**3. PCR detection of COVID-19 from saliva utilizing bead beating homogenization**

After months of nasopharyngeal swabbing for COVID-19 diagnostic testing, there was a push to look for equally sensitive testing methodologies which provided a more pleasant patient experience during sample collection [4, 6, 9]. By improving the patient experience with testing, the hope was to gain public cooperation with viral surveillance efforts [9, 13]. The high concentration of SARS-CoV-2 particles found throughout the upper respiratory tract led researchers to begin examining the utility of oral swabs or saliva in the current RT-qPCR testing strategies [13, 14].

Saliva samples were shown to have adequate viral loads for reliable RT-qPCR detection, however the high viscosity of the samples made them difficult to pipette preventing the utilization of the fully automated extraction machinery already in place in many large public health testing facilities [9, 13, 14]. Mechanical homogenization in the form of beat beating homogenization was introduced to saliva samples to break up the viscous structure and expose the viral particles [4, 15]. The beat beating strategy utilized ceramic bead media within a 2 mL screw capped sample tube and a mechanical homogenizer to apply rigorous kinetic energy to the saliva sample for 30 seconds to achieve complete dissociation (**Figure 2**) [4, 15]. It was shown that the kinetic energy transferred from the bead beading media homogenized in a sigmoidal pattern was highly effective in dissociating the sample to allow for pipettable lysate that could then be implemented into fully automated extraction-based PCR testing workflows [1, 4, 15]. With the addition of bead beating homogenization to this workflow, the throughput and sensitivity of the assay were dramatically increased [4, 15]. Prior to the implementation of beat beating homogenization, saliva-based testing demonstrated a sensitivity in the mid to low 80% range and throughput was limited to a few hundred samples per day via manual processing [4, 15]. Currently, saliva-based PCR testing utilizing mechanical homogenization prior to extraction procedures demonstrated a 95% sensitivity and 99% specificity, closely matching that of nasopharyngeal swab-based testing for COVID-19 [4, 13–15]. Additionally, with the capability of full automation integration, throughput of sample processing increased from hundreds to thousands of samples per day with the utilization of bead beating homogenization equipment [4, 15].

#### **Figure 2.**

*The methodology for saliva-based testing utilizing bead beating mechanical homogenization for adequate sample dissociation and viral lysis followed by RNA extraction for RT-qPCR viral detection.*

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

Given the similar sensitivity and specificity for SARS-CoV-2 testing, with the improvement in patient experience during sample collection, this method was implemented at universities across the state of Georgia as a community surveillance program [4, 15]. The difference in patient experience from obtaining a nasopharyngeal swab versus a saliva sample for weekly surveillance measures dramatically improved community compliance with testing, validating saliva testing as a viable public health surveillance strategy for COVID-19 propagation in a community [4, 6, 13–15]. Similar entities have now implemented saliva-based testing that utilize front-end mechanical homogenization across the world to improve compliance with public health testing efforts [4, 6, 15].
