COVID-19 Testing at the Broad Institute’s Clinical Research Sequencing Platform (CRSP)

Assay overview

The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal and oropharyngeal swabs collected from individuals who may have contracted the virus. Testing is limited to the Clinical Research Sequencing Platform at the Broad Institute which is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

The test was validated on 103 positive and 50 negative specimens representing nasopharyngeal (NP) and oropharyngeal (OP) swabs and found to have 100% accuracy with a lower limit of detection of 0.20 copies/µl.

Results are presumptive for the detection and identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in the nasopharynx and oropharynx during the acute phase of infection. Positive results are indicative of active infection with SARS-CoV-2 but do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. In addition, nucleic acid detection can persist following clearance of active viral replication. Laboratories within the United States and its territories are required to report all positive 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 treatment or other patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

Testing with the CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is intended for use by trained and competency-certified clinical laboratory personnel, specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. The CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay is only for use under the Food and Drug Administration’s Emergency Use Authorization.

Assay fact sheets

• for patients
• for providers

Required manifest file specifications

A manifest file is required for all options. Please include both:

1. a paper manifest in the shipping box.
   
2. an electronic manifest (.csv) with fields as specified below.

Prioritization tier

Although we do not expect this to happen any time soon, in the event that we become capacity constrained, we will follow the CDC and Massachusetts Department of Public Health guidelines for prioritization of testing. Tier A includes Categories 1-3 from the CDC and 1-6 from the MA DPH. Click on the links in the table below to obtain details from each source.

Please add a Tier level in your manifest for each sample according to the following criteria:

How to send us the manifest

We have deployed multiple options for transfer of manifest files. Please choose the version that best fits with your lab workflow.

Send us your .csv manifest file in one of the following ways:

• Through a CRSP provided link to a secure interface that you can upload your .csv directly into (will require a Gmail or G Suite account with 2 factor authentication).
• Through a secure SFTP site that you provide us access to.
• Onto an encrypted USB drive and ship (in a separate bag but within the same box) with your samples to the Broad.

How you will receive results

CRSP will provide a results file with the following format:

If PDF reports were required by the facility, you will receive PDFs that look similar to this:

Image of an example PDF of results.

Individuals designated to receive test results will be provided with one of the following options to access results:

• Through a CRSP provided link to a secure interface that you can download your .csv with results directly onto your system (will require a Gmail or G Suite account with 2 factor authentication).
• Through a secure SFTP site that you provide us access to.
• Onto an encrypted USB drive and shipped back to the requesting site.

Specimen rejection criteria

Regardless of option chosen above, samples may be rejected or significantly delayed if they arrive with any of the following nonconformances:

• Non-scannable barcodes
• Unlabeled, mislabeled or incomplete labeled specimen tubes
• Tubes with insufficient media
• Improperly capped tubes
• Tubes with media that has congealed
• Missing electronic manifest file
• Incomplete electronic manifest file
• Paper manifest not included in the box with samples
• Specimens(s) deemed unacceptable due to the requirements of test; i.e. unvalidated specimen type, wrong swab type or placement in specimen tube
• Suspected specimen contamination
• Pre-analytic handling and transport requirements not met

Unsatisfactory/TNP (test not performed) result code information*

*updated July 28, 2020

Unsatisfactory reason codes that only apply to samples submitted in media are NOT italicized.

Possible Unsatisfactory/“Test Not Performed (TNP)” Reason Codes for samples submitted using dry, anterior nasal swab collection (aka NO media) OR in media are italicized.

TNP reasons found in CareEvolve are shown in bold/italics.

When the laboratory issues an unsatisfactory or TNP result in a report, the individual must be tested again and a NEW sample must be submitted to the laboratory in order to receive a confirmed test result.

Reporting results

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 was validated on 103 positive and 50 negative specimens representing nasophryngeal (NP) and oropharyngeal (OP) swabs and found to have 100% accuracy with a lower limit of detection of 0.2 copies/µl. Subsequently, we validated oropharyngeal swabs (OP) which also showed 100% concordance with a second lab’s results. Further, we have validated the use of Saline as an alternate media (to VTM) for collection and transport of swabs.

Test limitations

Limitations include:

• Negative results do not rule out SARS-CoV-2 infection, particularly in those who have been in contact with the virus. Follow-up testing with a molecular diagnostic should be considered to rule out infection in these individuals.
• If the virus mutates in the RT-PCR target region, SARS-CoV-2 may not be detected or may be detected less predictably.
• 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.
• The test was validated for use with upper respiratory specimens obtained via nasopharyngeal or oropharyngeal swabs in VTM, UTM, M4, M5, M6, saline, and MTM media. The performance of this test has not been established for other specimens. Specimens collected using other FDA recommended Specimen Collection Materials listed in the FDA COVID-19 Diagnostic Technologies communication (March 26, 2020) are processed with the caveat that they were not all validated for use with this test and the result must be interpreted in this context. Furthermore, a false negative result may occur if a specimen is improperly collected, transported or handled.
• False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms 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.

Quality control (to avoid and detect any contamination in the assay)

1. Every qPCR run of 96 samples has NTC (no template control) in well H12.
2. When preparing taqman mastermix, work space and all pipettes are DNA-zapped and UV treated (3 min).
3. When preparing mastermix, empty PCR plates, empty qPCR plates, empty tubes, tips, and water for mastermix are UV-treated.
4. Mastermix preparation occurs in a PCR-free clean room (no amplified PCR product in this room, no Viral RNA template in this room, and no positive control sample in this room.)
5. Any opened positive control sample is prepared and physically stored in a separate room from where taqman mastermix plates are prepared.
6. PPE (gowns, booties, gloves) is changed between the mastermix prep room and the room where positive control is added to the plate.
7. No Template Controls (NTC) are prepared in a separate room from where the positive control is stored/prepared. NTC water and tubes are UV-treated before and after preparation.
8. qPCR preparation steps are performed on an automated liquid handling platform. All automation for qPCR preparation is done using disposable, filtered tips.
9. Before manually adding positive and negative controls to the RNA sample plates, bench space and pipettes are DNA-zapped.
10. Plate-seals on extracted RNA sample plates are gently pierced with tips on the automated liquid handler when aspirating RNA and dispensing into the qPCR plates. This eliminates the possibility of spray between sample wells that can occur during manual removal of seals.
11. qPCR occurs in a separate room from mastermix plate creation or RNA sample addition.
12. Extraction and qPCR automation scripts are visually monitored to ensure no unexpected spraying or droplets.
13. Each qPCR run is reviewed by a clinical supervisor before being reported out.

Contact and license information

Medical Director: Heidi Rehm Ph.D., FACMG
Project Manager: Maegan Harden, Ph.D. (617-714-7569)



Clinical Research Sequencing Platform, LLC
Broad Institute of MIT and Harvard
320 Charles St. Cambridge, MA 02141
CLIA ID: 22D2055652
CAP # 8707596

genomics@broadinstitute.org