COVID-19 Test Overview: SARS-CoV-2 amplification test

Assay Overview

The SARS-CoV-2 amplification test 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 performed by Clinical Research Sequencing Platform (CRSP) 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 (“CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay”) 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 in the reaction itself.

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.

The SARS-CoV-2 amplification test is only for use under the Food and Drug Administration’s Emergency Use Authorization.

Special Instructions

Nasopharyngeal and oropharyngeal swabs should be collected by qualified healthcare professionals, per FDA guidance. Color does not collect COVID-19 samples directly from patients.

Expected Turnaround Time

24-72 hours. Turnaround time is the usual number of hours from the time a specimen is accessioned at the laboratory to when the result is released to the ordering provider. Test results are most typically reported electronically, which generally allows for faster delivery. Schedules may vary.

Specimen Requirements
  • Specimen
    • Specimens should be collected by trained personnel.
    • This test has currently been validated for use with NP and OP swabs.
  • Media
    • Acceptable transport media include: DNA/RNA shield, VTM, UTM.
    • 1ml minimum
  • Storage and Transport Instructions
    • 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).
  • Causes for Rejection
    • Sample may be rejected or significantly delayed if they arrive with:
      • Insufficient, incompatible or congealed media
      • More than 48 hours after sample collection
      • Improperly capped or labeled tubes
      • Missing physician order
      • Incomplete or missing patient information
Test Details
  • Use: 

    • Detection of SARS-CoV-2 to assist in the diagnosis of COVID-2019 infections

  • Result types: 

    • Positive for SARS-CoV-2. Positive indicates that SARS-CoV-2 (the virus that causes COVID-19) was detected in the patient’s sample.

    • Negative for SARS-CoV-2. Negative indicates that SARS-CoV-2 (the virus that causes COVID-19) was not detected in the patient’s sample.

    • Inconclusive result for SARS-CoV-2. Inconclusive indicates that the test results were internally inconsistent and cannot be classified as either positive or negative.

    • Unsatisfactory sample. Test could not be completed.

  • Test method for CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay

    • 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, CAP #8707596. 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 nasopharyngeal (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.

    • Test 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.

      • Further details about CRSP SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Assay can be found here

 

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