Journal Article

Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR

Buonafine, Carolina Palamin, Fock, Ricardo Ambrosio, Barrientos, Anna Carlotta Mott, Viana, Amanda Oliveira, Ferreira, Luís Carlos Souza, Aguiar, Andressa Simões, Valério, Camila Araujo, Pinez, Célia Miranda Nunez, Leal, Fabyano Bruno, Monteiro, Cairo Oliveira, Matos, Samantha Faria, Oliveira, Danielle Bruna Leal, Santana, Rubia Anita Ferraz, Botosso, Viviane Fongaro, Telezynski, Bruna Larotonda, Machado, Rafael Rahal Guaragna, Soledade, Mariana Pereira, Dorlass, Erick Gustavo, Soares, Camila Pereira, Candido, Erika Donizette, Sucupira, Carolina, Sodré, Janaina Joice Martins, Mello, Ralyria, Araujo, Danielle Bastos, Pinho, João Renato Rebelo, Thomazelli, Luciano Matsumiya, Chalup, Vanessa Nascimento, Malta, Fernanda Mello, Ferreira, Sabrina Rodrigues, Pieroni, Leticia Nery Ferreira, Durigon, Edison Luiz, Sáfadi, Marco Aurélio Palazzi, Souza, Eloisa Corrêa, Silva, Daniella Gregorio Bonfim Prado, De Paulis, Milena, Almeida, Flavia Jaqueline

2020

Overview

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 10 7 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.

• Publisher: Springer International Publishing,Springer Nature B.V
• Subject: Adolescent / Adult / Animals / Assaying / Betacoronavirus - genetics / Betacoronavirus - isolation & purification / Biomedical and Life Sciences / Child / Chlorocebus aethiops / Clinical Microbiology - Research Paper / Coronavirus Infections - diagnosis / Coronavirus Infections - economics / Coronaviruses / COVID-19 / Cross Reactions / Cross-reactivity / Diagnosis / Dilution / E protein / Enzymes / Epidemics / Fluorescent Dyes - economics / Food Microbiology / Hospitals / Humans / Identification methods / Influenza / Laboratories / Life Sciences / Medical Microbiology / Microbial Ecology / Microbial Genetics and Genomics / Microbiology / Middle Aged / Mycology / Nasopharynx - virology / Organic Chemicals - economics / Oropharynx - virology / Pandemics / Pandemics - economics / Pneumonia, Viral - diagnosis / Pneumonia, Viral - economics / Polymerase chain reaction / Real time / Real-Time Polymerase Chain Reaction - economics / Real-Time Polymerase Chain Reaction - methods / RNA, Viral - isolation & purification / SARS-CoV-2 / Sensitivity and Specificity / Severe acute respiratory syndrome coronavirus 2 / Thermal cycling / Vero Cells / Viral diseases / Viruses / Young Adult