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

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 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.

SUBMITTER: Dorlass EG

PROVIDER: S-EPMC7411266 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Dorlass Erick Gustavo EG Monteiro Cairo Oliveira CO Viana Amanda Oliveira AO Soares Camila Pereira CP Machado Rafael Rahal Guaragna RRG Thomazelli Luciano Matsumiya LM Araujo Danielle Bastos DB Leal Fabyano Bruno FB Candido Erika Donizette ED Telezynski Bruna Larotonda BL Valério Camila Araujo CA Chalup Vanessa Nascimento VN Mello Ralyria R Almeida Flavia Jaqueline FJ Aguiar Andressa Simões AS Barrientos Anna Carlotta Mott ACM Sucupira Carolina C De Paulis Milena M Sáfadi Marco Aurélio Palazzi MAP Silva Daniella Gregorio Bonfim Prado DGBP Sodré Janaina Joice Martins JJM Soledade Mariana Pereira MP Matos Samantha Faria SF Ferreira Sabrina Rodrigues SR Pinez Célia Miranda Nunez CMN Buonafine Carolina Palamin CP Pieroni Leticia Nery Ferreira LNF Malta Fernanda Mello FM Santana Rubia Anita Ferraz RAF Souza Eloisa Corrêa EC Fock Ricardo Ambrosio RA Pinho João Renato Rebelo JRR Ferreira Luís Carlos Souza LCS Botosso Viviane Fongaro VF Durigon Edison Luiz EL Oliveira Danielle Bruna Leal DBL

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20200807 3

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) we ...[more]

PMID: 32767275

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Project description:Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/?L of RNA for RT-qPCR and 10 copies/?L of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.

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

Publications

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

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