Background

Polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-qPCR)-based methodologies are the current mainstay of detection for human viral infections (Elnifro et al., 2000). These PCR methodologies often rely on multiple chemical extraction steps, for lysis of the viral particles from the clinical sample, followed by a series of washing steps, which are meant to purify the exposed viral genetic material while removing other macromolecules, such as carbohydrates and proteins, that may impact downstream molecular applications (Elnifro et al., 2000). Once the purified genetic material has been isolated, it is then placed into the PCR mixture for detection of a specific targeted genetic sequence, thereby verifying the presence of the virus. This traditional extraction model for PCR detection of virus is highly sensitive and specific in confirming infection (Elnifro et al., 2000). However, as seen in the global pandemic caused by SARS-CoV-2, the stressed global supply chain could not always ensure availability of the chemical reagents needed for these extractions, driving the need for novel diagnostic techniques (Bustin et al., 2020).

Herein, we describe a methodology where mechanical homogenization was used to lyse viral particles, exposing RNA for accurate detection of the virus when placed directly into PCR, without undergoing further extraction steps or chemical washes (Morehouse et al., 2020). This methodology has been reported utilizing in vitro simulation of clinical viral samples with human coronavirus 229E (HCoV-229E) (Morehouse et al., 2020), as well as in a field validation study with the use of nasopharyngeal and oropharyngeal swabs for the detection of SARS-CoV-2 from patients (Morehouse et al., 2021). This homogenization-based direct-to-PCR technique allows for accurate viral detection of human coronaviruses, without the need for time consuming and costly chemical extraction techniques traditionally used in RT-qPCR detection workflows (Morehouse et al., 2020).

In an effort to improve access to cost effective diagnostics solutions, we demonstrate how human coronavirus can be accurately detected in clinical concentrations with similar sensitivity and specificity to the current gold standard extraction-based techniques (Morehouse et al., 2020 and Morehouse et al., 2021). It is critical that innovative diagnostic solutions such as this are continually developed with concern for the logistical challenges of providing accurate viral detection in resource-limited settings. Through the utilization of mechanical homogenization, we were able to detect HCoV-229E off spiked swabs, simulating clinically relevant concentrations of virus on nasopharyngeal swabs as an initial proof of concept study for this technique (Morehouse et al., 2021). The homogenization-based direct-to-PCR methodology was then further validated with patient samples screening for SARS-CoV-2 in a later study (Morehouse et al., 2021). In this manuscript, we lay out the detailed protocol utilized in the initial proof of concept testing of our homogenization-based direct-to-PCR technology, utilizing simulated nasopharyngeal swabs with spiked HCoV-229E to determine the limit of detection for this proposed methodology.