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

Author(s):Show less -

Dorlass, Erick Gustavo ^[1] ; Monteiro, Cairo Oliveira ^[1] ; Viana, Amanda Oliveira ^[1] ; Soares, Camila Pereira ^[1] ; Guaragna Machado, Rafael Rahal ^[1] ; Thomazelli, Luciano Matsumiya ^[1] ; Araujo, Danielle Bastos ^[1] ; Leal, Fabyano Bruno ^{[1, 2]} ; Candido, Erika Donizette ^[1] ; Telezynski, Bruna Larotonda ^[1] ; Valerio, Camila Araujo ^[1] ; Chalup, Vanessa Nascimento ^[1] ; Mello, Ralyria ^[1] ; Almeida, Flavia Jaqueline ^[3] ; Aguiar, Andressa Simoes ^{[4, 5, 6]} ; Mott Barrientos, Anna Carlotta ^[7] ; Sucupira, Carolina ^{[4, 5]} ; De Paulis, Milena ^[8] ; Palazzi Safadi, Marco Aurelio ^[3] ; Bonfim Prado Silva, Daniella Gregorio ^[6] ; Martins Sodre, Janaina Joice ^[7] ; Soledade, Mariana Pereira ^{[4, 5]} ; Matos, Samantha Faria ^[3] ; Ferreira, Sabrina Rodrigues ^[7] ; Nunez Pinez, Celia Miranda ^[9] ; Buonafine, Carolina Palamin ^[3] ; Ferreira Pieroni, Leticia Nery ^[7] ; Malta, Fernanda Mello ^[10] ; Ferraz Santana, Rubia Anita ^[10] ; Souza, Eloisa Correa ^[8] ; Fock, Ricardo Ambrosio ^{[11, 9]} ; Rebelo Pinho, Joao Renato ^{[10, 12]} ; Souza Ferreira, Luis Carlos ^[1] ; Botosso, Viviane Fongaro ^[2] ; Durigon, Edison Luiz ^{[1, 13]} ; Leal Oliveira, Danielle Bruna ^{[1, 2, 14]}

Affiliation:Show less -

^[1] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo - Brazil

^[2] Butantan Inst, Dev & Innovat Ctr, Lab Virol, Sao Paulo - Brazil

^[3] Santa Casa de Misericordia Sao Paulo, Dept Pediat, Sch Med Sci, Sao Paulo - Brazil

^[4] Municipal Childrens Hosp Candido Fontoura, Infect Control Serv, Sao Paulo - Brazil

^[5] Municipal Childrens Hosp Candido Fontoura, Epidemiol Hosp Nucleo, Sao Paulo - Brazil

^[6] Sao Luiz Gonzaga Hosp Santa Casa de Misericordia, Infect Control Serv, Sao Paulo - Brazil

^[7] Municipal Childrens Hosp Menino Jesus, Infect Control Serv, Sao Paulo - Brazil

^[8] Univ Sao Paulo, Univ Hosp, Sch Med, Dept Pediat, Sao Paulo - Brazil

^[9] Univ Sao Paulo, Univ Hosp, Pharm & Clin Lab Dept, Clin Lab Div, Sao Paulo - Brazil

^[10] Hosp Israelita Albert Einstein, Clin Lab, Sao Paulo - Brazil

^[11] Univ Sao Paulo, Sch Pharmaceut Sci, Dept Clin & Toxicol Anal, Sao Paulo - Brazil

^[12] Univ Sao Paulo, Clin Hosp LIM03 07, Sch Med, Sao Paulo - Brazil

^[13] Sci Platform Pasteur USP, Sao Paulo - Brazil

^[14] Univ Sao Paulo, Clin & Mol Virol Lab, Av Prof Lineu Prestes 1374, BR-05508900 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Brazilian Journal of Microbiology; v. 51, n. 3 AUG 2020.
• Web of Science Citations: 0

Abstract

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 averageCtvariation between SYBR Green and TaqMan was 1.92 and the highestCtdetected 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. (AU)

• FAPESP's process: 17/25123-9 - Monitoring of ectoparasites and viruses in bats in urban Atlantic Forest fragment
• Grantee: Amanda de Oliveira Viana
• Support type: Scholarships in Brazil - Master
• FAPESP's process: 16/20045-7 - Antigen discovery and development of serological diagnostic methods and vaccine approaches against the Zika Virus (ZIKV)
• Grantee: Luis Carlos de Souza Ferreira
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 18/23680-0 - Standardization of Sherlock technique (Specific High-Sensitivity Enzymatic Reporter Unlocking) using the Cas13a protein for the diagnosis of respiratory syncytial virus in pediatric patients
• Grantee: Camila Pereira Soares
• Support type: Scholarships in Brazil - Master
• FAPESP's process: 17/24769-2 - Zika virus in postpartum women and newborns: seroepidemiology and molecular characterization
• Grantee: Rafael Rahal Guaragna Machado
• Support type: Scholarships in Brazil - Doctorate (Direct)