Anais do Congresso Brasileiro de Virologia & Encontro de Virologia do Mercosul

A FAST MAGNETIC BEAD BASED RNA EXTRACTION METHOD FOR COVID-19 DIAGNOSE

Published in 04/01/2021 - ISBN: 978-65-5941-175-7

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LinkSubmission
DOI
10.29327/cbv.297770  
Paper Title
A FAST MAGNETIC BEAD BASED RNA EXTRACTION METHOD FOR COVID-19 DIAGNOSE
Authors
  • Leila Sabrina Ullmann
  • Fábio Sossai Possebon
  • Camila Dantas Malossi
  • Gabrielle Thaís Miodutzki
  • Evelyn Cristine da Silva
  • Eduardo Ferreira Machado
  • Iolanda Simões Braga
  • Isadora Fernanda Pelaquim
  • João Pessoa Araujo Junior
Modality
Resumo
Subject area
Humana
Publishing Date
04/01/2021
Country of Publishing
Brasil
Language of Publishing
Inglês
Paper Page
https://www.even3.com.br/anais/cbv/297770-a-fast-magnetic-bead-based-rna-extraction-method-for-covid-19-diagnose
ISBN
978-65-5941-175-7
Keywords
COVID, RT-qPCR, magnetic beads, RNA.
Summary
Coronavirus Disease 2019 (COVID-19), the pandemia caused by SARS-CoV-2, has an incubation period of 1 to 14 days, during which time the infected individual is contagious even without symptoms. Thus, rapid and accurate diagnostic methods have been of great importance. The gold standard diagnostic is the RT-qPCR using nasopharyngeal swabs, throat swabs or saliva samples. For nucleic acid purification, commercial RNA extraction kits are widely used. However, due the high demand there is a difficulty in obtaining these materials in addition to the high acquisition costs. Therefore, an in house extraction protocol was evaluated looking for a fast, easy to perform and repeatable purification in addition to a reduced cost. The present study aimed to compare and evaluate the adapted extraction protocol with commercially available MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific, Walthan, MA, USA) using SARS-CoV-2 positive nasopharyngeal and throat swabs. The in house extraction protocol developed for SARS-CoV-2 detection was adapted from the Bio-On-Magnetic-Beads (BOMB) platform, based on Guanidine Isothiocyanate cell lysis and nuclease inactivation and magnetic beads purification. Fifteen positive samples, with Cq ranging from 15 to 30, and one negative sample were selected. After a brief spin, 200 µL of sample was added into the first column of a previously prepared extraction plate for automated RNA extraction (UniXtractor™ deep well plates, Uniscience Corp., Miami, FL, USA). The wells that received the sample had 400 µL of Lysis Buffer. The beads solution were prepared with GE Healthcare Sera-Mag™Magnetic SpeedBeads™. After lysis and binding step, two washes were performed (150µL of isopropanol 100% and 200 µL of 70% ethanol, respectively) and RNA was eluted in 100 µL of elution buffer. Concomitantly, the same samples were extracted with the MagMAX™ CORE Nucleic Acid Purification commercial kit, according to the manufacturer instructions. The UniXtractor™ (Uniscience) was used to extract RNA from all the samples simultaneously. RNA samples were submitted to the RT-qPCR, using KiCqStart™ One-Step Probe RT-qPCR ReadyMix™ kit (Sigma-Aldrich, San Luis, MI, USA). Cycling conditions were 50°C for 10 minutes, 95°C for 3 minutes, 45 cycles at 95°C for 10 seconds and 60°C for 30 seconds. Cycle quantification (Cq) values for both extraction methods were compared. Paired T Test was used to evaluate the difference between the extraction methods. Pearson Correlation Coefficient (R) was also calculated. All positive samples revealed amplification in qPCR, which was not detected in the negative sample. The mean Cq values for the in-house protocol was 20.41 (95% CI = 17.86 - 22.96) and 21.02 (95% CI = 18.38 - 23.66) for the commercial kit. The in-house protocol produced a 0.61 lower mean Cq than the commercial kit (p=0.0009). Both methods had a strong correlation (r=0.99; p<0,0001). The in-house protocol revealed to be a robust RNA extraction method, with lower costs when compared to commercial kits, and a viable alternative for the detection of SARS-CoV-2 in nasopharyngeal and throat swabs.
Title of the Event
XXXI Congresso Brasileiro de Virologia & XV Encontro de Virologia do Mercosul
Title of the Proceedings of the event
Anais do Congresso Brasileiro de Virologia & Encontro de Virologia do Mercosul
Name of the Publisher
Even3
Means of Dissemination
Meio Digital
DOI

How to cite

ULLMANN, Leila Sabrina et al.. A FAST MAGNETIC BEAD BASED RNA EXTRACTION METHOD FOR COVID-19 DIAGNOSE.. In: Congresso Brasileiro de Virologia & Encontro de Virologia do Mercosul. Anais...Porto Alegre(RS) Online, 2020. Available in: https//www.even3.com.br/anais/cbv/297770-A-FAST-MAGNETIC-BEAD-BASED-RNA-EXTRACTION-METHOD-FOR-COVID-19-DIAGNOSE. Access in: 01/06/2025

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