Protocol Citation: Mammoth Biosciences, James P. Broughton, Wayne Deng, Clare L. Fasching, Jasmeet Singh, Charles Y. Chiu, Janice S. Chen 2020. A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR. protocols.io https://dx.doi.org/10.17504/protocols.io.bcmtiu6n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We used this protocol in our group and it is working
***DISCLAIMER: This protocol has not been approved by the FDA and should not be used as a clinical diagnostic***
Introduction
Given the global health emergency, rapid transmission, and severe respiratory disease associated with the outbreak of the 2019 novel coronavirus (SARS-CoV-2), Mammoth Biosciences has reconfigured our DETECTR platform to rapidly and accurately detect SARS-CoV-2 using a visual lateral flow strip format within 30 minutes from sample to result. To ensure specificity of detection, we selected a high-fidelity CRISPR detection enzyme and designed sets of gRNAs that can either 1) differentiate SARS-CoV-2 or 2) provide multi-coronavirus strain detection. SARS-CoV-2 DETECTR couples CRISPR detection with isothermal pre-amplification using primers based on protocols validated by the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). Currently in the United States, the CDC SARS-CoV-2 real-time RT-PCR diagnostic panel has a laboratory turnaround time of approximately 4-6 hours, with results that can be delayed for >24 hours after sample collection due to shipping requirements. In addition, these tests are only available in CDC-designated public health laboratories certified to perform high-complexity testing.
Mammoth is working to enable point of care testing (POCT) solutions that can be deployed in areas at greatest risk of transmitting SARS-CoV-2 infection, including airports, emergency departments, and local community hospitals, particularly in low-resource countries. Leveraging an “off-the-shelf” strategy to enable practical solutions within a short time frame, we describe here a protocol that is fast (<30 min), practical (available immediately from international suppliers), and validated using contrived samples.
Specifications
Targets
● N-gene (SARS-CoV-2 specific)
● E-gene (SARS-CoV, bat-SARS-like-CoV, and
SARS-CoV-2 coronaviruses)
● RNase P (human sample control)
Limit of detection
70-300 copies/μl input
Table 1: SARS-CoV-2 DETECTR assay workflow and specifications.
Acknowledgements: We thank Vikram Joshi, Nefeli Tsaloglou and Xin Miao for advice and helpful discussions in the preparation of this whitepaper.
Conflicts of Interest: JPB, CLF, JS and JSC are employees of Mammoth Biosciences, CYC is on the Scientific Advisory Board of Mammoth Biosciences, and JSC is a co-founder of Mammoth Biosciences. JPB, CLF, JS, CYC and JSC are co-inventors on CRISPR-related technologies.
While we were preparing this whitepaper, another protocol for SARS-CoV-2 detection using CRISPR diagnostics (SHERLOCK, v.20200214) was published. We compare the assay workflows and specifications between CRISPR diagnostics and established CDC/WHO protocols below. (Note: as of this publication, CRISPR diagnostics workflows have not yet been approved by the FDA)
SARS-CoV-2 DETECTR
SARS-CoV-2 SHERLOCK
CDC SARS-CoV2 qRT-PCR
Target
N gene & E gene (N gene gRNA compatible with CDC N2 amplicon, E gene compatible with WHO protocol)
S gene & Orf1ab gene
N-gene (3 amplicons)
Sample control
RNase P
None
RNase P
Limit of Detection
70-300 copies/μl input
10-100 copies/μl input
1 copy/μL input
Assay reaction time
~30 min
~60 min
~45-60 minutes
Assay components
RT-LAMP (62 °C, 290 min), Cas12 (37 °C, 10 min), Lateral flow (RT, 2 min)
RT-RPA (42 °C, 25 min), IVT + Cas13 (37 °C, 30 min), Lateral flow (RT, 2 min)
UDG digestion (25 °C, 2 min), reverse transcription (50 °C, 15 min), denature (95 °C, 2 min), amplification (95 °C, 3 min; 55 °C 30 sec; 45 cycles)
Heavy instrumentation required
No
No
Yes
FDA EUA approval
No
No
Yes
Appendix Tavle 1: Comparison of SARS-CoV-2 specifications for CRISPR diagnostic
protocols to the current CDC assay.
Materials
STEP MATERIALS
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
SARS-CoV-2 DETECTR Reagents
Step 1: Isothermal amplification (62°C, 20 min)
RT-LAMP Master Mix (Supplier: NEB)
WarmStart LAMP Kit (DNA and RNA) - 100 rxnsNew England BiolabsCatalog #E1700S
Allow the lateral flow strip to run for 00:02:00 at Room temperature and observe
the result.
Test interpretation
Test interpretation
N-gene
E-gene
RNase P
Interpretation
+
+
+/-
SARS-CoV-2 positive
+
-
+/-
Indeterminate
-
+
+/-
Indeterminate
-
-
+
SARS-CoV-2 negative
-
-
-
QC failure
Citations
Curtis KA, Morrison D, Rudolph DL, Shankar A, Bloomfield LSP, Switzer WM, Owen SM. A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood.