scholarly journals Extraction of HCV-RNA from Plasma Samples: Development towards Semiautomation

2015 ◽  
Vol 2015 ◽  
pp. 1-4 ◽  
Author(s):  
Imran Amin ◽  
Tania Jabbar ◽  
Fawad Niazi ◽  
Muhammad Saeed Akhtar
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Hcv Rna ◽  
Diagnostic Purpose ◽  
Test Results ◽  
Cost Performance ◽  
Vacuum Filtration ◽  
Assay Performance ◽  
Taqman Technology ◽  
Real Time Qpcr

A semiautomated extraction protocol of HCV-RNA using Favorgen RNA extraction kit has been developed. The kit provided protocol was modified by replacing manual spin steps with vacuum filtration. The assay performance was evaluated by real-time qPCR based on Taqman technology. Assay linearity was confirmed with the serial dilutions of RTA (Turkey) containing 1×(106, 105, 104, and 103) IU mL−1. Comparison of test results obtained by two extraction methods showed a good correlation (r=0.95,n=30) with detection limit of 102 IU mL−1. The semiautomated vacuum filtration based protocol demonstrated high throughput: 35 minutes for the extraction of a batch of 30 samples (150 µL each) with reduced labor, time, waste, and cost. Performance characteristics of semiautomated system make it suitable for use in diagnostic purpose and viral load determinations.

scholarly journals Rapid point-of-care (POC) testing for Hepatitis C antibodies in a very high prevalence setting: persons injecting drugs in Tallinn, Estonia

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Anneli Uusküla ◽  
Ave Talu ◽  
Jürgen Rannap ◽  
David M. Barnes ◽  
Don Des Jarlais
Keyword(s):  
Hepatitis C ◽  
Point Of Care ◽  
Antibody Test ◽  
Hcv Rna ◽  
Test Results ◽  
Blood Draw ◽  
Persons Who Inject Drugs ◽  
Oral Swab ◽  
High Prevalence ◽  
Hcv Antibody

Abstract Background Between December 2018 and January of 2019, we evaluated the accuracy of the point-of-care Hepatitis C (HCV) antibody test (POC; OraQuick HCV) used at a community-based needle and syringe exchange program serving persons who inject drugs in Tallinn, Estonia. Methods We compared the results of screening for HCV antibodies by OraQuick (oral swab) and enzyme immunoassay (EIA; blood draw) and assessed test results implications in a high prevalence setting. Findings Of the 100 participants, 88 (88%) had reactive POC test results, and 93 were HCV antibody positive on EIA testing. Sensitivity, specificity and negative predictive value (NPV) for the POC assay with EIA as the relevant reference test were as follows: 94.6% (95% CI 90.0–99.2%), 100% and 58.3% (95% CI 30.4–86.2%). Of the 12 testing, HCV-negative with the POC only 7 (58.3%) were true negatives. Conclusions Oral swab rapid testing HCV screening in this nonclinical setting was sensitive and specific but had unacceptably low NPV. In high prevalence settings, POC tests with high sensitivity and that directly measure HCV RNA may be warranted.

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 Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

2016 ◽  
Vol 50 (2) ◽  
pp. 108-115 ◽  
Author(s):  
Alves Mônica Ghislaine Oliveira ◽  
Mario Pérez-Sayáns ◽  
Maria-Elena Padín-Iruegas ◽  
Maria Dolores Reboiras-López ◽  
José Manuel Suarez-Peñaranda ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods ◽  
Oral Cytology

Enterohemorrhagic Escherichia coli Colony Check Assay for the Identification of Serogroups O26, O45, O103, O111, O121, O145, and O157 Colonies Isolated on Plating Media

2014 ◽  
Vol 77 (7) ◽  
pp. 1212-1218 ◽  
Author(s):  
BURTON BLAIS ◽  
MYLÈNE DESCHÊNES ◽  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
High Throughput Screening ◽  
Enterohemorrhagic Escherichia Coli ◽  
Test Results ◽  
Bacterial Strains ◽  
Enrichment Broth ◽  
Substrate Solution ◽  
E Coli ◽  
Assay Performance

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin–producing E. coli serogroups (all unreactive), and 33 non–E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

scholarly journals PERBANDINGAN IDENTIFIKASI PEMBICARA MENGGUNAKAN MFCC DAN SBC DENGAN CIRI PENCOCOKAN LBG-VQ

2018 ◽  
Author(s):  
Purwono Prasetyawan
Keyword(s):  
Feature Extraction ◽  
Human Behavior ◽  
Vector Quantization ◽  
Speaker Identification ◽  
Extraction Methods ◽  
Behavioral Characteristics ◽  
Physical Analysis ◽  
Test Results ◽  
Voice Signal ◽  
Signal Characteristics

Biometrics is a technology for physical analysis and human behavior used in authentication. One of the behavioral characteristics associated with a person is sound. A person's voice can be identified based on the person's voice signal characteristics. There are several methods in recognizing speaker sound, such as with Mel Frequency Cepstrum Calculation (MFCC) and Subband Based Cepstral (SBC). This study looked for the effectiveness of the use of MFCC and SBC feature extraction with LBG Vector Quantization matching characteristics. Effective feature extraction methods will be tested for realtime speaker identification. The results obtained from this research is the value of MFCC 32 coefficient more effective than the SBC accurately and the speed of the process of identification of both text-dependent and text-independent speaker. The test results of speaker identification in realtime using MFCC is still not satisfactory because the accuracy of recognition is still below 70%.

scholarly journals One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

scholarly journals Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

scholarly journals Optimal RNA Extraction Methods and Development of Synthetic Clones for Seven Strawberry Viruses

2020 ◽  
Vol 26 (3) ◽  
pp. 170-178
Author(s):  
Sun-Jung Kwon ◽  
Ju-Yeon Yoon ◽  
In-Sook Cho ◽  
Bong-Nam Chung
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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