Use of alternative RNA storage and extraction reagents and development of a hybrid PCR-based method for SARS-CoV-2 detection

The COVID-19 pandemic has presented multiple healthcare challenges, one of which is adequately meeting the need for large-scale diagnostic testing. The most commonly used assays for detection of SARS-CoV-2, including those recommended by the Center for Disease Control and Prevention (CDC), rely on a consistent set of core reagents. This has put a serious strain on the reagent supply chain, resulting in insufficient testing. It has also led to restricted animal testing, even though there are now multiple reports of animals, particularly cats, ferrets and minks, contracting the disease. We aimed to address the diagnostic bottleneck by developing a PCR-based SARS-CoV-2 detection assay for cats (and, potentially, other animals) which avoids the use of most common reagents, such as collection kits optimized for RNA stabilization, RNA isolation kits and TaqMan-based RT-PCR reagents. We demonstrated that an inexpensive solid-phase reversible immobilization (SPRI) method can be used for RNA extraction from feline samples collected with DNAGenoteks ORAcollect RNA OR-100 and PERFORMAgene DNA PG-100 sample collection kits, optimized for RNA or DNA stabilization, respectively. We developed a dual method SARS-CoV-2 detection assay relying on SYBR RT-PCR and Sanger sequencing, using the same set of custom synthesized oligo primers. We validated our tests specificity with a commercially available SARS-CoV-2 plasmid positive control, as well as two in-house positive control RNA samples. Our assays sensitivity was determined to be 10 viral copies per reaction. Our results suggest that a simple SPRI-dependent RNA extraction protocol and certain sample collection kits not specifically optimized for RNA stabilization could potentially be used in cases where reagent shortages are hindering adequate COVID-19 testing. These alternative reagents could be used in combination with our COVID-19 testing method, which relies on inexpensive and readily available SYBR RT-PCR and non-fluorescent PCR reagents. Depending on the detection goals and the laboratory setup available, the SYBR RT-PCR method and the Sanger sequencing based method can be used alone or in conjunction, for improved accuracy. Although the test is intended for animal use, it is, in theory, possible to use it with human samples, especially those with higher viral loads.