Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection

oleh: Dana Abdalghani AbuObead, Tasnim Khalid Alhomsi, Mahmoud Zhra, Bandar Alosaimi, Muaawia Hamza, Maaweya Awadalla, Osama Ezzeldin Abdelhadi, Joud Abdullah Alsharif, Liliane Okdah, Khaled AlKattan, Saeed Al Turki, Hana M. A. Fakhoury, Ahmad Aljada

Format: Article
Diterbitkan: MDPI AG 2022-12-01

Deskripsi

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the <i>RNA-dependent RNA polymerase</i> (<i>RdRp</i>) and <i>nucleocapsid</i> (<i>N</i>) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral <i>RdRP</i> and <i>N</i> genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.