WBT Viral Detection Technology Development and Application

Another innovative aspect of our proposed detection strategy is that measurements that use qRT-PCR will be compared with orthogonal, faster, and simpler colorimetric assays, including a novel fluorescence assay (FA) developed by members of our team at UM and a loop-mediated isothermal amplification (LAMP) assay (9a) developed by members of our team at WCM, which already has Emergency Use Authorization (EUA) approved by the FDA for human clinical testing. The major advantages of the FA are its simplicity and low cost. The cost of the assay is 10 to 20-fold lower than qRT-PCR, it is very simple to perform by an operator with minimal training, expensive equipment is not needed, and it can be completed in approximately one hour. In addition, the visual readout of positive versus negative reactions after PCR cycling is immediate and clear. Presently throughput is critical, and a laboratory with six relatively inexpensive thermocyclers could potentially perform approximately 500 tests per hour by an operator with basic training. While this approach can be used to test unprocessed saliva samples for the presence of SARS-CoV-2 RNA, this proposal would pioneer its first use with wastewater.

To enable more rapid utilization of the LAMP method, we have developed a portable instrument (called TINY – Tiny Isothermal Nucleic acid quantification sYstem) that can run the LAMP reaction when brought to wastewater facilities (Fig. A). The development of the TINY system came from our desire to create a field-portable, point-of-care, rugged, diagnostic tool for quantifying the nucleic acid content of suspected viruses in settings where little to no reliable infrastructure may be present (52a). The Mason lab worked with the Erickson lab at Cornell to perform extensive analysis of the performance of the TINY system and LAMP for COVID-19, performed under multiple operating conditions, in multiple locations, and by multiple user groups as reported in Snodgrass et al (52a). This includes laboratory-based analysis in which we showed near-identical sensitivities and specificities to qPCR, and field testing where we show near equivalent results independent of the training status of the user, energy source, or location. We also report approximately 30 minutes time to result (for amplification), a limit of detection around 20 viral copies/mL, and roughly equivalent quantification capabilities to qPCR above that limit of detection.

To further characterize the utility of the LAMP COVID-19 assay, a cohort of COVID-19 patient specimens was tested to demonstrate the efficacy of the LAMP assay as a diagnostic approach on clinical samples. The cohort consisted of 133 individuals that tested positive and 205 individuals that tested negative for SARS-CoV-2 by qRT-PCT, of which 182 had enough material available for testing. The CDC-recommended, maximum cycle threshold (Ct) for diagnostic positives was 40, with an average of 23.1 Ct in the positive cohort and undetectable in the negative cohort. By using the fluorescence measurement of the previously (qRT-PCR) established positive and negative samples as the gold standards, a Receiver Operator Characteristic (ROC) plot was then generated to estimate the diagnostic sensitivity and the specificity of the assay. After running the LAMP assay for 30 minutes, the resultant data showed an overall sensitivity of 96% and specificity of 100% as well as a positive predictive value (PPV) of 98% and a negative predictive value (NPV) of 100%. Also of note, with increasing viral load, as measured by Ct values, the LAMP assay shows an increased diagnostic sensitivity.