scholarly journals Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

2021 ◽  
Author(s):  
Cian Holohan ◽  
Sophia Hanrahan ◽  
Nathan Feely ◽  
Peng Li ◽  
John O'Connell ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Large Scale ◽  
Pcr Detection ◽  
Molecular Diagnostic ◽  
Pcr Analysis ◽  
Analytical Sensitivity ◽  
Freeze Thaw ◽  
Qrt Pcr ◽  
Viral Transport ◽  
The Stability

The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.

scholarly journals Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

Nanoscale ◽  
2021 ◽  
Author(s):  
Cian Holohan ◽  
Sophia Hanrahan ◽  
Nathan Feely ◽  
Peng Li ◽  
John O'Connell ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Nucleic Acids ◽  
Large Scale ◽  
Diagnostic Testing ◽  
Pcr Detection ◽  
Molecular Diagnostic ◽  
Freeze Thaw ◽  
Qrt Pcr ◽  
Viral Transport ◽  
Molecular Diagnostic Testing

Objective The events of the last year have highlighted the complexity of implementing molecular diagnostic testing for infectious diseases at a large scale. The purpose of this study was to...

scholarly journals The Effects of a Single Freeze-Thaw Cycle on Concentrations of Nutritional, Noncommunicable Disease, and Inflammatory Biomarkers in Serum Samples

2021 ◽  
Author(s):  
Ransi Ann Abraham ◽  
Garima Rana ◽  
Praween K. Agrawal ◽  
Robert Johnston ◽  
Avina Sarna ◽  
...  
Keyword(s):  
Large Scale ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Noncommunicable Disease ◽  
Noncommunicable Diseases ◽  
Serum Samples ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
The Stability

Abstract Background The stability of biological samples is vital for reliable measurements of biomarkers in large-scale survey settings, which may be affected by freeze-thaw procedures. We examined the effect of a single freeze-thaw cycle on 13 nutritional, noncommunicable diseases (NCD), and inflammatory bioanalytes in serum samples. Method Blood samples were collected from 70 subjects centrifuged after 30 minutes and aliquoted immediately. After a baseline analysis of the analytes, the samples were stored at − 70°C for 1 month and reanalyzed for all the parameters. Mean percentage differences between baseline (fresh blood) and freeze-thaw concentrations were calculated using paired sample t-tests and evaluated according to total allowable error (TEa) limits (desirable bias). Results Freeze-thaw concentrations differed significantly (p < 0.05) from baseline concentrations for soluble transferrin receptor (sTfR) (− 5.49%), vitamin D (− 12.51%), vitamin B12 (− 3.74%), plasma glucose (1.93%), C-reactive protein (CRP) (3.45%), high-density lipoprotein (HDL) (7.98%), and cholesterol (9.76%), but they were within respective TEa limits. Low-density lipoprotein (LDL) (− 0.67%), creatinine (0.94%), albumin (0.87%), total protein (1.00%), ferritin (− 0.58%), and triglycerides (TAG) (2.82%) concentrations remained stable following the freeze-thaw cycle. In conclusion, single freeze-thaw cycle of the biomarkers in serum/plasma samples after storage at − 70°C for 1 month had minimal effect on stability of the studied analytes, and the changes in concentration were within acceptable limit for all analytes.

scholarly journals Analysis of DNA interactions and GC content with energy decomposition in large-scale quantum mechanical calculations

2021 ◽  
Vol 23 (14) ◽  
pp. 8891-8899
Author(s):  
Han Chen ◽  
Chris-Kriton Skylaris
Keyword(s):  
Hydrogen Bonding ◽  
Nucleic Acids ◽  
Large Scale ◽  
Gc Content ◽  
Quantum Mechanical ◽  
Quantum Mechanical Calculations ◽  
Energy Decomposition ◽  
Dna Interactions ◽  
The Stability

GC content is a contributing factor to the stability of nucleic acids due to hydrogen bonding. HALMO-EDA scheme is used for decomposing the inter-strand interactions of dsDNA molecules.

scholarly journals Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jing Tong ◽  
Manman Hu ◽  
Beibei Han ◽  
Yanhai Ji ◽  
Baoju Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Transcriptome Profiling ◽  
Pcr Analysis ◽  
Allium Tuberosum ◽  
Qrt Pcr ◽  
Complex Processes ◽  
Chinese Chive ◽  
Gene Expression Studies ◽  
The Stability

AbstractChinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

scholarly journals Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

2021 ◽  
Vol 22 (5) ◽  
pp. 2569
Author(s):  
Xue Bai ◽  
Tao Chen ◽  
Yuan Wu ◽  
Mingyong Tang ◽  
Zeng-Fu Xu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Cyperus Esculentus ◽  
Transcriptome Data ◽  
Qrt Pcr ◽  
Below Ground ◽  
The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

scholarly journals Evaluation of Reference Genes for qRT-PCR Normalization in Angelica decursiva under Various Experimental Conditions

2020 ◽  
Author(s):  
Yuedong He ◽  
Yuan Zhong ◽  
Zhenzhen Bao ◽  
Weiqi Wang ◽  
Xiaoqing Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Binding Protein ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Angelica Decursiva ◽  
Qrt Pcr ◽  
Polypyrimidine Tract Binding Protein ◽  
Chinese Medicinal Plants ◽  
The Stability

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This qRT-PCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the qRT-PCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of qRT-PCR for an accurate and far-reaching gene expression analysis in this medicinal plant.

scholarly journals Longitudinal assessment and stability of long non-coding RNA gene expression profiles measured in human peripheral whole blood collected into PAXgene blood RNA tubes

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Lukasz S. Wylezinski ◽  
Guzel I. Shaginurova ◽  
Charles F. Spurlock III
Keyword(s):  
Whole Blood ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Molecular Diagnostic ◽  
Longitudinal Assessment ◽  
Total Rna ◽  
Freeze Thaw ◽  
Lncrna Expression ◽  
The Stability ◽  
The Impact

Abstract Objective Long non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at − 80 °C. Here we sought to determine the effects on lncRNA expression profiles following − 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze–thaw cycles on both total RNA and cDNA obtained from PAXgene tubes. Results We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at − 80 °C or up to ten total RNA or cDNA freeze–thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals ITRAQ-based quantitative proteomic analysis of Fusarium moniliforme (Fusarium verticillioides) in response to Phloridzin inducers

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rong Zhang ◽  
Weitao Jiang ◽  
Xin Liu ◽  
Yanan Duan ◽  
Li Xiang ◽  
...  
Keyword(s):  
Fusarium Moniliforme ◽  
Abiotic Factors ◽  
Fusarium Verticillioides ◽  
Protein Profile ◽  
Sugar Metabolism ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Differentially Expressed Proteins ◽  
Apple Replant Disease ◽  
Qrt Pcr

Abstract Background Apple replant disease (ARD) has been reported from all major fruit-growing regions of the world, and is often caused by biotic factors (pathogen fungi) and abiotic factors (phenolic compounds). In order to clarify the proteomic differences of Fusarium moniliforme under the action of phloridzin, and to explore the potential mechanism of F. moniliforme as the pathogen of ARD, the role of Fusarium spp. in ARD was further clarified. Methods In this paper, the quantitative proteomics method iTRAQ analysis technology was used to analyze the proteomic differences of F. moniliforme before and after phloridzin treatment. The differentially expressed protein was validated by qRT-PCR analysis. Results A total of 4535 proteins were detected, and 293 proteins were found with more than 1.2 times (P< 0.05) differences. In-depth data analysis revealed that 59 proteins were found with more than 1.5 times (P< 0.05) differences, and most proteins were consistent with the result of qRT-PCR. Differentially expressed proteins were influenced a variety of cellular processes, particularly metabolic processes. Among these metabolic pathways, a total of 8 significantly enriched KEGG pathways were identified with at least 2 affiliated proteins with different abundance in conidia and mycelium. Functional pathway analysis indicated that up-regulated proteins were mainly distributed in amino sugar, nucleotide sugar metabolism, glycolysis/ gluconeogenesis and phagosome pathways. Conclusions This study is the first to perform quantitative proteomic investigation by iTRAQ labeling and LC-MS/MS to identify differentially expressed proteins in F. moniliforme under phloridzin conditions. The results confirmed that F. moniliforme presented a unique protein profile that indicated the adaptive mechanisms of this species to phloridzin environments. The results deepened our understanding of the proteome in F. moniliforme in response to phloridzin inducers and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents to control ARD.

Sign in / Sign up

Export Citation Format

Share Document