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The surging COVID19 pandemic has underlined the need for quick, sensitive, and high-throughput SARS-CoV-2 detection assays. Although many different methods to detect SARS-CoV-2 particles in clinical material have been developed, none of these assays are successful in combining all three of the above characteristics into a single, easy-to-use method that is suitable for large-scale use. Here we report the development of a direct RT-PCR SARS-CoV-2 detection method that can reliably detect minute quantities of SARS-CoV-2 gRNA in nasopharyngeal swab samples as well as the presence of human genomic DNA. An extraction-less validation protocol was carried out to determine performance characteristics of the assay in both synthetic SARS-CoV-2 RNA as well as clinical specimens. Feasibility of the assay and analytical sensitivity was first determined by testing a dilution series of synthetic SARS-CoV-2 RNA in two different solvents (water and AMIES VTM), revealing a high degree of linearity and robustness in fluorescence readouts. Following analytical performance using synthetic RNA, the limit of detection was determined at equal to or less than 1 SARS-CoV-2 copy/ul of sample in a commercially available sample panel that contains surrogate clinical samples with varying SARS-CoV-2 viral load. Lastly, we benchmarked our method against a reference qPCR method by testing 87 nasopharyngeal swab samples. The direct endpoint ultra-fast RT-PCR method exhibited a positive percent agreement score of 98.5% and a negative percent agreement score of 100% as compared to the reference method, while RT-PCR cycling was completed in 27 minutes/sample as opposed to 60 minutes/sample in the reference qPCR method. In summary, we describe a rapid direct RT-PCR method to detect SARS-CoV-2 material in clinical specimens which can be completed in significantly less time as compared to conventional RT-PCR methods, making it an attractive option for large-scale SARS-CoV-2 screening applications.

Candida auris is a pathogenic yeast that causes invasive infections with high mortality. Infections most often occur in intensive care units of healthcare facilities. It is crucial to trace the source and prevent further spread of C. auris during an outbreak setting, therefore, genotyping of C. auris is required. To enable fast and cost-effective genotyping, we developed a microsatellite typing assay for C. auris.Short tandem repeats (STRs) in C. auris were identified, and a novel STR typing assay for C. auris was developed using 4 panels of three multiplex PCRs. Having shown that the microsatellite typing assay was highly reproducible and specific, a robust set of 444 C. auris isolates was investigated to identify genotypic diversity. In concordance with whole-genome sequencing (WGS) analysis we identified five major different C. auris clusters, namely, South-America, South-Asia, Africa, East-Asia and Iran. Overall, a total of 40 distinct genotypes were identified, with the largest variety in the East Asian clade. Comparison with WGS demonstrated that isolates with Altogether, a highly reproducible and specific microsatellite typing assay for C. auris was developed, which distinguishes the five different C. auris clades in identical fashion to WGS, while most isolates differing >20 SNPs, as determined via WGS, are also separated. This new C. auris specific genotyping technique is a rapid, reliable, cost-effective alternative to WGS analysis to speedily investigate outbreaks.ImportanceCandida auris is an emerging fungal pathogen now recognized as a threat to public health. The pathogen has spread worldwide and mainly causes hospital associated outbreaks. To track and trace outbreaks and to relate them to new introductions from elsewhere, whole genome sequencing and amplified fragment length polymorphism (AFLP) have been used for molecular typing. While the former is costly and only available in few centers, AFLP is a complicated technique and standardization is not possible. We describe a novel simple microsatellite genotyping technique based on small tandem repeats in the C. auris genome. Further we show that this microsatellite based genotyping technique has been proven comparable to WGS. Overall, this work provides a novel, rapid, reliable and cost-effective method of molecular outbreaks investigations of C. auris.