Commercially Available COVID-19 Tests:
The FDA has approved hundreds of COVID-19 tests for EUA usage, including molecular, antigen, and antibody tests. The following briefly describes three randomly selected tests.
A COVID-19 Molecular Test: The BioPerfectus Technologies COVID-19 Real-Time RT-PCR Kit
The BioPerfectus COVID-19 real-time RT-PCR kit is a real-time fluorescent RT-PCR-based assay used for the qualitative detection of SARS-CoV-2 nucleic acid in nasopharyngeal swabs, oropharyngeal (throat) swabs, anterior nasal swabs, mid-turbinate nasal swabs, nasal aspirates, nasal washes, bronchoalveolar lavage (BAL) fluid, and sputum.
The test kit includes a specific oligonucleotide primer and probes for specific detection of the SARS-CoV-2 from several regions of the genome, including the nucleocapsid gene. The kit also has primers and a probe to serve as internal controls for the assay. The assay begins by extracting and isolating the RNA from the collected specimens and is then reversed transcribed to cDNA and amplified in a real-time PCR instrument such as the Applied BioSystems 7500 or Roche LightCycler 480 PCR instruments.
The test has probes consisting of a reporter dye and a quenching dye. A fluorescent signal is emitted from the reporter dye, and the signal is absorbed by the quencher. During PCR amplification, a special polymerase (Taq DNA polymerase) is used to separate the reporter dye and the quencher, thereby generating a fluorescent signal that increases with each PCR cycle. Based on the changes in the fluorescent signals, the PCR instrument generates a real-time amplification curve, which is used to determine the presence or absence of SARS-CoV-2 RNA.
A positive and a blank control are used to monitor the validity of the test. If the test is valid, then a positive test is reported if one or two targeted gene nucleic acids are detected, whereas a negative test occurs when neither of the targeted gene nucleic acids are detected. A negative result may be due to the absence of the viral RNA or to the presence of viral RNA below the limit of detection of the assay.
The test manufacturer reports that validity studies have shown that for all types of collection specimens, the test has a positive agreement of 100% and a negative agreement of 95.1%. For studies on comparisons to clinical diagnosis, the test had a positive agreement of 94.9% and a negative agreement of 98.7%.
A COVID-19 Antigen Test: The Quidel Sofia® SARS Antigen FIA Test
The Sofia® SARS Antigen FIA test is available as a diagnostic kit from Quidel. The test is a lateral flow immunofluorescent sandwich assay that is used with the Sofia® or Sofia® 2 instruments. The test is used for the qualitative detection of the nucleocapsid protein antigen (N Protein) from SARS-CoV-2 in nasopharyngeal and nasal swab specimens either directly or after the swabs have been added to either Copan UTM or the CDC’s formulation of VTM from individuals who are suspected of COVID-19 by their healthcare provider.
The test is initiated by placing the patient sample either obtained by a direct swab collection or from samples collected in UTM or VTM special collection tubes. into the reagent tube and incubated for a short time. This incubation serves to disrupt the COVID-19 virus and exposes the N protein from inside the virus. From the reagent tube, an amount of sample is then pipetted into the test cassette sample well. The test cassette is then inserted into either a Sofia® or Sofia® 2 instrument, a small benchtop fluorescent immunoassay analyzer with an optical sensor to scan and collect data from the test strip inside the test cassette.
Before the analysis, the Sofia® or Sofia® 2 analyzer can be set to either a Walk Away or Read Now mode. In the Walk Away mode, the user immediately inserts the test cassette into the analyzer, whereby the test cassette is periodically scanned during the test development time. Positive or negative results are typically displayed within 15 minutes. In the Read Now mode, the user must allow the test to develop for 15 minutes by placing it on the countertop before inserting it into the analyzer. After 15 minutes, the user inserts the test cassette into the analyzer, the test strip is scanned, and the result is displayed within 1 minute.
The Sofia® or Sofia® 2 fluorescent analyzer, after scanning the test strip and measuring the fluorescent signal, uses a method-specific algorithm to produce a test result reported as positive, negative, or invalid. The analyzer displays the result of the procedural control as either “valid or invalid.” If the control result is invalid, then the test should be repeated with a new patient sample and a new test cassette. If the control is reported as valid, then the analyzer will report either a positive or negative result.
A positive test result indicates the presence of the SARS-CoV-2 nucleocapsid viral antigen; however, clinical correlation with patient history and other diagnostic information is necessary to determine infection status. Positive results do not rule out bacterial infection or co-infection with other viruses. A negative test result indicates the possible absence of the SARS-CoV-2 virus antigen; however, a negative result should be treated as presumptive and should be confirmed with a molecular-based assay such as RT-PCR. Therefore, a negative test result does not rule out COVID-19 and should not be used as the sole basis for treatment or patient management. In addition, negative test results should be considered in relationship with a patient’s recent exposures, history, and clinical signs and symptoms of COVID-19.
