A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA

Andrologia ◽  
2018 ◽  
Vol 50 (6) ◽  
pp. e13030 ◽  
Author(s):  
K. Vijayalakshmy ◽  
P. Kumar ◽  
M. Virmani ◽  
S. Pawaria ◽  
N. S. Lalaji ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Silica Colloid ◽  
Extraction Protocol

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

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 SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 863 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Müller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
N Gene ◽  
Extraction Protocol ◽  
Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

scholarly journals Comparison Between a Standard and SalivaDirect RNA Extraction Protocol for Molecular Diagnosis of SARS-CoV-2 Using Nasopharyngeal Swab and Saliva Clinical Samples

2021 ◽  
Vol 9 ◽  
Author(s):  
Sofía N. Rodríguez Flores ◽  
Luis Mario Rodríguez-Martínez ◽  
Bernardita L. Reyes-Berrones ◽  
Nadia A. Fernández-Santos ◽  
Elthon J. Sierra-Moncada ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Proteinase K ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Assay Performance ◽  
Two Samples ◽  
Oropharyngeal Swab

During the COVID-19 pandemic, a certified laboratory of Tamaulipas, Mexico has processed over 100,000 samples of COVID-19 suspected patients, working a minimum of 100 tests daily. Thus, it would be beneficial for such certified laboratories nationwide to reduce the time and cost involved in performing the diagnosis of COVID-19, from sample collection, transportation to local lab, processing of samples, and data acquisition. Here, 30 nasopharyngeal swab and saliva samples from the same COVID-19 individuals were assessed by a standard nucleic acid extraction protocol, including protein lysis with proteinase K followed by binding to column, washing, and elution, and by the SalivaDirect protocol based on protein lysis, skipping the other steps to reduce processing time and costs. The genomic RNA was amplified using a SARS-CoV-2 Real-Time PCR kit. A variation (P > 0.05) in the 95% CIs = 72.6%–96.7% was noted by using the SalivaDirect protocol and saliva samples (sensitivity of 88.2%) in comparison to those of standard protocol with oropharyngeal swab samples (95% CIs = 97.5%–100%; sensitivity of 100%) as reported elsewhere. However, when using nasopharyngeal swab samples in the SalivaDirect protocol (sensitivity of 93.6%; 95% CIs = 79.2%–99.2%), it was in concordance (P < 0.05) with those of the standard one. The logical explanation to this was that two samples with Ct values of 38, and 40 cycles for gene E produced two false negatives in the SalivaDirect protocol in relation to the standard one; thus, there was a reduction of the sensitivity of 6.4% in the overall assay performance.

scholarly journals SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP

2020 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Mueller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Large Scale ◽  
Magnetic Beads ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Extraction Protocol ◽  
Large Scale Testing ◽  
Commercial Kits

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and alternative, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

RNA Extraction Protocol for Shorea v2

2021 ◽  
Author(s):  
mpfsum not provided
Keyword(s):  
Rna Extraction ◽  
Extraction Protocol ◽  
Ctab Method

RNA extraction protocol using CTAB method optimized for leaf and bud samples from Shorea curtisii. Adapted from extraction protocol for Shorea beccariana (see attached publication).

QIAGEN RNeasy Plant RNA Extraction Protocol (Modified) v2

protocols.io ◽  
2021 ◽  
Author(s):  
Steven J
Keyword(s):  
Rna Extraction ◽  
Extraction Protocol

RNA extraction protocol (Trizol) v1 (protocols.io.ew7bfhn)

protocols.io ◽  
2016 ◽  
Author(s):  
Hebert F ◽  
Grambauer S ◽  
Barber I ◽  
Landry C ◽  
Aubin Horth
Keyword(s):  
Rna Extraction ◽  
Extraction Protocol

Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses

2019 ◽  
Vol 110 ◽  
pp. 104289 ◽  
Author(s):  
Gisele Silva Boos ◽  
Daniel Nobach ◽  
Klaus Failing ◽  
Markus Eickmann ◽  
Christiane Herden
Keyword(s):  
Rna Extraction ◽  
Extraction Protocol ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed
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