**2. Direct-to-PCR testing with shaker mill homogenization of nasopharyngeal swabs**

During the late spring of 2020, while SARS-CoV-2 was spreading exponentially and uncontrollably across the globe, testing for this disease was focused entirely on RT-qPCR detection of the virus using US CDC or WHO approved primers [5, 10]. The traditional method for these types of RT-qPCR tests involved two major components. First, the process of virus inactivation and RNA extraction completed through a series of chemical reactions that resulted in purified viral RNA from the provided patient sample [5, 6, 10]. The extracted RNA was then utilized in the second half of this method, amplification and detection [5, 6, 10]. Through RT-qPCR, the purified RNA from the patient sample was combined with the preapproved primers for attempted amplification of the targeted genes, indicating the presence or absence of SARS-CoV-2 depending on the level of amplification seen [5, 6, 10]. The RNA amplification was quantified and reported out as a Cq value, with any Cq less than 40 qualifying as a COVID-19 positive sample per the US CDC and WHO guidelines [10].

The necessity of testing drove up demand for all reagents, machines, and plastics utilized in the RT-qPCR testing method, overstressing the supply chain for these products [5, 8, 9]. Additionally, the need for cold storage of reagents involved in the extraction process and the high price tag on the automated machinery needed to complete both the extraction and detection phases of the traditional testing method, furthered the gap between resource challenged areas and the industrialized regions when it came to COVID-19 testing infrastructure [11, 12]. Areas with the capital needed to create multimillion dollar testing facilities were able to do so, improving their public health response to the pandemic, while those lacking that investment and infrastructure were left with reduced testing capabilities [11]. A critical need

#### *The Utility of Mechanical Homogenization in COVID-19 Diagnostic Workflows DOI: http://dx.doi.org/10.5772/intechopen.97110*

arose for a cost efficient, yet safe and effective testing methodology that could be implemented in these resources challenged settings [8, 11].

While the utility of mechanical homogenization in COVID-19 testing was already established as an effective adjunct to the extraction process, improving sensitivity through efficient viral lysis, this process was expanded upon in an attempt to remove the extraction process entirely allowing for direct detection of SARS-CoV-2 from lysed patient samples [2, 3]. The direct-to-PCR approach for COVID-19 testing arose out of necessity to reduce the use of costly reagents in a period where the strain on the supply chain made them difficult to come by [2, 3, 8]. Additionally, this proposed method dramatically reduces cost when compared with the fully automated extraction machinery [2, 3, 8].

In the direct-to-PCR method for viral detection, shaker mill mechanical homogenization was proposed to provide sufficient viral lysis off nasopharyngeal swabs to expose adequate amounts of RNA for RT-qPCR detection [2, 3]. This method was shown to lyse greater than 95% of virus off a nasopharyngeal swab, allowing the resultant lysate to be placed directly into the RT-qPCR reaction as denoted in **Figure 1** [2, 3].

Through proof-of-concept testing with a close relative of SARS-CoV-2, human coronavirus 229E (HCoV-229E), and direct comparison studies between the traditional extraction-based method and the direct-to-PCR method, it was shown that the two methods had above a 94% agreeability in the detection of positive samples [2, 3]. Utilizing the direct-to-PCR method diagrammed in **Figure 1**, shaker mill homogenization was proven to be a viable alternative to the traditional extraction-based method for RT-qPCR detection of SARS-CoV-2 off nasopharyngeal swabs [2, 3].

In addition to the quality of the matched proven efficacy with the traditional, extraction-based methodology, the direct-to-PCR method described utilizing mechanical homogenization also reduces the total cost and time per swab processed [2, 3, 8]. The traditional model for nasopharyngeal swab viral testing cost \$10 - \$40 USD per swab, when taking into account the extraction kits, automation equipment for extractions, and the RT-qPCR set up [8]. Compared to \$3 - \$5 USD per swab with the homogenization methodology, given that this workflow does not require additional reagents for viral nucleotide extraction and purification, the only reagent costs are associated with the final RT-qPCR testing [8]. The homogenization equipment utilized in this workflow is sold at a fraction of the cost of the large fully automated extraction machinery.

Along with reducing cost per sample the homogenization workflow reduces the total processing time per sample from approximately 3 hours to 1 hour and 15 minutes [2, 3]. This is accomplished through replacing the extraction and purification steps of the traditional workflow with a 30 sec homogenization step preceding the RT-qPCR [2, 3]. Further supporting the implementation of this workflow into

**Figure 1.** *The direct-to-PCR viral detection methodology using shaker mill homogenization off nasopharyngeal swabs.*

the COVID-19 testing repertoire to assist in increasing access to cost effective and timely viral detection methods that maintain sensitivity and specificity when compared to the traditional testing methodologies [8].
