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

• Published in: Current issues in molecular biology 2022-12, Vol.44 (12), p.6117-6131
• Main Authors: AbuObead, Dana Abdalghani, Alhomsi, Tasnim Khalid, Zhra, Mahmoud, Alosaimi, Bandar, Hamza, Muaawia, Awadalla, Maaweya, Abdelhadi, Osama Ezzeldin, Alsharif, Joud Abdullah, Okdah, Liliane, AlKattan, Khaled, Turki, Saeed Al, Fakhoury, Hana M A, Aljada, Ahmad
• Format: Article
• Language: English
• Subjects: COVID-19; in-house RNA isolation; magnetic beads RNA isolation; multiplex RT-PCR; SARS-CoV-2; silica-based RNA isolation
• ISSN: 1467-3045; 1467-3037; 1467-3045
• DOI: 10.3390/cimb44120417

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 ( ) and ( ) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral and 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.