the horses, whereas tetanus vaccination and fairs attendance were associated with lower seropositivity.
Conclusions
Almost half of the horses residing in Spain had antibodies against EP or circulating parasitaemia. Appropriate prevention measures and implementation of a EP surveillance programme should be considered in order to reduce and control the infection.
Aim
Serratia marcescens is an important multidrug‐resistant human pathogen. The pathogenicity of S. marcescens mainly depends on the quorum sensing (QS) mechanism, which regulates the virulence ...factors production and biofilm formation. Hence, targeting QS mechanism in S. marcescens will ultimately pave the way to combat its pathogenicity. Thus, the present study is intended to evaluate the efficacy of Vetiveria zizanioides root extract‐mediated silver nanoparticles (AgNPs) as a potent anti‐QS and antibiofilm agent against S. marcescens.
Methods and Results
The AgNPs were synthesized using V. zizanioides aqueous root extract and the physiochemical properties of V. zizanioides‐based AgNPs (VzAgNPs) were evaluated using analytical techniques such as ultraviolet‐visible absorption spectroscopy, X‐ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering and scanning and transmission electron microscopic techniques. VzAgNPs were found to attenuate the QS‐dependent virulence factors, namely prodigiosin, protease, lipase, exopolysaccharide productions and biofilm formation of S. marcescens, without inhibiting its growth. Further, the transcriptomic analysis confirmed the down‐regulation of QS‐dependent genes, which encode for the production of virulence factors and biofilm formation.
Conclusion
The current study confirms VzAgNPs as an ideal anti‐QS and antibiofilm agent against S. marcescens.
Significance and Impact of the Study
This is the first approach that validates the anti‐QS and antibiofilm potential of phytosynthesized VzAgNPs against the nosocomial pathogen, S. marcescens. As VzAgNPs exhibits potent antivirulent activities, it could be used to treat hospital‐acquired S. marcescens infections.
Early blight (EB), caused by the pathogen
, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted ...a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the
gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified
artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure
culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting
in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.
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