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

Abstract

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 RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N 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.

Keywords: COVID-19; SARS-CoV-2; in-house RNA isolation; magnetic beads RNA isolation; multiplex RT-PCR; silica-based RNA isolation.

Figure 1

Optimization of lysis buffers’ ingredients used in our in-house silica-column-based RNA isolation kits; (A) effect of ethanol concentration on RNA binding to silica columns; (B) comparison of Invitrogen Purelink kit with the in-house RNA isolation in the presence of two non-ionic detergents (Nonidet P40 and Tergitol solution type NP-40); and (C) the effect of glycogen as carrier RNA on RNA yield using Lysis Buffer II and 50% ethanol. The results are presented as mean ± SD, *: p < 0.05 compared with other methods.

Figure 2

Optimization of lysis buffers used in our in-house RNA isolation kits utilizing silica-coated magnetic beads (50 mg/mL); (A) DynaMag^TM-2 Magnet; (B) QuickPick; (C) SpiralPipet (M0022-S) Mix magnetic bead handling system; and (D) DynaMag^TM-2 Magnet with 2.5 µL proteinase K (40 mg/mL). The results are presented as mean ± SD, *: p < 0.05 when compared with 5 µL volume.

Figure 3

Comparison of the silica-based in-house RNA isolation kit and silica-coated magnetic beads (10 µL of 50 mg/mL) in-house RNA isolation method using either DynaMag^TM-2 Magnet or SpiralPipet (M0022-S) Mix magnetic bead handling system. Similar buffers were used in the three methods for RNA isolation. The results are presented as mean ± SD, *: p < 0.05 when compared with other methods.

Figure 4

Optimization of lysis buffers used in our in-house RNA/DNA isolation kit with Wash Buffer I and II for the isolation of RNA and DNA utilizing magnetic beads (50 mg/mL); (A) DynaMag^TM-2 Magnet and (B) SpiralPipet (M0022-S) Mix magnetic bead handling system. Proteinase K, 2.5 µL of 40 mg/mL, was added to Lysis Buffer II, and incubation was carried out at room temperature in both A and B. The results are presented as mean ± SD, *: p < 0.05 when compared with 5 µL volume.

Figure 5

Comparison of our in-house RNA/DNA isolation kit with Wash Buffer I and II (RNA and DNA) utilizing silica-coated magnetic beads (50 mg/mL) with QuickPick™ SML total RNA purification kit, which requires incubation of the sample with Proteinase K—2.5 µL (40 mg/mL) at 56 °C. DynaMag^TM-2 Magnets were used with both kits. The results are presented as mean ± SD, *: p < 0.05 when compared with the method at 56 °C incubation temperature.

Figure 6

Effect of RPP30 primers/probe designed for CDC protocol (N1 and RPP30) on NTC. (A) Both (N1 and RPP30) gave lower intensity and a high NTC in COVID-19 multiplex rRT-PCR; (B) the newly designed RPP30 primers/probe gave no NTC up to 50 cycles and increased RFU.