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

Sample collection and shipping

  • Specimens should be collected by trained personnel in accordance with manufacturer guidelines and in accordance with FDA approved collection devices (FAQs on Diagnostic Testing for SARS-CoV-2).
  • This test has currently been validated for use with NP and OP swabs.
    For a list of approved swabs, please visit FDA FAQ (Q: I am having trouble obtaining...).
  • VTM, UTM, M4, M5, M6, saline, and MTM are all acceptable transport media types.
  • Acceptable specimens should be transported in accordance with guidelines established by the Massachusetts state public health laboratory:
    • Refrigerate all samples at 2-8℃ prior to and during transport within 24 hrs.
    • If the specimen is to be submitted greater than 24 hrs post collection, freeze the specimens at -20℃ or below and then ship on dry ice.
    • Ship as a Category B (UN3373 - Biological Substances).
  • Specimen collection guidance is available here.
  • Samples should be shipped to the following address:
    CRSP Laboratory
    320 Charles Street
    Cambridge, MA 02141

How to send tubes to the Broad CRSP

We support 2 options for sending samples for testing to Broad. Please note that choice of tube submission may impact turnaround times and testing capacity. We are happy to discuss these options with you in more detail to figure out the most effective, safe way to transfer.

Option 1 - Matrix tubes Option 2 - Swab VTM tubes
For larger, automation savvy labs that have the ability to print scannable barcode labels to affix to the tube.
Tubes provided by CRSP: ThermoFisher catalog #3732
For smaller labs with lower test volume. If needed, we can provide scannable barcode labels.
We do NOT supply swabs or collection tubes.

Requires a matrix tube labeled with:

  • a scannable barcode label of the site’s sample_id; the new label may cover the barcode on the tube
  • matrix_id (on bottom of tube)

Requires a swab collected in a VTM tube labeled with:

  • a scannable barcode label of the site’s sample_id
  • patient_id and/or patient_name
  • Please provide at least 150-300µL of media in the tubes for processing.
  • Due to regulations, sample tubes MUST include 2 identifiers that are also present on the manifest.

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.

Required fields:

  • sample_id (must be equivalent to the sample barcode on the tube)
  • patient_id and/or patient_name*
  • institution_id
  • time_collected
  • tier
  • matrix_id (if using option 1)
  • *Note: if your site will require pdf reports, please include patient_name in manifest so it can be included in the report.

Optional fields could include:

  • physician
  • specimen_type
  • other additional fields if desired

The file should be named as follows: SITENAME_ManifestNumber_Date.csv, e.g. st.elsewhere_Manifest203_3.25.20.csv

Image of an example manifest with required columns. Order is not important, text in headers is and should match.

Nonconformances

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 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

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:

Category Description
Tier A CDC Priorities 1-3 or MA DPH Categories 1-6
Tier B All other symptomatic
Tier C All other non-symptomatic

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:

How you will receive results

CRSP will provide a results file with the following format:

Image of an example results. "matrix_id" field will be empty if swab VTM tubes were used.

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

Image of an example PDF of results. Click here to download it.

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

Result codes

The test result code (column I of the report) may be any of the following:

Result Description ("reason" or column J in the results file)
NEG 2019-novel Coronavirus (2019-nCoV) not detected by the qRT-PCR assay. Consider testing for other respiratory viruses or re-collecting for 2019-nCoV testing. Note: Optimum timing for peak viral levels during infections caused by 2019-nCoV have not been determined. Collection of multiple specimens from the same patient may be necessary to detect the virus.
INCONCLUSIVE Inconclusive for 2019-novel Coronavirus (2019-nCoV) by qRT-PCR with one of the two viral probes (either N1 or N2) being positive while the other is not detected after two attempts on this specimen. Consider re-collection of specimen. Note: Optimum timing for peak viral levels during infections caused by 2019-nCoV have not been determined. Collection of multiple specimens from the same patient may be necessary to detect the virus.
POS Positive for detection of 2019-novel Coronavirus (2019-nCoV) by qRT-PCR.
INVALID This specimen failed to produce a valid result after two attempts. An invalid result means no nucleic acids (viral or human) were detected by qRT-PCR. Consider re-collection of specimen.
UNSATISFACTORY_1 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: improper specimen transport medium.
UNSATISFACTORY_2 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: inappropriate timing of collection relative to specimen receipt. Specimens must be received within 72 hrs of collection unless frozen.
UNSATISFACTORY_3 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: sample tube unlabeled.
UNSATISFACTORY_4 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: insufficient volume.
UNSATISFACTORY_5 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: tube sample data does not match the submission form.
UNSATISFACTORY_6 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: laboratory accident.
UNSATISFACTORY_7 Unsatisfactory for 2019-novel Coronavirus (2019-nCoV) testing by PCR: leaking/broken tube.
CANCELED Testing canceled per submitter.

Reporting results

Results are typically returned within 24 hours from receipt.

Reporting of results to the Commonwealth of Massachusetts is the responsibility of the collection site. Broad CRSP lab will not be reporting results for your samples back to the state.

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:

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