Abstract
The continuous cropping of cucumber in the same potting soils may result in a reduction of yield and quality of the crop, a phenomenon described as soil sickness. The changes of soil ...microbial communities as affected by continuous cropping and the link between these changes and soil sickness of cucumber are still not clear. In the present study, cucumber was cropped in pots under greenhouse conditions for nine successive cropping cycles. Structures and sizes of rhizosphere fungal and Fusarium (Ascomycota, Fungi) communities, both ubiquitous and ecologically important in soils, were analysed with PCR-denaturing gradient gel electrophoresis and quantitative reverse transcription PCR, respectively. Cucumber showed retarded growth in the seventh cropping cycle. The RNA- and DNA-based fungal community structures derived from the same sample differed from each other, and the active soil fungal communities were more sensitive to continuous cropping. The RNA-based fungal and Fusarium community sizes were larger in the seventh cropping cycle than in the other cropping cycles. Overall, the findings of this study indicate that the population sizes rather than the diversity of fungi and Fusarium communities are linked to the soil sickness associated with cucumber cultivation.
The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their ...accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing.
Graphical abstract
There are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.
Different studies have shown that HPV16‐positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the ...levels of viral genes, nor those of virally disrupted human genes, a genome‐wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty‐three fresh‐frozen HPV‐16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo‐keto‐reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV‐positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non‐hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV‐positive (p ≤0.001) and ‐negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV‐negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.
What's new?
Oropharyngeal squamous cell carcinoma (OPSCC) is often associated with high‐risk human papillomavirus infection, especially HPV16, but the clinical relevance of viral integration in a subset of these tumors remains unclear. Here the authors describe transcriptional signatures of metabolic reprogramming including increased expression of the aldo‐keto‐reductases AKR1C1 and AKR1C3 proteins in OPSCC cells regardless of HPV status. Tumors with upregulation of these proteins were strongly associated with poor prognosis suggesting prognostic as well as therapeutic implications for OPSCC patients beyond HPV16 infection.
The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on ...milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.
Most persons infected with enterically transmitted viruses shed large amounts of virus in feces for days or weeks, both before and after onset of symptoms. Therefore, viruses causing gastroenteritis ...may be detected in wastewater, even if only a few persons are infected. In this study, the presence of eight pathogenic viruses (norovirus, astrovirus, rotavirus, adenovirus, Aichi virus, parechovirus, hepatitis A virus HAV, and hepatitis E virus) was investigated in sewage to explore whether their identification could be used as an early warning of outbreaks. Samples of the untreated sewage were collected in proportion to flow at Ryaverket, Gothenburg, Sweden. Daily samples collected during every second week between January and May 2013 were pooled and analyzed for detection of viruses by concentration through adsorption to milk proteins and PCR. The largest amount of noroviruses was detected in sewage 2 to 3 weeks before most patients were diagnosed with this infection in Gothenburg. The other viruses were detected at lower levels. HAV was detected between weeks 5 and 13, and partial sequencing of the structural VP1protein identified three different strains. Two strains were involved in an ongoing outbreak in Scandinavia and were also identified in samples from patients with acute hepatitis A in Gothenburg during spring of 2013. The third strain was unique and was not detected in any patient sample. The method used may thus be a tool to detect incipient outbreaks of these viruses and provide early warning before the causative pathogens have been recognized in health care.
Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, ...improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This minireview will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology, and considerations for implementation of the method in a clinical laboratory.
Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of water microbiology. This paper reviews the fundamental principles of dPCR ...and its application for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) - the virus that causes Coronavirus Disease 2019 (COVID-19). The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic material from microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in water microbiology. With dPCR platforms, the sample analysis cost and processing time are typically greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is critical, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.
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•dPCR application for water microbiology is accelerating.•dPCR may improve analytical performance for microbial targets in complex aqueous matrices.•Increased costs, processing time, and need for specialized instruments constrain widespread adoption of dPCR.•dPCR relies on fundamental assumptions and should not be viewed as a panacea for water microbiology.
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