Assay overview
The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay (Version 3) is intended for the qualitative detection of nucleic acid from SARS-CoV-2 in dry nasal swabs from individuals suspected of COVID-19 by their healthcare provider.
The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay (Version 3) targets two portions of the nucleocapsid (N) gene SARS-CoV2 genome with a set of primers and probes designed and first validated by the US CDC. These primer/probe combinations are known as N1 and N2. The test also targets a gene in the human genome, RNaseP, as a control in every test to ensure that a successful swabbing event has occurred. All three targets are assayed in a single well reaction (known as a multiplex).
All testing is limited to the Clinical Research Sequencing Platform (CRSP), LLC at the Broad Institute of MIT and Harvard, located at 320 Charles Street, Cambridge, MA 02141 which is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, and meets requirements to perform high complexity tests.
Results are for the detection and identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in upper respiratory specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease.
Laboratories within the United States and its territories are required to report all results to the appropriate public health authorities.
Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.
Sample collection and shipping:
This test has currently been validated for use with nasal swabs placed into an empty tube.
Test method and limitations
Test method
This RT-PCR test, a high throughput version of the CDC 2019-nCoV Realtime RT-PCR test, was performed by the Clinical Research Sequencing Platform at the Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02141, CLIA #22D2055652. This test is not FDA-cleared but its performance characteristics were established by a CLIA-certified high-complexity laboratory in accordance with CLIA regulations. The test performance was validated on 36 positive and 30 negative clinical specimens that were also run at another laboratory with a comparable test that has been deemed to have high sensitivity by the FDA. The lower limit of detection (LOD) of the Version 3 assay was determined to be 60 total copies of the virus which equates to 1600 copies/mL. Our Version 3 assay was shown to have 100% positive sample concordance and 96.7% negative sample concordance to the comparator laboratory. We also observed 100% positive concordance with 36 positive samples diluted to close to the limit of detection of the comparator lab assay. Further, we observed 100% concordance across 36 positive samples run on both the Version 2 (non-mulitplexed) and Version 3 assays within our own lab.
Test Limitations include
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Negative results do not rule out SARS-CoV-2 infection.
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If the virus mutates in the RT-PCR target region, SARS-CoV-2 may not be detected or may be detected less predictably.
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Inhibitors or other types of interference may produce a false negative result. An interference study evaluating the effect of common cold medications was not performed.
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False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of virus are present in the specimen. Optimum specimen types and timing for peak viral levels during infections caused by SARS-CoV-2 have not been fully determined. Collection of multiple specimens (types and time points) from the same patient may be necessary to detect the virus.