Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To ...avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR®Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii.
In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii.
Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples.
We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems.
Fusarium species, which can produce mycotoxins, are the predominant pathogens causing maize ear rot, a disease that results in severe economic losses and serves as a potential health risk for humans ...and animals. A survey was conducted in 2012 to investigate the contamination of maize by Fusarium species and fumonisins B
1
and B
2
. A total of 250 maize samples were randomly collected from nine provinces (Hebei, Shanxi, Inner Mongolia, Yunnan, Sichuan, Guizhou, Heilongjiang, Liaoning and Ningxia) in China. Fusarium species were isolated and identified using morphological (electron microscope) and molecular methods (polymerase chain reaction (PCR) and sequencing). Fumonisins B
1
and B
2
were analysed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with OPA (2-Mercaptoethanol, o-phthaldialdehyde) post-column derivatisation. A total of 2321 Fusarium isolates (20.7%) were obtained from all the samples. These isolates included nine Fusarium species, namely, F. graminearum, F. verticillioides, F. subglutinans, F. proliferatum, F. temperatum, F. oxysporum, F. equiseti, F. meridionale and F. chlamydosporum. The incidence of occurrence of Fusarium species in Guizhou was the highest, while in Inner Mongolia it was the lowest. F. verticillioides was the dominant species of maize ear rot in Liaoning, Sichuan, Hebei and Ningxia. F. graminearum was the dominant species in Yunnan, Guizhou and Shanxi. F. subglutinans was the dominant species in Heilongjiang. F. verticillioides and F. graminearum percentages were the same in Inner Mongolia. The incidence of fumonisins in Liaoning was high (up to 81.0%) and in Heilongjiang low (up to 10.3%). Except Shanxi, more than 50% of maize samples from other provinces were contaminated with fumonisins, with concentrations less than 500 ng g
−1
. About 33% of maize samples from Yunnan were contaminated with high levels of fumonisins, and average of fumonisin levels were 5191 ng g
−1
. Fusarium species causing maize ear rot in different areas in China were highly diverse and such areas with exposure to high levels of fumonisin contamination have a potential health risk for human and animals.
Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized ...into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.
The phototrophic dinoflagellate Biecheleriopsis adriatica is a small suessioid species characterized by a fragile thin wall. Although the morphology of this dinoflagellate is well established, there ...is currently little information available on its distribution and the environmental factors that influence this distribution. Thus, to investigate the spatial and seasonal distributions of the vegetative cells of B. adriatica in Korean waters, surface water samples were collected on a seasonal basis from 28 stations in the East, West, and South Sea of Korea and Jeju Island from April 2015 to October 2018, and abundances of the vegetative cells of B. adriatica were quantifled using quantitative real-time polymerase chain reactions, for which we developed the species-speciflc primer and probe set. Simultaneously, major environmental parameters, including temperature, salinity, nutrient concentrations, and dissolved oxygen concentrations were measured. The vegetative cells of B. adriatica were detected at 20 of the 28 sampling stations: 19 stations in summer and 6 in autumn, although from no stations in either spring or winter. The ranges of water temperature and salinity at sites where this species was detected were 17.7-26.4°C and 9.9-34.3, respectively, whereas those of nitrate and phosphate concentrations were not detectable-96.2 and 0.18-2.66 pM, respectively. Thus, the sites at which this species is found are characterized by a narrow range of temperature, but wide ranges of salinity and concentrations of nitrate and phosphate. The highest abundance of the vegetative cells of B. adriatica was 41.7 cells mL-1, which was recorded in Jinhae Bay in July 2018. In Jinhae Bay, the abundance of vegetative cells was significantly positively correlated with the concentration of nitrate, but was negatively correlated with salinity. On the basis of these flndings, it appears that the abundance of B. adriatica vegetative cells shows strong seasonality, and in Jinhae Bay, could be affected by the concentrations of nitrate.
Background: In recent years, use of powdered infant formula (PIF) milk for neonates feed is increasing; therefore, the quality control (QC) of PIF products is very important. The aim of present study ...was detection of toxigenic Bacillus cereus species in PIF milk using PCR assay. Methods: The cross-sectional study was carried out on 125 samples of powdered infant formula milk (PIF) purchased between March 2015 and April 2016 in Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Briefly, 0.1 dilutions were prepared and inoculated on Bacillus cereus selective media (MYP) and incubated at 30 °C for 24 hours. The suspicious colonies were verified using biochemical tests based on standard methods. Final confirmation of studied isolates was carried out by ITS gene detection using polymerase chain reaction (PCR) assay. Presence of nonhemolytic enterotoxin (NHE) (linked to diarrhoea syndrome) and emetic toxin (EM) (linked to emetic syndrome) virulence genes were investigated using polymerase chain reaction assay. Results: In this study, of 125 PIF samples, 84 (67.2%) were contaminated. Of various recovered bacteria from these samples, 110 bacterial isolates were suspected to be Bacillus spp. using phenotypic methods. The ITS PCR results showed that 91.8% of the isolates were B. cereus. Respectively, 53.63 and 79% of B. cereus isolates possessed NHE and EM virulence genes. Conclusion: Our data revealed that near 80% of Bacillus cereus isolates have emetic toxin (EM) gene, as result virulence potency of this isolates is very high. However, the low number of this organisms in foods is very important and food safety protocols for these opportunistic toxigenic bacteria should be revised. Since the pasteurization process is ineffective on B. cereus spores; therefore, spores can remain in PIF milk and the vegetative bacterial cells can cause food poisoning in neonates. Therefore, modification of foods quality control protocols is essential in order to identify virulence genes in this bacterium.
DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and ...spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis.
The pseudo-molecular method is employed to obtain analytical expressions for the elastic constants of an ensemble of anisotropic particles, in both disc-like and rod-like geometries. These particles ...interact via a phenomenological pair potential constructed from the non-spherical correction to the dispersion forces between two identical molecules. The molecular shape appears in the calculations of the elastic constants in two different cases. The first case considers a molecular volume of ellipsoidal shape continuously deformed from a positive (prolate spheroid, rod-like molecule) to large negative (oblate spheroid, disc-like molecule) values of a parameter describing some kind of eccentricity. The second one considers a molecular volume shape continuously deformed from a cylinder (calamitic molecule) to a plane disc by changing the ratio between the diameter of the cylinder and its long axis. The particular cases of Maier-Saupe and Nehring-Saupe interactions are obtained as simple limiting cases of the general pair potential interaction. These general results may be helpful to understand the limits of the pseudo-molecular method, and to understand the origin of elastic constants in discotic liquid crystals from a molecular perspective.
Introduction: The Rh system is very complex, polymorphous and the most significant for clinical practice, along with the ABO blood group system. The D antigen is the most important antigen in the Rh ...system and the most immunogenic one, following the ABO antigens. The D antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. Most people are either RhD positive or RhD negative, but there is a certain number of people who have a variation of the D antigen, which are called weak D, partial D and DEL phenotypes. Aim of the Study: The objective is to use molecular methods to determine whether blood donors in the Republic of Srpska (with whom a serological weak D antigen has been detected) really have the weak D antigen, partial D, a combination of these two variants or if their D antigen is normally present, but the used anti-D serum tests did not have the avidity needed to prove the presence of this antigen in blood donors. Patients and Methods: Blood samples were used from regular blood donors, who had been determined as persons with a weaker D antigen (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. To determine the blood groups and red blood cell/erythrocyte antigen typing, the following methods were applied: a) test tube method or agglutination in an aqueous environment, b) gel method, c) microplate method and d) molecular determination of blood groups. Results: Blood group samples were collected from April 2016 to February 2017 in the Institute for Transfusion Medicine of Republika Srpska. During this period, blood was collected from 8153 voluntary donors. It was serologically proved that 40 donors (0.49%) had the weak D antigen. All results where the weak D antigen was determined serologically were confirmed by molecular testing. 23 respondents were proved to have weak D type 3 (0.28%), while 17 had weak D type 1 (0.20%). Conclusion: The results from the first molecular testing of our population is in accordance with the results of frequency of weak D antigen in the populations of other European countries, though it did show a small advantage of weak D type 3 over weak D type 1.
This study aimed to find candidate strains of Lactobacillus isolated from sheep dairy products (yogurt and ewe colostrum) with probiotic and anticancer activity. A total of 100 samples were randomly ...collected from yogurt and colostrum and 125 lactic acid bacteria were isolated. Of these, 17 Lactobacillus strains belonging to five species (L. delbrueckii, L. plantarum, L. rhamnosus, L. paracasei, and L. casei) were identified. L. plantarum 17C and 13C, which isolated from colostrums, demonstrated remarkable results such as resistant to low pH and high concentrations of bile salts, susceptible to some antibiotics and good antimicrobial activity that candidate them as potential probiotics. Seven strains (1C, 5C, 12C, 13C, 17C, 7M, and 40M), the most resistant to simulated digestion, were further investigated to evaluate their capability to adhere to human intestinal Caco‐2 cells. L. plantarum 17C was the most adherent strain. The bioactivity assessment of L. plantarum 17C showed anticancer effects via the induction of apoptosis on HT‐29 human cancer cells and negligible side effects on one human epithelial normal cell line (FHs 74). The metabolites produced by this strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells.
Probiotics modify the gut microbial composition/homeostasis. Probiotics bind to mutagens and eliminate them from the body. Probiotics enhances the mucosal and system immune responses. Probiotics can control the cell proliferation and apoptosis. Probiotics protect the intestinal barrier from deleterious.
PDF
