scholarly journals Singleseed detection of an uncharacterised Betaflexiviridae virus in Actinidia

2016 ◽  
Vol 69 ◽  
pp. 325-325
Author(s):  
P.T. Austin ◽  
R.C. Van_den_Brink ◽  
M.B. Horner ◽  
A.M. Duffy ◽  
P.M. Datson ◽  
...  
Keyword(s):  
Internal Control ◽  
Extraction Method ◽  
Rna Extraction ◽  
Seed Infection ◽  
Fresh Leaf ◽  
The Family ◽  
Partial Rdrp ◽  
Virus Isolates ◽  
Initial Results ◽  
Efficient Transmission

A sensitive conventional RTPCR assay has been developed for an uncharacterised virus within the family Betaflexiviridae prompted by detection of the virus in Actinidia seedlings held in postentry quarantine that had been grown from openpollinated seed Infection is asymptomatic and presently assessed as lowtozero biosecurity risk The assay targets the RNAdependent RNA polymerase (RdRP) of the virus primer sequences (BetaF2 and BetaR2 278bp amplicon) being based on a partial RdRP sequence for the virus The assay detects the nine virus isolates found to date in a range of sample types (fresh leaf stem cambium archived RNA) The assay which includes a multiplexed internal control has been accredited by International Accreditation New Zealand for Actinidia testing To support management of the virus the assay and associated RNA extraction method have been applied to testing dried Actinidia seed directly They have been found sufficiently sensitive to detect the virus in individual seeds Initial results show efficient transmission of the virus from infected pollen to the seed

scholarly journals Molecular detection of SARS-CoV-2 using a reagent-free approach

2020 ◽  
Author(s):  
Ronan Calvez ◽  
Andrew Taylor ◽  
Leo Calvo-Bado ◽  
Colin Fink
Keyword(s):  
Internal Control ◽  
Extraction Method ◽  
Molecular Detection ◽  
Rna Extraction ◽  
Heat Processing ◽  
Virus Transport ◽  
Validation Process ◽  
Treatment Conditions ◽  
Different Temperatures ◽  
Alternative Approaches

AbstractShortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Fomsgaard et al 20205 recently presented results using heat-processing of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost.Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this technique. We first investigated various treatments using different temperatures, incubation times and sample volumes based on the above study to optimise the heat-treatment conditions. Although the initial data confirmed the published results, further investigations revealed unexpected inhibitory properties of some commonly used virus transport media (VTMs) on some commercially available RT-qPCR mixes, emphasising the critical importance of a thorough validation process to determine the most adapted reagents to be used depending on the sample types to be tested.In conclusion, although the method works, with very consistent Ct values and an excellent sensitivity when compared to a conventional RNA extraction method, it is critical to include an internal control to check each sample for potential inhibition.

scholarly journals RNA Extraction Alternative Method for SARS-CoV-2 Molecular Diagnosis

2021 ◽  
Author(s):  
Elisa Bianchini ◽  
Francesco Rossignolo ◽  
Melissa Perfranceschi ◽  
Chiara Cazzin ◽  
Ronaldo Silva ◽  
...  
Keyword(s):  
Internal Control ◽  
Extraction Method ◽  
Target Genes ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Molecular Test ◽  
Control And Prevention ◽  
Set Up

Background: the devastating outbreak of COVID-19 poses serious challenges for the diagnostics laboratories, which are often facing global shortage of reagents and equipment. With the aim of increasing the diagnostic throughput for SARS-CoV-2 molecular test, the purpose of this study was to validate an additional RNA extraction method respect to those already recommended by WHO and the US Centers for Disease Control and Prevention (CDC). Methods: a new protocol for RNA extraction from nasopharyngeal swab was set up, adapting the Qiagen RNeasy 96 plate and validated on a set of 100 clinical samples analyzed in parallel by Roche-Magnapure method (already recommended by CDC guidelines). Results: the internal control and target genes analysis showed a good agreement between the two extraction methods indicating that the two methods can be considered equivalent and that the RNeasy-adapted method can be applied for the SARS-CoV-2 diagnostics. The addition of this new extraction method resulted in a throughput increase for SARS-CoV-2 molecular test of about 2000 samples/month during the initial months of the pandemic emergency in which the lack of reagents for the extraction led to an insufficient sample processing throughput of the analysis of the swabs.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

scholarly journals Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 240
Author(s):  
Alison Woodward ◽  
Alina Pandele ◽  
Salah Abdelrazig ◽  
Catherine A. Ortori ◽  
Iqbal Khan ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Extraction Method ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Serum Starvation ◽  
Extraction Techniques ◽  
Metabolic Genes ◽  
Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

scholarly journals Detection of Schmallenberg Virus RNA in Bull Semen in Poland

2016 ◽  
Vol 19 (3) ◽  
pp. 655-657 ◽  
Author(s):  
J. Kęsik-Maliszewska ◽  
M. Larska
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Schmallenberg Virus ◽  
Rt Pcr ◽  
Bovine Semen ◽  
Bull Semen ◽  
Virus Rna ◽  
Virus Excretion ◽  
Venereal Transmission

Abstract The detection of Schmallenberg virus (SBV) in the breeding bull semen raised the question of the possibility of venereal transmission of SBV which could result in cost-intensive restrictions in the trade of bovine semen. In order to evaluate the presence of SBV RNA in bovine semen, 131 bull semen samples from four locations in Poland collected between 2013 and 2015 were analysed by RT-PCR for viral RNA. SBV RNA was detected in 5.3% of the samples. The study has revealed that application of an appropriate RNA extraction method is crucial to detect virus excretion via semen.

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr

scholarly journals Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia

2003 ◽  
Vol 84 (10) ◽  
pp. 2685-2689 ◽  
Author(s):  
J. C. Osborne ◽  
C. E. Rupprecht ◽  
J. G. Olson ◽  
T. G. Ksiazek ◽  
P. E. Rollin ◽  
...  
Keyword(s):  
Virus Isolation ◽  
Public Health Problem ◽  
Distinct Group ◽  
Genetic Data ◽  
Geographic Range ◽  
South East Asia ◽  
Rt Pcr ◽  
The Family ◽  
Geographic Separation ◽  
Virus Isolates

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2·6–3·2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

Rapid Virus Detection Procedure for Molecular Tracing of Shellfish Associated with Disease Outbreaks

2007 ◽  
Vol 70 (4) ◽  
pp. 967-974 ◽  
Author(s):  
ANA MARIA de RODA HUSMAN ◽  
FROUKJE LODDER-VERSCHOOR ◽  
HAROLD H. J. L. van den BERG ◽  
FRANÇOISE S. LE GUYADER ◽  
HILDE van PELT ◽  
...  
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Virus Detection ◽  
Disease Outbreaks ◽  
Extraction Methods ◽  
Virus Rna ◽  
Pathogenic Viruses ◽  
Rna Extraction Methods ◽  
Digestive Glands ◽  
Outbreak Situation

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III–spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

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