A multiplexed Cas13-based assay with point-of-care attributes for simultaneous COVID-19 diagnosis and variant surveillance (preprint)

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection, such as multiplexed detection for viral variant surveillance, may limit their widespread adoption. Herein, we developed a robust multiplexed CRISPR-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose SARS-CoV-2 infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool, CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance which can be locally manufactured, may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Rapid SARS-CoV-2 testing in primary material based on a novel multiplex LAMP assay (preprint)

Background: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. Methods: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including loop-mediated isothermal amplification (LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). Results: Whilst specificity of standard LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel LAMP assays with SHERLOCK. Conclusion: In summary, this approach reveals one-step multiplexed LAMP assays as a prime-option for the development of easy and cheap POC test kits.

Point-of-care testing for COVID-19 using SHERLOCK diagnostics (preprint)

The recent outbreak of the novel coronavirus SARS-CoV-2, which causes COVID-19, can be diagnosed using RT-qPCR, but inadequate access to reagents and equipment has slowed disease detection and impeded efforts to mitigate viral spread. Alternative approaches based on combinations of isothermal amplification and CRISPR-mediated detection, such as the SHERLOCK ( S pecific H igh S ensitivity E nzymatic R eporter Un LOCK ing) technique, offer reduced dependence on RT-qPCR equipment, but previously reported methods required multiple fluid handling steps, complicating their deployment outside clinical labs. Here we developed a simple test chemistry called STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use. This simplified test, STOPCovid, provides sensitivity comparable to RT-qPCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns result in 70 minutes, and using fluorescence readout, the test returns result in 40 minutes. Moreover, we validated STOPCovid using nasopharyngeal swabs from COVID-19 patients and were able to correctly diagnose 12 positive and 5 negative patients out of 3 replicates. We envision that implementation of STOPCovid will significantly aid "test-trace-isolate" efforts, especially in low-resource settings, which will be critical for long-term public health safety and effective reopening of the society.

A 5-min RNA preparation method for COVID-19 detection with RT-qPCR (preprint)

RNA extraction has become a bottleneck for detection of COVID-19, in part because of reagent shortages. We present here a rapid protocol that circumvents the need for RNA extraction that is compatible with RT-qPCR-based detection methods.