Manual Silane magnetic bead-based high throughput protocol for SARS-CoV-2 RNA extraction v2 (protocols.io.bfmajk2e)

Manual Silane magnetic bead-based high throughput protocol for SARS-CoV-2 RNA extraction v1 (protocols.io.be8bjhsn)

protocols.io ◽

10.17504/protocols.io.be8bjhsn ◽

2020 ◽

Author(s):

Ainhoa Go ◽

Jose Antonio ◽

Nicholas Weber ◽

Juan Pablo ◽

Josepmaria Argemi ◽

...

Keyword(s):

High Throughput ◽

Rna Extraction ◽

Magnetic Bead

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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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

Virology Journal ◽

10.1186/s12985-021-01523-1 ◽

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.

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Development of a multi-channel magnetic bead micro-probe assay for high-throughput detection of zearalenone in edible and medicinal Coix seed

Food Chemistry ◽

10.1016/j.foodchem.2020.128977 ◽

2021 ◽

pp. 128977

Author(s):

Xiaofei Liu ◽

Xiaofang Liao ◽

Boyu Jia ◽

Chaonan Sun ◽

Lidong Zhou ◽

...

Keyword(s):

High Throughput ◽

Magnetic Bead ◽

Coix Seed ◽

High Throughput Detection ◽

Probe Assay

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Assessing high-throughput environmental DNA extraction methods for meta-barcode characterization of aquatic microbial communities

Journal of Water and Health ◽

10.2166/wh.2018.108 ◽

2018 ◽

Vol 17 (1) ◽

pp. 37-49 ◽

Cited By ~ 6

Author(s):

Abdolrazagh Hashemi Shahraki ◽

Subba Rao Chaganti ◽

Daniel Heath

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

High Throughput ◽

Environmentally Friendly ◽

Environmental Dna ◽

Magnetic Bead ◽

Extraction Methods ◽

Environmental Research ◽

Rrna Gene

Abstract The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

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Finding diabetic nephropathy biomarkers in the plasma peptidome by high-throughput magnetic bead processing and MALDI-TOF-MS analysis

PROTEOMICS - CLINICAL APPLICATIONS ◽

10.1002/prca.200900169 ◽

2010 ◽

Vol 4 (8-9) ◽

pp. 697-705 ◽

Cited By ~ 17

Author(s):

Henning G. Hansen ◽

Julie Overgaard ◽

Maria Lajer ◽

Frantisek Hubalek ◽

Peter Højrup ◽

...

Keyword(s):

Diabetic Nephropathy ◽

High Throughput ◽

Magnetic Bead ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Ms Analysis ◽

Tof Ms

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A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

Malaria Journal ◽

10.1186/s12936-020-03436-w ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Michal Kucharski ◽

Jaishree Tripathi ◽

Sourav Nayak ◽

Lei Zhu ◽

Grennady Wirjanata ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Rna Extraction ◽

Transcriptome Data ◽

High Quality ◽

Diverse Range ◽

Human Haemoglobin ◽

Infected Blood ◽

Blood Samples ◽

Total Rna

Abstract Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest 2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

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An Automated and High-Throughput Immunoaffinity Magnetic Bead-Based Sample Clean-Up Platform for the Determination of Aflatoxins in Grains and Oils Using UPLC-FLD

Toxins ◽

10.3390/toxins11100583 ◽

2019 ◽

Vol 11 (10) ◽

pp. 583 ◽

Cited By ~ 3

Author(s):

Zhihong Xuan ◽

Jin Ye ◽

Bing Zhang ◽

Li Li ◽

Yu Wu ◽

...

Keyword(s):

High Throughput ◽

High Performance ◽

Cost Effective ◽

Magnetic Bead ◽

The Novel ◽

Analysis Method ◽

Automated Method ◽

Accuracy And Precision ◽

Optimized Conditions

Sample clean-up remains the most time-consuming and error-prone step in the whole analytical procedure for aflatoxins (AFTs) analysis. Herein, an automated and high-throughput sample clean-up platform was developed with a disposable, cost-effective immunoaffinity magnetic bead-based kit. Under optimized conditions, the automated method takes less than 30 min to simultaneously purify 20 samples without requiring any centrifugation or filtering steps. When coupled to ultra-high performance liquid chromatography with fluorescence detection, this new analysis method displays excellent accuracy and precision as well as outstanding efficiency. Furthermore, an interlaboratory study was performed in six laboratories to validate the novel protocol. Mean recovery, repeatability, reproducibility, and Horwitz ratio values were within 91.9%–107.4%, 2.5%–7.4%, 2.7%–10.6%, and 0.26%–0.90, respectively. Results demonstrate that the developed sample clean-up platform is a reliable alternative to most widely adopted clean-up procedures for AFTs in cereals and oils.

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

Viruses ◽

10.3390/v12080863 ◽

2020 ◽

Vol 12 (8) ◽

pp. 863 ◽

Cited By ~ 2

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.

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