It was about a year ago that I highlighted here some neat graphene technology out of Cardea Biosciences, and in that context I mentioned CRISPR-based diagnostics. (If interested, you can find the post here.)
The FDA recently approved under Emergency Use Authorization (EUA) Sherlock BioSciences’ SARS-CoV-2 Rapid diagnostic (FDA EUA page is here) and let’s take a little closer look at how it works.
Principle of operation
The FDA posted the Instructions for Use (aka the ‘IFU document’) that has a lot of detail around how to use the testing kit, for a high -complexity CLIA laboratory. For background, the original SHERLOCK paper (here in Science from 2018) combined a multiplexing method, a modified endonuclease, and a lateral-flow device for inexpensive and fast readout. Notably their Appendix points out that these lateral flow immuno-assays (LFIAs) cost about $0.62 to manufacture at scale.
The existing kit has a multiplexing feature (a two-plex) but does not have lateral-flow chromogenic readout; it has a fluorescent microplate reader format. (96-well / 384-well microplate fluorescent readers are common laboratory pieces of instrumentation). A thermoplate is required, however no thermal cycler is required, because their assay uses RT-LAMP (reverse transcriptase loop-mediated isothermal amplification) (Wikipedia).
The kit is comprised of two steps: starting from purified viral SARS-CoV-2 RNA, there is a single incubation step at 61C for 40 minutes, and then a CRISPR-Cas detection step in a 384-well plate reader for 10 minutes (four measurements every 2.5 minutes).
For purification, the kit specifies the Thermo Fisher Scientific PureLink Viral RNA/DNA Mini Kit (this is a manual, spin-column based method) and the samples are from nasopharyngeal swabs (or bronchial lavage) samples. in 2 or 3 mL of viral transport media.
Limit of detection down to 0.9 copies / uL
Using 200 uL of the VTM (viral transport media), that is 10% the amount from the swab; eluted in 30 uL from the purification kit, and 8 uL going into the diagnostic test reaction, this represents another 26% of the original sample. Thus 2.6% of the original swab (assuming 100% went from the swab into the media) goes into the assay.
Limit of Detection (LoD) is a critical parameter in measurement of test performance. Below is Table 17 from the Instructions for Use (available here as a PDF).
The two targets in the two-plex diagnostic assay are called ORF1ab and N respectively. ORF1ab is Open Reading Frame 1ab, and the N stands for Nucleocapsid gene. RNase P is a common ‘housekeeping’ RNA gene long-used in the real-time PCR research and diagnostics worlds, and serves as the positive control.
As you can see, the LoD for ORF1ab is 4.5 copies / uL; for N is 0.9 copies / uL. Doing the math, these represent 2.6% of the total virus from the original swab sample: 173 copies for ORF1ab, and 35 copies for N. At the bottom is the NTC (negative template control, just purified water).
Specificity very high against SARS-CoV-2 and not related coronaviruses
Additional data provided in Table 17 is reproduced from the IFU below. The data testing four human coronaviruses, influenza and RSV is shown below.
Not a PONT but new technology shows promise
While not the Point Of Need Test (PONT also known as Point of Care Test or POCT) like a lateral-flow cassette, this CRISPR approach shows promise as an easy-to-implement technology for SARS-CoV-2 detection without the requirement for expensive equipment, whether a real-time PCR instrument or a proprietary reader such as the GeneXpert from Cepheid / Danaher. All that is needed is a heatblock and a microplate reader, and the two-hour turnaround time is another plus.
The high sensitivity is very similar to quantitative RT-PCR (typically 8 copies / uL). There is a pre-print that came out last week, showing the performance of Abbott Laboratories’ ID-NOW had a 30% false-negative rate (BioRxiv preprint server reference, titled “Performance of the rapid Nucleic Acid Amplification by Abbott ID NOW COVID-19 in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution”).
How can a school re-open?
A friend asked me last week how a school of say 450 students could re-open, and one idea would be to pool samples as a screening test for a group of students. Ideally, it would not require PPE and swabs, but rather saliva, which has been shown recently to be as effective as swabs (MedRxiv preprint server reference, “Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs”).
Challenges remain however – not the least of which how to validate a screening test with 450 pooled saliva samples, how to get a test like this approved under EUA and implemented widely, how to get the testing done quickly (preferably at the local level for speed), and how it would be paid for.
This last point is worth elaborating on – the healthcare system in the US is not setup for this scenario. The economist Paul Romer elegantly wrote about this recently ‘If virus tests were sodas’, where a can of soda costs $10, and only 100,000 cans were provided for the entire country, and the country starts to get very thirsty.
Yes there are cheaper ways to get plenty of soda, but due to regulation and reimbursement, millions of cans of sodas do not appear. Romer accurately points out that the Rutger’s saliva-based automated test is not being reimbursed for their efforts. “For now, they do them because they are good sports.”