Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Validation of the performance of a comprehensive genotyping assay panel of single nucleotide polymorphisms in drug metabolism enzyme genes.

A class of genes, known as drug metabolism enzymes (DMEs) are responsible for the metabolism and transport of drugs and other xenobiotics. Variation in DME genes most likely accounts for a proportion of the variability in drug response in humans, and may contribute to complex diseases such as cancer (Nebert DW, Dieter MZ. Pharmacology 2000;61:124-135). To date, assessing the extent of this variation has proven difficult, especially because of sequence paralogy issues that cause difficulty when attempting to genotype polymorphisms in very closely-related gene families (Murphy MP. Pharmacogenomics 2000;1:115-123; Ingelman-Sundberg M. Drug Metab Rev 1999;31:449-459). We have developed and genotyped a panel of N=2,325 individual TaqMan genotyping assays for polymorphisms in >200 DME genes; many of the variants in the panel are single nucleotide polymorphisms (SNPs) that are of known or putative function (e.g., missense, nonsense or frameshift). Using these assays, we have examined genetic variation among several groups of populations, including: 1) the two SNP500 Cancer population panels (http://snp500cancer.nci.nih.gov; last accessed: 11 December 2007); and 2) the panel used in the International HapMap Project panel (www.hapmap.org; last accessed: 11 December 2007). We have developed a comprehensive validation strategy to ensure reproducibility and accuracy of the assays and estimated minor allele frequencies. Here, we present the results of these analyses, which strongly suggest that this panel of DME assays are of extremely high quality and produce robust, accurate, and reproducible results.

Recovery efficiencies for Burkholderia thailandensis from various aerosol sampling media.

Burkholderia thailandensis is used in the laboratory as a surrogate of the more virulent B. pseudomallei. Since inhalation is believed to be a natural route of infection for B. pseudomallei, many animal studies with B. pseudomallei and B. thailandensis utilize the inhalation route of exposure. The aim of the present study was to quantify the recovery efficiency of culturable B. thailandensis from several common aerosol sampling devices to ensure that collected microorganisms could be reliably recovered post-collection. The sampling devices tested included 25 mm gelatin filters, 22 mm stainless steel disks used in Mercer cascade impactors, and two types of glass impingers. The results demonstrate that while several processing methods tested resulted in significantly lower physical recovery efficiencies than other methods, it was possible to obtain culturable recovery efficiencies for B. thailandensis and physical recovery efficiencies for 1 µm fluorescent spheres of at least 0.95 from all of the sampling media tested given an appropriate sample processing procedure. The results of the present study also demonstrated that the bubbling action of liquid media in all-glass impingers (AGIs) can result in physical loss of material from the collection medium, although additional studies are needed to verify the exact mechanisms involved. Overall, the results of this study demonstrate that the collection mechanism as well as the post-collection processing method can significantly affect the recovery from and retention of culturable microorganisms in sampling media, potentially affecting the calculated airborne concentration and any subsequent estimations of risk or dose derived from such data.