Conventional quantification of L. helveticus, in presence of other lactobacilli species, using classical plate method employing low selective media is very inaccurate. Determination of L. helveticus using quantitative PCR (qPCR) was performed in six artisanal Kazakh soft cheeses made from cow’s milk or from a mixture of cow’s and goat’s milk. L. helveticus was quantified by species-specific qPCR, monitoring the presence of genes encoding for peptidoglycan hydrolases. Quantification of L. helveticus based on qPCR ranged from 2.6×106 to 4.1×108 CFU·g−1 according to the type of the cheese. The microflora of cheese consisted of a mixture of starter and non-starter lactic acid bacteria.
Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.
Objective of this study was to assess the quantification of osteocalcin (OCN) expression by ovine osteoblasts cultured with different concentrations of sodium fluoride (F) and sodium selenite (Se) to evaluate the interaction of these agents on OCN expression in vitro . We wanted to demonstrate a possible protective effect of selenium on the toxic effect of fluoride. Osteoblasts were isolated by complete trypsin and collagenase digestion from ovine calvarial bone and cultured in DMEM supplemented with 15% FBS at 37 °C in a humidified 5% CO 2 incubator. Identified osteoblasts were divided into one control group (C) and eight experimental groups, which were exposed to different concentrations of sodium fluoride (F; 0, 0.5, 1 mM) sodium selenite (Se; 0, 0.1, 1 μM). At different time points after treatment total RNA was extracted and reverse transcribed into first-strand cDNA. OCN mRNA was indirectly measured by real-time fluorescent quantitative PCR (qPCR). OCN mRNA expression in F 1 mM with Se 1 μM group was found to have a high peak at day seven and was lower before and afterwards. Expression of OCN mRNA in all groups except control could be promoted by F and/or Se showing a general upregulation. Furthermore, the toxicity from excessive exposure of osteoblast with F could be circumvented by usage of moderate concentration of Se. Osteoblasts cultured in vitro may have stressful responses to F and Se at the first few days. Low concentrations of Se inhibit the toxic effects of high concentrations of F. Therefore, F and Se could be used as antagonistic factors, which could regulate osteocalcin expression.
Zymoseptoria tritici, a globally distributed pathogen, is responsible of Septoria tritici blotch (STB), one of the most damaging wheat diseases. In Italy the incidence of STB has increased during the past few years. The presence of Z. tritici on flag leaves of susceptible durum wheat plants, cultivar San Carlo, after a single artificial inoculation with two inoculum concentrations at different vegetative stages has been evaluated in the plain of Bologna (North of Italy), in a two year field study (2012–2013). The pathogen presence was also assessed in natural infection conditions after a fungicide application in the second year (2013). The results obtained, by visual examination (Incidence, Disease Severity) and DNA quantification by Real time PCR, demonstrated that BBCH 39 (flag leaf stage) is the most susceptible vegetative stage, independently of inoculum concentration and climatic conditions. A good correlation between Disease Severity and DNA quantity was observed in either sampling methods, entire flag leaves and flag leaf discs. Thereafter the most suitable period to obtain the best crop protection with only one fungicide treatment is the flag leaf stage.
Saprophytic microflora and non-toxin producing Microdochium spp. capable of causing Fusarium head blight (FHB) have been suggested to affect the development of FHB caused by Fusarium spp., the occurrence of mycotoxins and the efficacy of fungicides for the control of the disease. The effects of metconazole and azoxystrobin on the interactions between Fusarium culmorum and Microdochium spp., Alternaria tenuissima or Cladosporium herbarum on FHB symptom development, Tri5 DNA concentration and deoxynivalenol (DON) production were studied under glasshouse conditions. Results indicated that the sequence of infection of wheat heads and the relative timing of fungicide application can significantly affect FHB severity and the resulting mycotoxin contamination of harvested grain. Introduction of A. tenuissima, C. herbarum or Microdochium spp. to wheat heads at GS 57 before inoculation with F. culmorum at GS 65 generally resulted in increased FHB severity, Tri5 DNA and DON concentration in harvested grain. The greatest increases of FHB severity (266%), Tri5 DNA (79%) and DON (152%) were observed when Microdochium spp. were introduced first at GS 57 and F. culmorum inoculation followed at GS 65. Metconazole generally reduced FHB severity, Tri5 DNA and DON concentration in grain but azoxystrobin was most efficient at reducing DNA of Microdochium spp. in grain.
Porcine circoviruses (PCV) are widespread in domestic pigs worldwide and there is growing information about the presence of PCV in other suid species. Based on serological studies with sera of wild boars, it was established that PCV1 was present in these animals and antibodies specific to PCV2 were also detected in wild boars living in captivity or in sylvatic areas, both with or without clinical signs of PMWS. Studies including PCV2 genome or antigen detection confirmed the previous findings. This is the first report about the presence of PCV in Transylvanian wild boar populations. Four hundred and sixty-nine samples were collected and grouped according to geographic origin, tested for the presence of PCV DNA using a real-time quantitative polymerase chain reaction assay, and 13.52% of the animals proved to be positive for one or in three cases both of the PCV genotypes. PCV2 was detected in all of the PCV-positive samples.
fraction was observed. In the case of serum with immunoreactivity against some bands considered non-diagnostic of low molecular weight between 20 and 46 kDa, these bands were classified as indeterminate. A quantitative real-time PCR (qPCR) was used
gene of Campylobacter jejuni , Twhip2 gene of T. whipplei and invA gene of Salmonella spp. [ 17 ]. Identification of resistance genes Real-time PCR (qPCR) amplifications were carried out using a C1000 Touch™ Thermal Cycle (Bio-Rad, USA) with
PCR (RT-qPCR) is a standard tool for gene expression profiling. Several factors may hinder the accuracy of RT-qPCR assays, such as fluctuations in RNA integrity, inter-sample variation ( Taylor et al., 2019 ). Reference genes (RGs) allow accurate RT-qPCR
, and the rats in each group were sacrificed and the skulls were opened to take out the hippocampus. Collected tissues were snap frozen and stored in a −80 °C refrigerator or liquid nitrogen. qPCR detection