Taqman qPCR Quantification and Fusarium Community Analysis to Evaluate Toxigenic Fungi in Cereals

Fusarium head blight (FHB) is an economically important plant disease. Some Fusarium species produce mycotoxins that cause food safety concerns for both humans and animals. One especially important mycotoxin-producing fungus causing FHB is Fusarium graminearum. However, Fusarium species form a disease complex where different Fusarium species co-occur in the infected cereals. Effective management strategies for FHB are needed. Development of the management tools requires information about the diversity and abundance of the whole Fusarium community. Molecular quantification assays for detecting individual Fusarium species and subgroups exist, but a method for the detection and quantification of the whole Fusarium group is still lacking. In this study, a new TaqMan-based qPCR method (FusE) targeting the Fusarium-specific elongation factor region (EF1α) was developed for the detection and quantification of Fusarium spp. The FusE method was proven as a sensitive method with a detection limit of 1 pg of Fusarium DNA. Fusarium abundance results from oat samples correlated significantly with deoxynivalenol (DON) toxin content. In addition, the whole Fusarium community in Finnish oat samples was characterized with a new metabarcoding method. A shift from F. culmorum to F. graminearum in FHB-infected oats has been detected in Europe, and the results of this study confirm that. These new molecular methods can be applied in the assessment of the Fusarium community and mycotoxin risk in cereals. Knowledge gained from the Fusarium community analyses can be applied in developing and selecting effective management strategies for FHB.

Enhanced fluorescence signal using stray light shutter in a quantitative PCR chip

Quantitative polymerase chain reaction (qPCR) is the most important quantitative sensing technique for pathogens, especially for emerging pandemics such as coronavirus outbreak this year. The qPCR chip and device were investigated to meet the unmet needs of ultrafast inspection time, high accuracy, and small system volume. Therein, the fluorescence intensity was the most important signal in qPCR quantification of DNA amplifications, which is essential not only in the confirmative diagnosis of positive or negative infection, but also in the assessment of viral load for therapeutic and quarantine decision making. As the target DNAs got amplified, the interaction of fluorescence dye and double strand DNA will generate fluorescence signal proportional to amplified DNA in the intensity when excited by certain wavelength. A miniature spectro-detector was employed to receive the fluorescence scattering for digital output of the intensity in the qPCR chip in this study, and the optical simulation and actual experimental design and results according to the optical simulation results were performed to study the effect of the stray light shutter (SLS) in the improvement of the signal in fluorescence detection. The analysis results showed that the signal-to-noise ratio (SNR) of the fluorescence can be enhanced significantly for 5 times of the control using the SLS with a shape of extended component aperture, where the protruding structure was positioned away from the center. The experimental results showed that fluorescence intensity can be enhanced by 15.50% and 9.86% when adding the above shape of SLS in resin- and in glass-based chip, respectively. The results also demonstrated that the optical setup had good stability and repeatability in fluorescence detection, and variation was less than 1.00 %. Our results can provide important reference to the development of qPCR chip to obtain the high SNR fluorescence signal in DNA quantification process.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1β, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, − 20 °C and − 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes

Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.

Soybean microbiome recovery after disruption is modulated by the seed and not the soil microbiome

Endophytic microbiome of healthy seeds forms a symbiotic relationship with their host. Seeds and environment are sources of microbes that colonise the developing plant, however, the influence of each remains unclear. Here, using irradiation combined with surface sterilisation to generate near-axenic seeds with disrupted and reduced microbiome, we contrasted colonisation potential of seed and soil microbiome. We hypothesised that the seed microbiome would be the primary coloniser of the plant endophytic compartments. Our experimental design comprised four treatments, using soybean as a model plant: 1) nearly axenic seeds growing in a sterile environment, 2) non-axenic seeds inoculated with a microbial soil extract, 3) nearly axenic seeds inoculated with a microbial seed extract, and 4) nearly axenic seeds inoculated with a microbial soil extract. After 14 days of growth, plants were harvested, and DNA was extracted from the shoot, roots, rhizosphere, and subjected to 16S rRNA gene amplicon sequencing, qPCR quantification of the total community and functional genes involved in the N-cycle. Community dynamics were similar for most treatments within their respective compartments, except for the soil treatment, where rhizosphere and root microbiome differed from other treatments, suggesting that the soil microbiome colonises the belowground compartment efficiently only when the seed microbiome is severely disrupted. For the shoot, all treatments resembled the seed microbiome treatment, suggesting that the seed-borne bacteria colonise the aboveground compartment preferentially. Our results highlight the primacy of the seed microbiome over the soils during early colonisation, putting seed microbes as potential candidates of microbiome engineering efforts.

Somatic mutation landscapes at single-molecule resolution

Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85–97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103–107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. Key points • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential

Comparison of parasite load by qPCR and histopathological changes of inner and outer edge of ulcerated cutaneous lesions of cutaneous leishmaniasis

Background Cutaneous leishmaniasis (CL) is an infectious vector-borne disease caused by protozoa of the Leishmania genus that affects humans and animals. The distribution of parasites in the lesion is not uniform, and there are divergences in the literature about the choice of the better sampling site for diagnosis–inner or outer edge of the ulcerated skin lesion. In this context, determining the region of the lesion with the highest parasite density and, consequently, the appropriate site for collecting samples can define the success of the laboratory diagnosis. Hence, this study aims to comparatively evaluate the parasite load by qPCR, quantification of amastigotes forms in the direct exam, and the histopathological profile on the inner and outer edges of ulcerated CL lesions. Methods Samples from ulcerated skin lesions from 39 patients with confirmed CL were examined. We performed scraping of the ulcer inner edge (base) and outer edge (raised border) and lesion biopsy for imprint and histopathological examination. Slides smears were stained by Giemsa and observed in optical microscopy, the material contained on the smears was used to determine parasite load by quantitative real-time PCR (qPCR) with primers directed to the Leishmania (Viannia) minicircle kinetoplast DNA. The histopathological exam was performed to evaluate cell profile, tissue alterations and semi-quantitative assessment of amastigote forms in inner and outer edges. Principal findings Parasite loads were higher on the inner edge compared to the outer edge of the lesions, either by qPCR technique (P<0.001) and histopathological examination (P< 0.003). There was no significant difference in the parasite load between the imprint and scraping on the outer edge (P = 1.0000). Conclusion/Significance The results suggest that clinical specimens from the inner edge of the ulcerated CL lesions are the most suitable for both molecular diagnosis and direct parasitological examination.

Infection by Zika Vírus in human cells alters the expression profile of miRNA-15 and activation of apoptotic caspases

Objective: Evaluate the miRNA-15 expression profile involved in cellular apoptotic regulation factors. Methodology: We used the H818308 Asian strain of ZIKV without neurological damage. The inoculations occurred in human embryonic kidney cells (HEK-293). After inoculation, samples were extracted for RT-qPCR quantification of viral RNA and miR-15. The level of activation of caspases 1, 3/7 and 8 of cells was performed using chemofluorescence. Results: The ZIKV infection alters the expression of genes and their regulators, affecting several cellular physiological processes such as apoptosis. Conclusion: Therefore, it is important to emphasize that renal progenitor cells (HEK-293) are susceptible to VZIK infection. The genetic deregulation resulting from infection directly affects important cellular processes such as apoptosis from the disordered miRNA-15 expression during the infection period.