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Development and validation of an in-house, low-cost SARS-CoV-2 detection assay.
Alhamlan, Fatimah S; Al-Qahtani, Ahmed A; Bakheet, Dana M; Bohol, Marie F; Althawadi, Sahar I; Mutabagani, Maysoon S; Almaghrabi, Reem S; Obeid, Dalia A.
  • Alhamlan FS; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Al-Qahtani AA; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; College of Medicine, Alfaisal University, Riyadh, Saudi Arabia.
  • Bakheet DM; College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Bohol MF; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Althawadi SI; Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Mutabagani MS; Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Almaghrabi RS; Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
  • Obeid DA; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
J Infect Public Health ; 14(9): 1139-1143, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1338435
ABSTRACT

BACKGROUND:

One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol.

METHODS:

We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory.

RESULTS:

The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89).

CONCLUSIONS:

These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Clinical Practice Guide / Health economic evaluation Limits: Humans Language: English Journal: J Infect Public Health Journal subject: Communicable Diseases / Public Health Year: 2021 Document Type: Article Affiliation country: J.jiph.2021.07.009

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Clinical Practice Guide / Health economic evaluation Limits: Humans Language: English Journal: J Infect Public Health Journal subject: Communicable Diseases / Public Health Year: 2021 Document Type: Article Affiliation country: J.jiph.2021.07.009