The Sherlock™ CRISPR SARS-CoV-2 kit it is the first US FDA emergency use authorization (EUA) CRISPR-based diagnostic test intended for the qualitative detection of nucleic acid from SARS-CoV-2. This kit provides specific and sensitive detection of the SARS-CoV-2 virus in upper respiratory tract samples from individuals suspected of having COVID-19 by their healthcare provider.
IDT is an OEM supplier and distributor for Sherlock Biosciences.
The Sherlock™ CRISPR SARS-CoV-2 kit is designed to detect fragments in the Open Reading Frame (ORF1ab) gene and the Nucleocapsid (N) gene of the SARS-CoV-2 virus. An internal control targets the human RNase P POP7 gene to confirm clinical sample extraction in the absence of a positive SARS-CoV-2 result.
The assay (Figure 1) is comprised of two steps. Step one is reverse transcription, loop mediated isothermal amplification (RT-LAMP), where targeted SARS-CoV-2 genomic RNA is reverse transcribed to DNA then amplified by a strand-displacing DNA polymerase. Step two involves transcription of the amplified DNA to activate collateral cleavage activity of a CRISPR complex programmed to the target RNA sequence. During the second step, cleavage of nucleic acid reporters results in a fluorescent readout detectable by a standard plate reader.
To determine the Limit of Detection (LoD), limiting dilutions of quantified, extracted genomic RNA were spiked into a clinical matrix composed of pooled nasopharyngeal swabs after the initial lysis step. The LoD for the Sherlock CRISPR SARS-CoV-2 kit was determined to be the lowest concentration of genomic viral RNA (copies/µL of Viral Transport Media) at which ≥95% of all replicates tested were positive. As the assay contains two SARS-CoV-2 targets, ORF1ab and Nucleocapsid (N), the LoD for each target was independently determined and confirmed. The LoD claimed for the kit is the higher of the two values: 6.75 copies/µL VTM. Confirmatory LoD testing is demonstrated in Table 1. For more information on how the LoD was determined, refer to the Sherlock SARS-CoV-2 kit Instructions for Use in the Resources section. (Data courtesy of Sherlock Biosciences.)Table 1. Confirmatory LoD testing.
|Target||Viral copies in sample (copies/µL VTM)||Number of samples||Number detected||Detection rate (%)|
The clinical evaluation was performed on 30 contrived positive and 30 contrived negative nasopharyngeal specimens. Positive samples were contrived by spiking with quantitated SARS-CoV-2 viral genomic RNA to a concentration of 2X LoD (20 specimens), 3X LoD (5 specimens), or 5X LoD (5 specimens). The samples were randomized, then processed using the Sherlock CRISPR SARS-CoV-2 kit workflow. The results, as presented in Table 2, showed 100% agreement with the expected results for both the positive and negative specimens. For more information on how the clinical evaluation was performed, refer to the Sherlock SARS-CoV-2 kit Instructions for Use in the Resources section. (Data courtesy of Sherlock Biosciences.)Table 2. Contrived clinical sample evaluation.
|Sample concentration||Number of samples||Number detected||% agreement (95% confidence interval)|
|5X LoD||5||5||100% (NA*)|
|3X LoD||5||5||100% (NA*)|
|2X LoD||20||20||100% (83.9–100%)|
|Negative specimens||30||0||100% (88.6–100%)|
* NA = not applicable, confidence intervals not calculated for sample sizes of 5 or less