Summary
Telomeres play a fundamental role in the protection of chromosomal DNA and in the regulation of cellular senescence. Recent work in human epidemiology and evolutionary ecology suggests adult ...telomere length (TL) may reflect past physiological stress and predict subsequent morbidity and mortality, independent of chronological age.
Several different methods have been developed to measure TL, each offering its own technical challenges. The aim of this review is to provide an overview of the advantages and drawbacks of each method for researchers, with a particular focus on issues that are likely to face ecologists and evolutionary biologists collecting samples in the field or in organisms that may never have been studied in this context before.
We discuss the key issues to consider and wherever possible try to provide current consensus view regarding best practice with regard to sample collection and storage, DNA extraction and storage, and the five main methods currently available to measure TL.
Decisions regarding which tissues to sample, how to store them, how to extract DNA, and which TL measurement method to use cannot be prescribed, and are dependent on the biological question addressed and the constraints imposed by the study system. What is essential for future studies of telomere dynamics in evolution and ecology is that researchers publish full details of their methods and the quality control thresholds they employ.
Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available qPCR-based methods for miRNA ...expression analysis and discuss their advantages and disadvantages. Existing techniques involve the use of stem–loop reverse transcriptase–PCR, polyadenylation of RNAs, ligation of adapters or RT with complex primers, using universal or miRNA-specific qPCR primers and/or probes. Many of these methods are oriented towards the expression analysis of mature miRNAs and few are designed for the study of pre-miRNAs and pri-miRNAs. We also discuss findings from articles that compare results from existing methods. Finally, we suggest key points for the improvement of available techniques and for the future development of additional methods.
According to the evidence, the coronavirus disease 19 (COVID‐19) is caused by a zoonotic pathogen named respiratory syndrome coronavirus 2 (SARS‐CoV‐2). This virus can spread through personal ...contact, respiratory droplets, and also through airborne transmission. A rapid, low‐cost, and effective biosensor platform is essential to diagnose patients with COVID‐19 infection, predominantly the asymptomatic individuals, and prevent the spread of the SARS‐CoV‐2 via transmission routes. The objective of this review is to provide a comparative view among current diagnostic methods, focusing on recently suggested biosensors for the detection of SARS‐CoV2 in clinical samples. A capable SARS‐CoV‐2 biosensor can be designed by the holistic insights of various biosensor studies.
Schematic representation for various detection methods for COVID‐19 infection.
•A specific real-time PCR assay (Mit1C) was developed for the detection of C. cayetanensis.•The Mit1C assay targets a conserved region of the C. cayetanensis mitochondrial genome.•A single laboratory ...validation was performed in spiked produce samples.•Evaluation against closely related organisms revealed specificity to C. cayetanensis.•As few as 5 oocysts were detected in spiked produce samples, demonstrating sensitivity.
Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.
In order to reveal the inhibitory effects of cinnamaldehyde, citral, and eugenol on aflatoxin biosynthesis, the expression levels of 5 key aflatoxin biosynthetic genes were evaluated by real‐time ...PCR. Aspergillus flavus growth and AFB1 production were completely inhibited by 0.80 mmol/L of cinnamaldehyde and 2.80 mmol/L of citral. However, at lower concentration, cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L) significantly reduced AFB1 production with inhibition rate of 68.9%, 95.4%, and 41.8%, respectively, while no effect on fungal growth. Real‐time PCR showed that the expressions of aflR, aflT, aflD, aflM, and aflP were down‐regulated by cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L). In the presence of cinnamaldehyde, AflM was highly down‐regulated (average of 5963 folds), followed by aflP, aflR, aflD, and aflT with the average folds of 55, 18, 6.5, and 5.8, respectively. With 0.80 mmol/L of eugenol, aflP was highly down‐regulated (average of 2061‐folds), followed by aflM, aflR, aflD, and aflT with average of 138‐, 15‐, 5.2‐, and 4.8‐folds reduction, respectively. With 0.56 mmol/L of citral, aflT was completely inhibited, followed by aflM, aflP, aflR, and aflD with average of 257‐, 29‐, 3.5‐, and 2.5‐folds reduction, respectively. These results suggest that the reduction in AFB1 production by cinnamaldehyde, eugenol, and citral at low concentration may be due to the down‐regulations of the transcription level of aflatoxin biosynthetic genes. Cinnamaldehyde and eugenol may be employed successfully as a good candidate in controlling of toxigenic fungi and subsequently contamination with aflatoxins in practice.
Practical Application
In our study, we gained some information about the inhibitory effects of cinnamaldehyde, eugenol, and citral on Aspergillus flavus growth and AFB1 production, the effects of them on the expression levels of the 5 key aflatoxin biosynthetic genes. At lower concentrations, cinnamaldehyde, eugenol, and citral could down regulate the expression of aflR, aflT, aflD, aflM, and aflP. In the large scale approach to control A. flavus and aflatoxin contaminated in wheat, maize, and peanut, the dose of natural compounds estimated to be 100 g/ton with the cost around $1.28 to 1.60/ton, which is less expensive in Chinese market. As a result, we conclude that cinnamaldehyde and eugenol can be potential biocontrol agents against aflatoxins contamination in grains and other agro‐products.
Background
Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune‐related complications, and eventually to customize ...immunosuppression.
Methods
In this study, 327 blood samples were tested using two real‐time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in‐house rtPCR developed by our group, the second one was the recently marketed TTV R‐GENE assay.
Results
In the validation study, the TTV R‐GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in‐house rtPCR. The Bland‐Altman analysis showed that the mean difference between the two methods was −0.3 log copies/mL. In the comparison study, 69% and 72% of samples were detected positive by rtPCR and TTV R‐GENE, respectively (94% concordance, κ = 0.88). Performances did not differ between the two rtPCRs by type of TTV group examined. When a newly‐developed in‐house digital droplet PCR was applied for TTV quantification and used as an alternative method of comparison on 94 samples, the results strongly correlated with those obtained by the two rtPCR methods (99% concordance).
Conclusion
In summary, all the molecular methods assayed are highly sensitive and accurate in quantitation of TTV DNA in blood samples.
Highlight
TTV monitoring is a promising strategy for quantification of the immunocompetence in transplant patients.
PCR methods actually in use have comparable performances in TTV quantification
Background and purpose: The aim is to investigate the change in the expression level of miR-32 and PTEN genes in tumor samples with melanoma. Materials and methods: This study collected tumor samples ...from 50 patients and 50 samples from healthy marginal tissue around the tumor as a control sample. Following that, the RNA related to the samples was extracted and the expression of the desired gene was evaluated using real-time polymerase chain reaction. Appropriate statistical tests assessed differences in expression levels of the two groups. In statistical tests. Results: The expression of PTEN was lower in tumor samples than in healthy samples. In contrast, the expression of miR-32 was higher in tumor samples and the difference was significant in both cases (P<0.05). Conclusion: Our findings indicated that the miR-32 and PTEN genes can be used as diagnostic or predictive biomarkers in melanoma. However, it is recommended that further research be conducted in different communities with larger sample sizes. Similar tests can contribute to the immediate diagnosis of melanoma. Using the potential of these biomarkers for diagnosis, early detection of melanoma and better treatment will become attainable and more successful.
Aim
The aim of this study was to identify efficient plant‐beneficial rhizobacterium that has the potential to be developed as biocontrol agent for the control of wheat soil‐borne diseases.
Methods ...and Results
Rhizosphere soil samples were collected from a wheat field located in Taian City. Numerous bacteria were isolated and screened for antagonistic activity against soil‐borne plant pathogenic fungi by performing dual‐culture assays. Among them, XH‐9 was selected for its highly antagonistic activity and others growth‐promoting characteristics. Subsequently, the strain was identified as Bacillus amyloliquefaciens subsp. plantarum based on phylogenetic analysis of 16S rDNA sequence. Pot experiment indicated that XH‐9 has good capacities for wheat, corn, and chili root colonization and considerably increased the biometric parameters of wheat seedlings. Quantitative real‐time polymerase chain reaction experiments showed that the amount of Fusarium oxysporum associated with the XH‐9 after treatment significantly decreased compared with control group.
Conclusions
Bacillus amyloliquefaciens subsp. plantarum XH‐9 has the potential as biocontrol agent when applied in local arable land to prevent damage caused by F. oxysporum and other phytopathogens.
Significance and Impact of the Study
The development of biocontrol strategies for reducing the damage caused by plant pathogens is fully in accord with the current principles of sustainability.
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