Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR green-based RT-qPCR
Dorlass, Erick Gustavo; Cairo Oliveira Monteiro; Viana, Amanda Oliveira; Soares, Camila Pereira; Machado, Rafael Rahal Guaragna; Thomazelli, Luciano Matsumiya; Araujo, Danielle Bastos; Leal, Fabyano Bruno ; Candido, Erika Donizette; Telezynski, Bruna Larotonda; Valério, Camila Araujo; Chalup, Vanessa Nascimento; Mello, Ralyria; Almeida, Flavia Jaqueline; Aguiar, Andressa Simões; Barrientos, Anna Carlotta Mott; Sucupira, Carolina; Paulis, Milena De; Sáfadi, Marco Aurélio Palazzi; Silva, Daniella Gregorio Bonfim Prado; Sodré, Janaina Joice Martins; Soledade, Mariana Pereira; Matos, Samantha Faria; Ferreira, Sabrina Rodrigues; Pinez, Célia Miranda Nunez; Buonafine, Carolina Palamin; Pieroni, Leticia Nery Ferreira; Malta, Fernanda Mello; Santana, Rubia Anita Ferraz; Souza, Eloisa Corrêa; Fock, Ricardo Ambrosio; Pinho, João Renato Rebelo; Ferreira, Luís Carlos Souza; Botosso, Viviane Fongaro ; Durigon, Edison Luiz; Oliveira, Danielle Bruna Leal de
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 107 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.
COVID-19 pandemic; Molecular diagnoses; Conventional PCR; SYBR Green assay; SARS-CoV-2
Dorlass EG, COM, Viana AO, Soares CP, Machado RRG, Thomazelli LM, et al. Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR green-based RT-qPCR. Braz. J. Microbiol.. 2020 Aug;51:1117–1123. doi:10.1007/s42770-020-00347-5.
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