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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; Monteiro, Cairo Oliveira; 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; De Paulis, Milena; 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.
  • Dorlass EG; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Monteiro CO; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Viana AO; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Soares CP; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Machado RRG; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Thomazelli LM; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Araujo DB; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Leal FB; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Candido ED; Development and Innovation Center, Laboratory of Virology, Butantan Institute, São Paulo, Brazil.
  • Telezynski BL; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Valério CA; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Chalup VN; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Mello R; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Almeida FJ; Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Aguiar AS; Department of Pediatrics, School of Medical Sciences, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil.
  • Barrientos ACM; Infection Control Service, São Luiz Gonzaga Hospital of Santa Casa de Misericordia of São Paulo, São Paulo, Brazil.
  • Sucupira C; Infection Control Service and Epidemiological Hospital Nucleo, Municipal Children's Hospital Candido Fontoura, São Paulo, Brazil.
  • De Paulis M; Infection Control Service, Municipal Children's Hospital Menino Jesus, São Paulo, Brazil.
  • Sáfadi MAP; Infection Control Service and Epidemiological Hospital Nucleo, Municipal Children's Hospital Candido Fontoura, São Paulo, Brazil.
  • Silva DGBP; Department of Pediatrics, University Hospital, School of Medicine, University of São Paulo, São Paulo, Brazil.
  • Sodré JJM; Department of Pediatrics, School of Medical Sciences, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil.
  • Soledade MP; Infection Control Service, São Luiz Gonzaga Hospital of Santa Casa de Misericordia of São Paulo, São Paulo, Brazil.
  • Matos SF; Infection Control Service, Municipal Children's Hospital Menino Jesus, São Paulo, Brazil.
  • Ferreira SR; Infection Control Service and Epidemiological Hospital Nucleo, Municipal Children's Hospital Candido Fontoura, São Paulo, Brazil.
  • Pinez CMN; Department of Pediatrics, School of Medical Sciences, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil.
  • Buonafine CP; Infection Control Service, Municipal Children's Hospital Menino Jesus, São Paulo, Brazil.
  • Pieroni LNF; Clinical Laboratory Division, Pharmacy and Clinical Laboratory Department, University Hospital, University of São Paulo, São Paulo, Brazil.
  • Malta FM; Department of Pediatrics, School of Medical Sciences, Santa Casa de Misericórdia de São Paulo, São Paulo, Brazil.
  • Santana RAF; Infection Control Service, Municipal Children's Hospital Menino Jesus, São Paulo, Brazil.
  • Souza EC; Clinical Laboratory, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Fock RA; Clinical Laboratory, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Pinho JRR; Department of Pediatrics, University Hospital, School of Medicine, University of São Paulo, São Paulo, Brazil.
  • Ferreira LCS; Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
  • Botosso VF; Clinical Laboratory Division, Pharmacy and Clinical Laboratory Department, University Hospital, University of São Paulo, São Paulo, Brazil.
  • Durigon EL; Clinical Laboratory, Hospital Israelita Albert Einstein, São Paulo, Brazil.
  • Oliveira DBL; LIM03/07 Clinics Hospital, School of Medicine, University of São Paulo, São Paulo, Brazil.
Braz J Microbiol ; 51(3): 1117-1123, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-695574
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Organic Chemicals / Pneumonia, Viral / Coronavirus Infections / Real-Time Polymerase Chain Reaction / Fluorescent Dyes / Betacoronavirus Type of study: Diagnostic study / Randomized controlled trials Limits: Adolescent / Adult / Animals / Child / Humans / Middle aged / Young adult Language: English Journal: Braz J Microbiol Year: 2020 Document Type: Article Affiliation country: S42770-020-00347-5

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Organic Chemicals / Pneumonia, Viral / Coronavirus Infections / Real-Time Polymerase Chain Reaction / Fluorescent Dyes / Betacoronavirus Type of study: Diagnostic study / Randomized controlled trials Limits: Adolescent / Adult / Animals / Child / Humans / Middle aged / Young adult Language: English Journal: Braz J Microbiol Year: 2020 Document Type: Article Affiliation country: S42770-020-00347-5