scholarly journals Comparison of RNA extraction methods for the detection of SARS-CoV-2 by RT-PCR

2020 ◽  
Author(s):  
Ossia M Eichhoff ◽  
Elisa Bellini ◽  
Reto Lienhard ◽  
Wendelin J. Stark ◽  
Philippe Bechtold ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Diagnostic Quality ◽  
Viral Spread ◽  
Significant Difference ◽  
Rna Extraction Methods

Objectives: The SARS-CoV-2 pandemic outbreak has stressed health care systems as well as medical supply chains, but diagnostic testing is an essential public health measure to control viral spread. Here we test the suitability of different RNA extraction methods for integration into a diagnostic workflow for coronavirus testing. Methods: We applied six RNA extraction methods on the same 24 SARS-CoV-2 positive patient samples and quantified their results by subsequent reverse-transcriptase PCR (RT-PCR) of three viral genes. These methods included a) column-based extraction, b) phenol-chloroform extraction, as well as c) extraction using magnetic beads (i.e., one commercial kit as well as three different magnetic beads in combination with home-brewed buffers and solutions). Results: We achieved diagnostic-quality RT-PCR results with all methods, and there was no significant difference between the tested methods, except for one magnetic bead protocol with home-brewed buffers, in which the number of positive tested genes was significantly lower. Conclusions: Five of the six RNA extraction methods are interchangeable in a diagnostic workflow. Since some methods are more scalable than others, and have comparable results on RT-PCR quantitation, they may be more amenable to high-throughput sample processing pipelines.

Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR

2015 ◽  
Vol 160 (7) ◽  
pp. 1791-1796 ◽  
Author(s):  
Xiaoli Zhao ◽  
Qi Zhou ◽  
Lijie Zhang ◽  
Wenlong Yan ◽  
Ning Sun ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Rna Extraction Methods

scholarly journals Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

2005 ◽  
Vol 17 (6) ◽  
pp. 574-578 ◽  
Author(s):  
Ming Y. Deng ◽  
He Wang ◽  
Gordon B. Ward ◽  
Tammy R. Beckham ◽  
Thomas S. McKenna
Keyword(s):  
Real Time ◽  
Classical Swine Fever Virus ◽  
Rna Extraction ◽  
Classical Swine Fever ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcription Pcr ◽  
Rna Extraction Methods ◽  
Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

scholarly journals COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

2016 ◽  
Vol 38 (1) ◽  
pp. 226-232 ◽  
Author(s):  
ANNY CAROLYNE DA LUZ ◽  
IRANY RODRIGUES PRETTI ◽  
MARIA DO CARMO PIMENTEL BATITUCCI
Keyword(s):  
Rna Extraction ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Passiflora Edulis ◽  
Genomic Analyses ◽  
Oxidative Processes ◽  
Rna Extraction Methods ◽  
Polymerase Chain ◽  
Low Levels

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

2016 ◽  
Vol 38 (3) ◽  
pp. 382-388 ◽  
Author(s):  
Nur D. Roslan ◽  
Ong A. Meilina ◽  
Intan-Nur A. Mohamed-Azni ◽  
Idris A. Seman ◽  
Shamala Sundram
Keyword(s):  
Oil Palm ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Rna Extraction Methods

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

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.

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.

Sign in / Sign up

Export Citation Format

Share Document