The TaqPath™ COVID-19 Diagnostic PCR Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens from individuals with signs and symptoms of respiratory tract infection. The TaqPath™ COVID-19 Diagnostic PCR Kit is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical observations, epidemiological information and laboratory findings. The SARS-CoV-2 RNA is generally detectable in upper respiratory (anterior nasal and nasopharyngeal swabs) specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not rule out bacterial infection or co-infection with other pathogens. The agent detected may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. The TaqPath™ COVID-19 Diagnostic PCR Kit is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.
Device Story
The TaqPath COVID-19 Diagnostic PCR Kit is a multiplex real-time RT-PCR assay for SARS-CoV-2 detection. Input: nasopharyngeal or anterior nasal swab specimens. Process: manual addition of samples/IPC to plates; automated nucleic acid extraction via KingFisher Flex system using magnetic-bead technology; RT-PCR amplification of ORF1ab, S, and N genes plus MS2 internal control on Applied Biosystems 7500 Fast Dx or QuantStudio 5 Dx instruments. Output: fluorescent signal measured per cycle. Data analysis: COVID-19 Interpretive Software IVD Edition calculates Ct values and determines results based on predefined cutoffs. Clinical use: aid in COVID-19 diagnosis by laboratory professionals. Benefit: provides qualitative SARS-CoV-2 detection to support clinical decision-making.
Clinical Evidence
Prospective multi-site clinical study (n=1,055 NP, n=1,052 AN specimens) compared the device against a composite comparator of three FDA-authorized molecular assays. For NP swabs, PPA was 98.9% and NPA was 98.4%-98.7% across instruments. For AN swabs, PPA was 98.8% and NPA was 97.8%-98.0%.
Technological Characteristics
Multiplex real-time RT-PCR assay. Targets: ORF1ab, S gene, N gene, and MS2 bacteriophage IPC. Instrumentation: Applied Biosystems 7500 Fast Dx or QuantStudio 5 Dx. Extraction: Magnetic-bead based (MagMAX Viral/Pathogen II). Software: COVID-19 Interpretive Software IVD Edition for automated Ct-based result calling. Prescription use only.
Indications for Use
Indicated for qualitative detection of SARS-CoV-2 RNA in nasopharyngeal and anterior nasal swabs from symptomatic individuals. For prescription use by trained clinical laboratory personnel.
Regulatory Classification
Identification
A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
*e.g.,* BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
*e.g.,* when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
*e.g.,* recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
*e.g.,* regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
*e.g.,* saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (*e.g.,* how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads if infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to State or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
*i.e.,* H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
## I Background Information:
A 510(k) Number
K233453
B Applicant
Life Technologies Corporation
C Proprietary and Established Names
Applied Biosystems TaqPath COVID-19 Diagnostic PCR Kit
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QQX | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The Sars-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology |
## II Submission/Device Overview:
A Purpose for Submission:
To obtain market clearance for the Applied Biosystems TaqPath COVID-19 Diagnostic PCR Kit (referred to as TaqPath COVID-19 Diagnostic PCR Kit below).
B Measurand:
SARS-CoV-2 RNA
C Type of Test:
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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The TaqPath COVID-19 Diagnostic PCR Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test.
## III Intended Use/Indications for Use:
### A Intended Use(s):
See Indications for Use below.
### B Indication(s) for Use:
The TaqPath COVID-19 Diagnostic PCR Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens from individuals with signs and symptoms of respiratory tract infection.
The TaqPath COVID-19 Diagnostic PCR Kit is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical observations, epidemiological information, and laboratory findings. The SARS-CoV-2 RNA is generally detectable in upper respiratory (anterior nasal and nasopharyngeal swabs) specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not rule out bacterial infection or co-infection with other pathogens. The agent detected may not be the definite cause of disease.
Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions.
The TaqPath COVID-19 Diagnostic PCR Kit is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.
### C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
IVD - For In Vitro Diagnostic Use Only
### D Special Instrument Requirements:
This assay is to be used with one of the following real-time PCR instruments only:
- Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument
- Applied Biosystems QuantStudio 5 Dx Real-Time PCR Instrument
## IV Device/System Characteristics:
### A Device Description:
The TaqPath COVID-19 Diagnostic PCR Kit is a multiplex real-time RT-PCR test for the qualitative detection of RNA from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens. Each TaqPath COVID-19 Diagnostic PCR Kit includes:
K233453 - Page 2 of 25
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- TaqPath COVID-19 Diagnostic PCR Assay Multiplex: Primer and probe sets to detect RNA from three specific genomic regions in SARS-CoV-2: Open Reading Frame (ORF) 1ab, Spike (S) protein, Nucleocapsid (N) protein and primers/probes for MS2 bacteriophage.
- MS2 bacteriophage, an internal processing control (IPC) for nucleic acid extraction.
- TaqPath COVID-19 Diagnostic PCR Control as a positive RNA control that contains targets specific to the SARS-CoV-2 genomic regions targeted by the assays.
- TaqPath COVID-19 Diagnostic PCR Control Dilution Buffer for diluting the TaqPath COVID-19 Diagnostic PCR Control to a working concentration.
Patient samples and the IPC are manually added to KingFisher 96 Deep-Well plates and loaded onto the KingFisher Flex Purification System along with reagents from the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit for automated processing. In this process, nucleic acids from the patient samples are recovered using magnetic-bead technology. Unbound substances and impurities are removed with automated wash steps and purified nucleic acid is eluted from the beads with Elution Buffer.
The extracted sample RNA is subsequently mixed with TaqPath COVID-19 Diagnostic PCR Assay Multiplex and the TaqPath 1-Step Multiplex Master Mix (No ROX) in 96-well reaction plates and loaded onto one of the following instruments for reverse transcription into cDNA followed by amplification of the SARS-CoV-2 target sequences and the MS2 IPC target sequence:
- Applied Biosystems 7500 Fast Dx Real Time PCR instrument
- Applied Biosystems QuantStudio 5 Dx Real-Time PCR instrument
Following amplification, the data from the test run is imported into COVID-19 Interpretive Software IVD Edition for analysis and interpretation. The COVID-19 Interpretive Software IVD Edition analyzes the run data, performs quality check analysis, and calculates the interpretive results for each sample and control based on the cycle threshold (Ct) value obtained for each of the targets. The imported data and interpretive results for each run are saved as a batch in the software. Results can be exported as CSV files and reports can be generated in PDF format.
## B Principle of Operation:
The TaqPath COVID-19 Diagnostic PCR Kit contains primer and probe sets to detect RNA from three specific genomic regions in SARS-CoV-2: Open Reading Frame (ORF) 1ab, Spike (S) protein, Nucleocapsid (N) protein and primers/probes for MS2 bacteriophage IPC. During PCR amplification, the probes anneal to the target sequences located between the forward and reverse primers for the target genes. During the PCR extension phase, the 5' nuclease activity of the Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity, which is measured at each PCR cycle by the real-time PCR instrument. Validation of the results is performed automatically by the COVID-19 Interpretive Software IVD Edition based on the performance of positive and negative controls (see Table 1 below). The patient sample results are automatically generated using the calling rules, plate validity and the Ct cutoff values for assay targets as shown in Table 2 below.
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Table 1: Interpretation of Positive and Negative Controls
| ORF1ab | N gene | S gene | MS2 | Status | Result | Action |
| --- | --- | --- | --- | --- | --- | --- |
| Negative Control | | | | | | |
| NEG | NEG | NEG | POS | VALID | NA | REPORT
Report the results to the healthcare provider. |
| All other scenarios | | | | INVALID | NA | RETEST
1. In the Samples pane of the Home screen, ensure that none of the patient samples are incorrectly assigned as a control sample. If a patient sample has been incorrectly assigned: In the instrument software, correct the Task assignment, for EDS files change the experiment name, save the file with a new file name, import the corrected file into the interpretive software, then review the results.
2. If the patient sample assignments are correct and the status is still INVALID, check the target calls:
• If MS2 is reported as NEGATIVE, the extraction failed. Repeat the extraction, ensuring that MS2 is correctly added to the negative control.
• If any viral gene targets are reported as POSITIVE, contamination occurred. Decontaminate the equipment, replace the extraction reagents and qPCR reagents, then repeat the extraction and PCR. |
| Positive Control | | | | | | |
| POS | POS | POS | NEG | VALID | NA | REPORT
Report the results to the healthcare provider. |
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Table 2: Result Interpretation for Patient Samples and Required Action
| ORF1ab | N gene | S gene | MS2 | Status | Result | Action |
| --- | --- | --- | --- | --- | --- | --- |
| NEG | NEG | NEG | NEG | INVALID | NA | RETEST Repeat test by re-extracting the original sample and repeating the RT-PCR. If the repeat result remains invalid, consider collecting a new specimen. |
| NEG | NEG | NEG | POS | VALID | SARS-CoV-2 Not Detected | REPORT Report the results to the healthcare provider. |
| Only one SARS-CoV-2 target = POS | | | POS or NEG | VALID | SARS-CoV-2 Inconclusive | RETEST/REPORT 1. Repeat the test by re-extracting the original sample and repeating the RT-PCR. IMPORTANT! Samples with a result of SARS-CoV-2 Inconclusive shall be retested one time. 2. After retesting one time, report the results to the healthcare provider. 3. If the repeat result remains inconclusive, the healthcare |
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| | | | | provider should conduct additional confirmation testing with a new specimen, if clinically indicated. |
| --- | --- | --- | --- | --- |
| Two or more SARS-CoV-2 targets = POS | POS or NEG | VALID | Positive SARS-CoV-2 | REPORT Report the results to the healthcare provider. |
# C Instrument Description Information:
1. **Instrument Name:**
- Applied Biosystems 7500 Fast Dx Real Time PCR Instrument
- Applied Biosystems QuantStudio 5 Dx Real-Time PCR Instrument
2. **Specimen Identification:**
Specimen identification can be configured in an automated fashion or entered manually.
3. **Specimen Sampling and Handling:**
Nasopharyngeal or anterior nasal swab specimen collected in transport media.
4. **Quality Control:**
Following controls are included in the TaqPath COVID-19 Diagnostic PCR Kit:
a) MS2 bacteriophage internal processing control (IPC) for the nucleic acid extraction. MS2 IPC is added to the sample wells before RNA extraction.
b) External Positive Control (TaqPath COVID-19 Diagnostic PCR Control) is required for each plate run and contains targets specific to the SARS-CoV-2 genomic regions targeted by the assays.
c) A Negative Control (molecular-grade, nuclease-free, non-DEPC-treated water) is required for each plate run to monitor non-specific amplification, cross-contamination during experimental setup and nucleic acid contamination of reagents.
# V Substantial Equivalence Information:
A **Predicate Device Name(s):**
BioFire COVID-19 Test 2
B **Predicate 510(k) Number(s):**
K211079
C **Comparison with Predicate(s):**
Table 3: TaqPath COVID-19 Diagnostic Kit Substantial Equivalence Comparison
| Device & Predicate Device | K233453 | K211079 |
| --- | --- | --- |
| Device Trade Name | TaqPath COVID-19 Diagnostic Kit | BioFire COVID-19 Test 2 |
| Regulation Number | 21 CFR 866.3981 | Same |
| Regulation Name | Multi-Target Respiratory Specimen Nucleic Acid Test Including Sars-Cov-2 And Other Microbial Agents | Same |
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K233453 - Page 7 of 25
| Product Code | QQX | QQX |
| --- | --- | --- |
| General Device Characteristic | | |
| Organisms Detected | SARS-CoV-2 | SARS-CoV-2 |
| Analyte | RNA | RNA |
| Technological Principles | RT-PCR | RT-PCR |
| Condition for use | For prescription use
For in vitro diagnostic use only. | Same |
| Specimen Types | nasopharyngeal swabs and anterior nasal swabs | nasopharyngeal swabs |
| Test Interpretation | Automated test interpretation | Automated test interpretation |
| Target Genes | ORF1ab, S gene, N Gene | ORF1ab, S gene, N Gene, ORF8 |
| Intended Use/ Indications For Use | The TaqPath COVID-19 Diagnostic PCR Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens from individuals with signs and symptoms of respiratory tract infection.
The TaqPath COVID-19 Diagnostic PCR Kit is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical observations, epidemiological information, and laboratory findings. The SARS-CoV-2 RNA is generally detectable in upper respiratory (anterior nasal and nasopharyngeal swabs) specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not rule out bacterial infection or co-infection with other pathogens. The agent detected may not be the definite cause of disease.
Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions.
The TaqPath COVID-19 Diagnostic PCR Kit is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. | The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic. test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems.
The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from individuals suspected of COVID-19 by their healthcare provider. Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS 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 co-infection with other pathogens. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities.
The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional. |
| Instrumentation | 7500 Fast Dx, or QuantStudio 5 Dx | FilmArray 2.0 or FilmArray Torch |
| Controls | One negative control and one positive control are run for each assay. | Two internal controls are included in each reagent pouch for quality control of sample processing and both PCR stages and melt analysis. |
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VI Standards/Guidance Documents Referenced:
Class II Special Controls as per 21 CFR 866.3981.
VII Performance Characteristics:
A Analytical Performance:
1. Precision/Reproducibility
The reproducibility of the TaqPath COVID-19 Diagnostic PCR Kit was assessed across three sites utilizing both the 7500 Fast Dx Real-Time PCR (RT-PCR) instrument and the QuantStudio 5 Dx RT-PCR instrument at each site. A panel of three samples was created using inactivated SARS-CoV-2 virus (USA-WA1/2020) spiked into pooled negative nasopharyngeal (NP) matrix at concentrations to target 5x limit of detection (LoD), 1.5x LoD, and 0x LoD (i.e., no analyte). Each site conducted two runs per day involving two operators each performing one run per day for five non-consecutive days for each of the three lots of assay reagents. Each run tested three replicates of each sample, resulting in a total of 270 observations per panel member for each RT-PCR instrument (3 sites × 2 operators/site × 3 lots/operator × 5 days/lot × 3 replicates/day = 270 observations). Samples were blinded and randomized before testing. All samples were extracted using the KingFisher Flex and the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit.
Qualitative results obtained from testing TaqPath COVID-19 Diagnostic PCR Kit across the three sites are summarized in Table 4. There was 100% agreement with the expected results observed across all three testing sites on both the 7500 Fast Dx RT-PCR instrument and the QuantStudio 5 Dx RT-PCR instrument.
The reproducibility of TaqPath COVID-19 Diagnostic PCR Kit for the panel members was also assessed based on the standard deviation (SD) and coefficient of variation (%CV) of the mean cycle threshold (Ct) value for each potential source of variability. This assessment, presented in Tables 5 and 6 for each of the RT-PCR instruments, is stratified by the assay target. An analysis will have less than 270 replicates if there are samples tested that do not have amplification detected for an assay target. The within-laboratory precision of the TaqPath COVID-19 Diagnostic PCR Kit by site was also evaluated and is presented in Table 7 and 8 for each of the RT-PCR instruments.
Table 4: Summary of the Qualitative Results for TaqPath COVID-19 Diagnostic PCR Kit
| Instrument | Panel Member | Site | N^{[1]} | Agreement with the Expected Results |
| --- | --- | --- | --- | --- |
| 7500 Fast Dx RT-PCR Instrument | 1.5x LoD | 1 | 90 | 100% |
| | | 2 | 90 | 100% |
| | | 3 | 90 | 100% |
| | 5x LoD | 1 | 90 | 100% |
| | | 2 | 90 | 100% |
| | | 3 | 90 | 100% |
| | | 1 | 90 | 100% |
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Table 5: Summary of Reproducibility for 7500 Fast Dx RT-PCR Instrument (Ct signal analysis)
| Panel Member | Target | Detected (n/N)[1] | Mean Ct | Between Lots | Between Sites | Between Days | Between Operators/Run | Repeatability (Within-Run) | Total Reproducibility |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1.5x LoD | ORF1ab | 270/270 | 31.82 | 0.02 | 0.07 | 0.56 | 1.76 | 0.14 | 0.43 | 0.15 | 0.48 | 0.55 | 1.72 | 0.81 | 2.54 |
| N gene | 270/270 | 32.27 | 0.02 | 0.06 | 0.43 | 1.34 | 0.00 | 0.00 | 0.19 | 0.60 | 0.58 | 1.81 | 0.75 | 2.33 |
| S gene | 264/270 | 32.27 | 0.07 | 0.20 | 0.05 | 0.15 | 0.00 | 0.00 | 0.26 | 0.82 | 0.80 | 2.47 | 0.84 | 2.61 |
| MS2 | 270/270 | 24.48 | 0.14 | 0.58 | 0.26 | 1.06 | 0.28 | 1.14 | 0.23 | 0.92 | 0.18 | 0.73 | 0.50 | 2.04 |
| 5x LoD | ORF1ab | 270/270 | 30.14 | 0.06 | 0.21 | 0.62 | 2.07 | 0.06 | 0.21 | 0.14 | 0.45 | 0.39 | 1.31 | 0.75 | 2.50 |
| N gene | 270/270 | 30.57 | 0.01 | 0.04 | 0.48 | 1.58 | 0.16 | 0.53 | 0.00 | 0.00 | 0.34 | 1.12 | 0.61 | 2.01 |
| S gene | 270/270 | 30.41 | 0.08 | 0.25 | 0.26 | 0.87 | 0.08 | 0.28 | 0.05 | 0.17 | 0.36 | 1.19 | 0.47 | 1.53 |
| MS2 | 270/270 | 24.46 | 0.13 | 0.52 | 0.33 | 1.33 | 0.33 | 1.33 | 0.27 | 1.09 | 0.22 | 0.92 | 0.59 | 2.42 |
| 0x LoD | MS2 | 270/270 | 24.50 | 0.11 | 0.46 | 0.18 | 0.75 | 0.27 | 1.09 | 0.30 | 1.21 | 0.17 | 0.69 | 0.48 | 1.97 |
[1]n is number of tests which contribute Ct values to the analysis. N is the total number of valid tests
$\mathrm{Ct} =$ cycle threshold, $\mathrm{LoD} =$ limit of detection, $\mathrm{SD} =$ standard deviation, $\% \mathrm{CV} =$ percent coefficient of variation
Table 6: Summary of Reproducibility for QuantStudio 5 Dx RT-PCR Instrument (Ct signal analysis)
| Panel Member | Target | Detected (n/N)[1] | Mean Ct | Between Lots | | Between Sites | | Between Days | | Between Operators/Run | | Repeatability (Within-Run) | | Total Reproducibility | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1.5x LoD | ORF1ab | 270/270 | 32.56 | 0.11 | 0.35 | 0.10 | 0.29 | 0.00 | 0.00 | 0.17 | 0.53 | 0.51 | 1.56 | 0.56 | 1.71 |
| | N gene | 269/270 | 33.07 | 0.00 | 0.00 | 0.15 | 0.44 | 0.23 | 0.70 | 0.10 | 0.30 | 0.61 | 1.84 | 0.67 | 2.04 |
| | S gene | 264/270 | 33.35 | 0.09 | 0.26 | 0.10 | 0.31 | 0.00 | 0.00 | 0.37 | 1.12 | 0.77 | 2.31 | 0.87 | 2.60 |
| | MS2 | 270/270 | 25.55 | 0.05 | 0.19 | 0.12 | 0.46 | 0.30 | 1.17 | 0.40 | 1.58 | 0.17 | 0.65 | 0.54 | 2.13 |
| 5x LoD | ORF1ab | 270/270 | 30.94 | 0.11 | 0.34 | 0.00 | 0.00 | 0.00 | 0.00 | 0.13 | 0.43 | 0.30 | 0.99 | 0.35 | 1.13 |
| | N gene | 270/270 | 31.34 | 0.10 | 0.31 | 0.05 | 0.15 | 0.07 | 0.22 | 0.14 | 0.44 | 0.35 | 1.10 | 0.39 | 1.25 |
| | S gene | 270/270 | 31.35 | 0.11 | 0.34 | 0.11 | 0.35 | 0.00 | 0.00 | 0.19 | 0.59 | 0.27 | 0.87 | 0.36 | 1.16 |
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Table 7: Summary of Within-Laboratory Precision by Site for 7500 Fast Dx RT-PCR Instrument (Ct signal analysis)
| Site | Panel Member | Target | Detected (n/N) | Mean Ct | Between Lots | | Between Days | | Between Operators/Runs | | Repeatability (Within-run) | | Within-lab Precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Site 1 | 1.5x LoD | ORF1ab | 90/90 | 32.19 | 0.00 | 0.00 | 0.14 | 0.42 | 0.14 | 0.44 | 0.54 | 1.69 | 0.58 | 1.80 |
| | | N gene | 90/90 | 32.56 | 0.00 | 0.00 | 0.00 | 0.00 | 0.19 | 0.58 | 0.49 | 1.50 | 0.52 | 1.61 |
| | | S gene | 89/90 | 32.38 | 0.00 | 0.00 | 0.14 | 0.43 | 0.00 | 0.00 | 0.57 | 1.76 | 0.59 | 1.81 |
| | | MS2 | 90/90 | 24.64 | 0.42 | 1.70 | 0.22 | 0.89 | 0.12 | 0.50 | 0.18 | 0.75 | 0.52 | 2.12 |
| | 5x LoD | ORF1ab | 90/90 | 30.39 | 0.06 | 0.18 | 0.12 | 0.41 | 0.00 | 0.00 | 0.38 | 1.24 | 0.40 | 1.32 |
| | | N gene | 90/90 | 30.90 | 0.15 | 0.48 | 0.11 | 0.35 | 0.00 | 0.00 | 0.34 | 1.11 | 0.39 | 1.26 |
| | | S gene | 90/90 | 30.65 | 0.17 | 0.55 | 0.08 | 0.27 | 0.00 | 0.00 | 0.34 | 1.10 | 0.38 | 1.26 |
| | | MS2 | 90/90 | 24.55 | 0.48 | 1.94 | 0.08 | 0.32 | 0.18 | 0.74 | 0.20 | 0.83 | 0.55 | 2.26 |
| | 0x LoD | MS2 | 90/90 | 24.48 | 0.51 | 2.08 | 0.11 | 0.46 | 0.19 | 0.78 | 0.21 | 0.87 | 0.60 | 2.43 |
| Site 2 | 1.5x LoD | ORF1ab | 90/90 | 31.17 | 0.00 | 0.00 | 0.19 | 0.60 | 0.06 | 0.18 | 0.53 | 1.71 | 0.57 | 1.82 |
| | | N gene | 90/90 | 31.76 | 0.00 | 0.00 | 0.00 | 0.00 | 0.22 | 0.69 | 0.58 | 1.83 | 0.62 | 1.95 |
| | | S gene | 86/90 | 32.18 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.74 | 2.30 | 0.74 | 2.30 |
| | | MS2 | 90/90 | 24.17 | 0.10 | 0.40 | 0.26 | 1.06 | 0.22 | 0.93 | 0.14 | 0.59 | 0.38 | 1.58 |
| | 5x LoD | ORF1ab | 90/90 | 29.42 | 0.05 | 0.16 | 0.12 | 0.41 | 0.18 | 0.60 | 0.44 | 1.48 | 0.49 | 1.66 |
| | | N gene | 90/90 | 30.02 | 0.08 | 0.26 | 0.11 | 0.36 | 0.00 | 0.00 | 0.36 | 1.21 | 0.39 | 1.29 |
| | | S gene | 90/90 | 30.12 | 0.04 | 0.12 | 0.10 | 0.34 | 0.00 | 0.00 | 0.36 | 1.21 | 0.38 | 1.26 |
| | | MS2 | 90/90 | 24.09 | 0.15 | 0.61 | 0.17 | 0.69 | 0.27 | 1.12 | 0.27 | 1.12 | 0.44 | 1.83 |
| | 0x LoD | MS2 | 90/90 | 24.30 | 0.23 | 0.94 | 0.00 | 0.00 | 0.32 | 1.34 | 0.13 | 0.52 | 0.42 | 1.71 |
| Site 3 | 1.5x LoD | ORF1ab | 90/90 | 32.11 | 0.00 | 0.00 | 0.12 | 0.39 | 0.22 | 0.67 | 0.56 | 1.75 | 0.62 | 1.92 |
| | | N gene | 90/90 | 32.47 | 0.18 | 0.57 | 0.00 | 0.00 | 0.13 | 0.39 | 0.67 | 2.07 | 0.71 | 2.18 |
| | | S gene | 89/90 | 32.27 | 0.00 | 0.00 | 0.00 | 0.00 | 0.54 | 1.66 | 1.01 | 3.13 | 1.14 | 3.55 |
| | | MS2 | 90/90 | 24.64 | 0.17 | 0.71 | 0.12 | 0.47 | 0.30 | 1.20 | 0.20 | 0.83 | 0.42 | 1.69 |
| | 5x LoD | ORF1ab | 90/90 | 30.60 | 0.11 | 0.38 | 0.00 | 0.00 | 0.23 | 0.74 | 0.36 | 1.19 | 0.44 | 1.45 |
| | | N gene | 90/90 | 30.80 | 0.14 | 0.46 | 0.15 | 0.49 | 0.00 | 0.00 | 0.32 | 1.03 | 0.38 | 1.23 |
| | | S gene | 90/90 | 30.44 | 0.10 | 0.32 | 0.00 | 0.00 | 0.15 | 0.49 | 0.38 | 1.26 | 0.42 | 1.39 |
| | | MS2 | 90/90 | 24.75 | 0.23 | 0.91 | 0.32 | 1.30 | 0.33 | 1.33 | 0.19 | 0.77 | 0.55 | 2.21 |
| | 0x LoD | MS2 | 90/90 | 24.71 | 0.10 | 0.42 | 0.15 | 0.61 | 0.35 | 1.42 | 0.16 | 0.64 | 0.43 | 1.72 |
[1]n is number of tests which contribute Ct values to the analysis. N is the total number of valid tests
$\mathrm{Ct} =$ cycle threshold, $\mathrm{LoD} =$ limit of detection, $\mathrm{SD} =$ standard deviation, $\% \mathrm{CV} =$ percent coefficient of variation
Table 8: Summary of Within-Laboratory Precision Results by Site for QuantStudio 5 Dx RT-PCR Instrument
| Site | Panel Member | Target | Detected (n/N) | Mean Ct | Between Lots | | Between Days | | Between Operators/Runs | | Repeatability (Within-run) | | Within-lab Precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Site 1 | 1.5x LoD | ORF1ab | 90/90 | 32.50 | 0.14 | 0.42 | 0.00 | 0.00 | 0.20 | 0.62 | 0.51 | 1.58 | 0.57 | 1.75 |
| | | N gene | 90/90 | 33.11 | 0.00 | 0.00 | 0.27 | 0.82 | 0.00 | 0.00 | 0.56 | 1.69 | 0.62 | 1.88 |
| | | S gene | 89/90 | 33.20 | 0.11 | 0.33 | 0.23 | 0.71 | 0.00 | 0.00 | 0.53 | 1.59 | 0.59 | 1.77 |
| | | MS2 | 90/90 | 25.36 | 0.41 | 1.62 | 0.19 | 0.75 | 0.18 | 0.71 | 0.14 | 0.55 | 0.51 | 2.00 |
| | 5x LoD | ORF1ab | 90/90 | 30.93 | 0.10 | 0.33 | 0.00 | 0.00 | 0.07 | 0.22 | 0.30 | 0.97 | 0.33 | 1.05 |
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| | | N gene | 90/90 | 31.41 | 0.12 | 0.38 | 0.11 | 0.34 | 0.00 | 0.00 | 0.35 | 1.10 | 0.38 | 1.21 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | S gene | 90/90 | 31.47 | 0.15 | 0.47 | 0.00 | 0.00 | 0.09 | 0.29 | 0.27 | 0.87 | 0.32 | 1.03 |
| | | MS2 | 90/90 | 25.31 | 0.45 | 1.77 | 0.09 | 0.37 | 0.16 | 0.63 | 0.14 | 0.56 | 0.51 | 2.00 |
| | 0x LoD | MS2 | 90/90 | 25.21 | 0.46 | 1.81 | 0.10 | 0.39 | 0.22 | 0.89 | 0.12 | 0.47 | 0.53 | 2.11 |
| Site 2 | 1.5x LoD | ORF1ab | 90/90 | 32.69 | 0.06 | 0.17 | 0.00 | 0.00 | 0.32 | 0.98 | 0.42 | 1.27 | 0.53 | 1.61 |
| | | N gene | 90/90 | 33.22 | 0.00 | 0.00 | 0.22 | 0.67 | 0.06 | 0.19 | 0.61 | 1.84 | 0.65 | 1.97 |
| | | S gene | 86/90 | 33.43 | 0.26 | 0.77 | 0.00 | 0.00 | 0.34 | 1.02 | 0.85 | 2.55 | 0.95 | 2.85 |
| | | MS2 | 90/90 | 25.63 | 0.06 | 0.22 | 0.18 | 0.70 | 0.36 | 1.40 | 0.12 | 0.46 | 0.42 | 1.65 |
| | 5x LoD | ORF1ab | 90/90 | 30.98 | 0.09 | 0.28 | 0.00 | 0.00 | 0.00 | 0.00 | 0.31 | 1.01 | 0.32 | 1.05 |
| | | N gene | 90/90 | 31.33 | 0.07 | 0.23 | 0.00 | 0.00 | 0.00 | 0.00 | 0.36 | 1.14 | 0.37 | 1.17 |
| | | S gene | 90/90 | 31.24 | 0.10 | 0.31 | 0.00 | 0.00 | 0.17 | 0.56 | 0.27 | 0.86 | 0.34 | 1.07 |
| | | MS2 | 90/90 | 25.58 | 0.13 | 0.52 | 0.05 | 0.21 | 0.27 | 1.05 | 0.28 | 1.08 | 0.41 | 1.61 |
| | 0x LoD | MS2 | 90/90 | 25.70 | 0.17 | 0.68 | 0.06 | 0.25 | 0.27 | 1.06 | 0.13 | 0.51 | 0.35 | 1.38 |
| Site 3 | 1.5x LoD | ORF1ab | 90/90 | 32.48 | 0.16 | 0.49 | 0.21 | 0.65 | 0.00 | 0.00 | 0.58 | 1.78 | 0.64 | 1.96 |
| | | N gene | 90/90 | 32.89 | 0.12 | 0.36 | 0.24 | 0.72 | 0.27 | 0.83 | 0.65 | 1.97 | 0.75 | 2.28 |
| | | S gene | 89/90 | 33.44 | 0.00 | 0.00 | 0.00 | 0.00 | 0.61 | 1.82 | 0.89 | 2.65 | 1.08 | 3.22 |
| | | MS2 | 90/90 | 25.65 | 0.25 | 0.98 | 0.15 | 0.58 | 0.58 | 2.27 | 0.22 | 0.86 | 0.69 | 2.68 |
| | 5x LoD | ORF1ab | 90/90 | 30.91 | 0.16 | 0.52 | 0.03 | 0.09 | 0.23 | 0.74 | 0.30 | 0.97 | 0.41 | 1.33 |
| | | N gene | 90/90 | 31.28 | 0.21 | 0.68 | 0.00 | 0.00 | 0.25 | 0.79 | 0.33 | 1.06 | 0.47 | 1.49 |
| | | S gene | 90/90 | 31.34 | 0.07 | 0.22 | 0.00 | 0.00 | 0.26 | 0.82 | 0.28 | 0.88 | 0.38 | 1.22 |
| | | MS2 | 90/90 | 25.72 | 0.35 | 1.37 | 0.25 | 0.96 | 0.45 | 1.74 | 0.25 | 0.96 | 0.67 | 2.60 |
| | 0x LoD | MS2 | 90/90 | 25.66 | 0.29 | 1.11 | 0.22 | 0.86 | 0.36 | 1.39 | 0.17 | 0.67 | 0.54 | 2.09 |
[1]n is number of tests which contribute Ct values to the analysis. N is the total number of valid tests
$\mathrm{Ct} =$ cycle threshold, $\mathrm{LoD} =$ limit of detection, $\mathrm{SD} =$ standard deviation, $\% \mathrm{CV} =$ percent coefficient of variation
# 2. Linearity:
Not applicable; this is a qualitative assay.
# 3. Analytical Specificity/Interference:
# a) Cross-Reactivity and Microbial Interference
This study evaluated the analytical specificity of the TaqPath COVID-19 Diagnostic PCR Kit by testing samples containing organisms that are commonly found in the respiratory tract and might cause similar clinical symptoms as SARS-CoV-2. A panel of 46 potentially cross-reactive organisms (viruses, bacteria and fungi) were spiked into pooled negative nasopharyngeal (NP) clinical matrix at a high, clinically relevant concentration $(10^{6}\mathrm{CFU / mL}$ or higher for bacteria/fungi and $10^{5}\mathrm{PFU / mL}$ or $\mathrm{TCID}_{50} / \mathrm{mL}$ or higher for viruses) in the absence or presence of SARS-CoV-2 target (spiked at $\sim 3\mathrm{x}$ LoD). Organisms that did not meet the target testing concentration above were tested at the highest possible concentration. Each microorganism was tested in triplicate. For all organisms tested, no cross-reactivity or microbial interference was observed at the concentrations tested, as shown below.
Table 9: Summary of the Microorganisms and Concentrations Tested in Cross-Reactivity Studies
| Type | Microorganism | Concentration Tested |
| --- | --- | --- |
| Virus | Human coronavirus 229E[1] | 2.8×104TCID50/mL |
| Virus | Human coronavirus OC43 | 1.0×105TCID50/mL |
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| Virus | Human coronavirus NL63[1] | 2.3×10^{4} TCID_{50}/mL |
| --- | --- | --- |
| Virus | MERS-CoV | 29.4 Ct |
| Virus | Coronavirus-SARS | 28.8 Ct |
| Virus | Adenovirus Type 1 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Mastadenovirus B Type 7 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Human Metapneumovirus (hMPV) | 1.0×10^{5} TCID_{50}/mL |
| Virus | Parainfluenza virus 1 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Parainfluenza virus 2 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Parainfluenza virus 3 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Parainfluenza virus 4 | 1.0×10^{5} TCID_{50}/mL |
| Virus | Influenza A[1] | 8.3×10^{4} TCID_{50}/mL |
| Virus | Influenza B[1] | 8.3×10^{4} TCID_{50}/mL |
| Virus | Respiratory syncytial virus A | 1.0×10^{5} TCID_{50}/mL |
| Virus | Respiratory syncytial virus B | 1.0×10^{5} TCID_{50}/mL |
| Virus | Rhinovirus[1] | 2.8×10^{4} TCID_{50}/mL |
| Virus | Measles | 1.0×10^{5} TCID_{50}/mL |
| Virus | Mumps | 1.0×10^{5} TCID_{50}/mL |
| Virus | Cytomegalovirus[1] | 3.2×10^{4} TCID_{50}/mL |
| Virus | Epstein-Barr virus | 1.6×10^{7} copies/mL (cross-reactivity)
5.9×10^{6} copies/mL (microbial interference) |
| Virus | Human Immunodeficiency Virus type 1[1] | 1.0×10^{4} International Units (IU)/mL |
| Virus | Human coronavirus (HKU1)[1][2] | 9.99 × 10^{4} copies/mL |
| Virus | Enterovirus (EV-D68)[1][2] | 9.88 × 10^{4} copies/mL |
| Bacteria | Chlamydia pneumoniae | 1.0×10^{6} Infectious Units (IFU)/mL |
| Bacteria | Haemophilus influenzae | 1.0×10^{6} CFU/mL |
| Bacteria | Legionella pneumophila | 1.0×10^{6} CFU/mL |
| Bacteria | Mycobacterium tuberculosis | 5.4×10^{7} GCE/mL (cross-reactivity)
8.6×10^{7} GCE/mL (microbial interference) |
| Bacteria | Streptococcus pneumoniae | 1.0×10^{6} CFU/mL |
| Bacteria | Fusobacterium necrophorum | 1.0×10^{6} CFU/mL |
| Bacteria | Pseudomonas aeruginosa | 1.0×10^{6} CFU/mL |
| Bacteria | Staphylococcus epidermis, MRSE | 1.0×10^{6} CFU/mL |
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| Bacteria | Streptococcus salivarius | 1.0×106CFU/mL |
| --- | --- | --- |
| Bacteria | Corynebacterium diphtheriae | 1.0×106CFU/mL |
| Bacteria | Escherichia coli | 1.0×106CFU/mL |
| Bacteria | Lactobacillus acidophilus | 1.0×106CFU/mL |
| Bacteria | Moraxella catarrhalis | 1.0×106CFU/mL |
| Bacteria | Neisseria meningitidis | 1.0×106CFU/mL |
| Bacteria | Neisseria elongata | 1.0×106CFU/mL |
| Bacteria | Staphylococcus aureus, MRSA | 1.00×106CFU/mL |
| Bacteria | Streptococcus pyogenes | 1.0×106CFU/mL |
| Bacteria | Bordetella pertussis | 1.0×106CFU/mL |
| Bacteria | Bordetella parapertussis | 1.0×106CFU/mL |
| Bacteria | Mycoplasma pneumoniae | 1.0×106Colony Changing Units (CCU)/mL |
| Bacteria | Mycoplasma genitalium[1] | 4.0×105Bacteria/mL |
| Fungi | Candida albicans | 1.0×106CFU/mL |
| Fungi | Aspergillus flavus | 1.0×106CFU/mL |
| Fungi | Pneumocystis carinii | 1.0×106Cells/mL |
| Biofluid | Pooled human nasal wash[3] | Neat |
[1]A lower concentration was tested due to inability to obtain stock material with high titer.
[2] Microbial interference was not evaluated.
[3]Used to represent diverse microbial flora in the human respiratory tract.
In silico cross-reactivity analysis of the primer sequences was conducted in March 2023 for the SARS-CoV-2 and MS2 assay components of the TaqPath COVID-19 Diagnostic PCR Kit and common respiratory microorganisms to demonstrate specificity of the primers to their targeted sequences.
A total of 40,811 genome sequences (31,011 virus isolates, 9,996 bacterial isolates, and 14 fungal isolates) were downloaded from GenBank using the National Center for Biotechnology Information (NCBI) Genome (https://www.ncbi.nlm.nih.gov/data-hub/genome) and NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/). As complete genomes for Candida albicans and Pneumocystis jirovecii where not on NCBI, their reference sequences (RefSeq) were tested. Rhinovirus Type 1A did not have any complete genomes or a RefSeq, so all 18 partial genomes downloaded from NCBI were tested. These sequences were aligned to the TaqPath COVID-19 Diagnostic PCR Kit assay primer and probe components using Basic Local Alignment Search Tool (BLAST) software v2.12.0 (see table below). Microorganisms were considered non cross-reactive if at least one of the SARS-CoV-2 and MS2 assay primers and probes have $\leq 80\%$ homology or is predicted to produce a qPCR amplicon of $\geq 10,000$ bases.
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None of the isolates analyzed in the study are predicted to cross-react with the TaqPath COVID-19 Diagnostic PCR Kit assay.
Table 10: Microorganisms Assessed for in silico Cross-Reactivity Analysis
| Type | Respiratory Pathogen | Number of Isolates Tested |
| --- | --- | --- |
| Bacteria | Bordetella parapertussis | 90 |
| Bordetella pertussis | 830 |
| Chlamydophila pneumoniae | 12 |
| Corynebacterium sp | 445 |
| Escherichia coli | 3368 |
| Fusobacterium necrophorum | 5 |
| Haemophilus influenzae | 103 |
| Klebsiella pneumoniae | 2053 |
| Lactobacillus plantarum | 210 |
| Lactobacillus acidophilus | 16 |
| Legionella pneumophila | 117 |
| Moraxella catarrhalis | 12 |
| Mycobacterium tuberculosis | 351 |
| Mycoplasma genitalium | 5 |
| Mycoplasma pneumoniae | 80 |
| Neisseria meningitidis | 127 |
| Neisseria sp. | 310 |
| Pseudomonas aeruginosa | 665 |
| Staphylococcus aureus MRSA | 605 |
| Staphylococcus epidermidis | 154 |
| Streptococcus pneumoniae | 151 |
| Streptococcus pyogenes | 268 |
| Streptococcus salivarius | 19 |
| Fungi | Aspergillus sp. | 12 |
| Candida albicans | 1 |
| Pneumocystis jirovecii | 1 |
| Viruses | Adenovirus Type 7 | 284 |
| Cytomegalovirus | 341 |
| Enterovirus | 6556 |
| Epstein-Barr virus | 485 |
| Hepatitis B Virus | 10963 |
| Hepatitis C Virus | 1091 |
| Herpes Simplex Virus-1 | 109 |
| Human coronavirus 229E | 90 |
| Human coronavirus HKU1 | 56 |
| Human coronavirus NL63 | 81 |
| Human coronavirus OC43 | 251 |
| Human Immunodeficiency Virus type 1 | 6740 |
| Human metapneumovirus | 385 |
| Measles virus | 759 |
| MERS coronavirus | 657 |
| Mumps virus | 1332 |
| Parainfluenza virus 1 | 88 |
| Parainfluenza virus 2 | 74 |
| Parainfluenza virus 3 | 415 |
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b) Interfering Substances Study:
The potential for interference for the TaqPath COVID-19 Diagnostic PCR Kit was evaluated with substances that may be present in respiratory specimens. A total of 27 potentially interfering endogenous and exogenous substances were tested at or above clinically relevant levels in negative pooled nasopharyngeal clinical matrix in absence (contrived negative sample) and presence (contrived positive sample) of SARS-CoV-2 target (spiked at $\sim 3\mathrm{x}$ LoD). Each sample was tested in triplicate. The FluMist nasal vaccine was not tested.
Two of the substances tested in the study, nasal corticosteroid Triamcinolone and nasal spray or drops containing Oxymetazoline (e.g., Afrin - No Drip, Extra Moisturizing) gave false results at concentrations greater than $5\%$ (v/v). Additional testing at lower test substance concentrations were performed to determine the concentration where interference is no longer observed. None of the 27 substances tested were determined to be inhibitory to the TaqPath COVID-19 Diagnostic PCR Kit at the concentrations shown in Table 11 below.
Table 11: Potential Interfering Substances Tested on the TaqPath COVID-19 Diagnostic PCR Kit
| Interfering Substance | Concentration |
| --- | --- |
| Leukocytes (human) | 1% (v/v) |
| Mucin: bovine submaxillary gland, type I-S | 0.1 mg/mL |
| Blood (human) | 1% (v/v) |
| Throat lozenges: Benzocaine (7.5 mg), Dextromethorphan HBr (5 mg) | 1% (w/v) |
| Throat lozenges: Menthol (5.4 mg) | 2.2 mg/mL |
| Nasal sprays or drops: Phenylephrine | 10% (v/v) |
| Nasal sprays or drops: Oxymetazoline (Afrin - Allergy Sinus) | 10% (v/v) |
| Nasal sprays or drops: Oxymetazoline (Afrin - No Drip, Extra Moisturizing) | 5% (v/v)[1] |
| Nasal sprays or drops: Sodium chloride with preservatives | 10% (v/v) |
| Bronchodilator: Albuterol | 0.83 mg/mL |
| Nasal corticosteroids: Beclomethasone | 2 mg/mL |
| Nasal corticosteroids: Dexamethasone | 1.5 mg/mL |
| Nasal corticosteroids: Flunisolide | 2 mg/mL |
| Nasal corticosteroids: Budesonide | 1% (v/v) |
| Nasal corticosteroids: Mometasone | 1 mg/mL |
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# 4. Analytical Reactivity (Inclusivity):
The inclusivity of the TaqPath COVID-19 Diagnostic PCR Kit for detection of SARS-CoV-2 was confirmed by testing fifteen variants of SARS-CoV-2. Positive samples were contrived by spiking each SARS-CoV-2 virus strain into negative pooled nasopharyngeal clinical matrix at a concentration of $3\mathrm{x}$ LoD (150 GCE/mL), then tested in triplicate in a blinded and randomized fashion.
All 15 strains of SARS-CoV-2 tested generated $100\%$ positivity rate demonstrating analytical inclusivity. Results are summarized in the table below.
Table 12: TaqPath Diagnostic PCR Kit Inclusivity Study Results
| Variant | Source | Catalog Number | Isolate ID | Positivity Rate (#Positive/#Tested) |
| --- | --- | --- | --- | --- |
| N/A | BEI Resources | NR-52287 | USA-WA1/2020 | 100% (3/3) |
| N/A | ZeptoMetrix | 0810589CFHI | Italy-INMI | 100% (3/3) |
| N/A | | 0810590CFHI | Hong Kong/VM20001061/2020 | 100% (3/3) |
| Alpha; B.1.1.7 | | NATSars(CoV2)- VP | England/204820464/2020 | 100% (3/3) |
| Beta; B.1.351 | | NATSars(CoV2)- VP | South Africa/KRISP-K005325/2020 | 100% (3/3) |
| Gamma; P.1 | | NATSars(CoV2)- VP | Japan/TY7-503/2021 | 100% (3/3) |
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| Delta; B.1.617.2 | NATSars(CoV2)- VP | USA/PHC658/2021 | 100% (3/3) |
| --- | --- | --- | --- |
| Lambda; C.37 | 0810640CFHI | Peru/UN-CDC-2-4069945/2021 | 100% (3/3) |
| Kappa; B.1.617.1 | 0810623CFHI | USA/CA-Stanford-15_S02/2021 | 100% (3/3) |
| Omicron; B.1.1.529 | 0810642CFHI | USA/MD-HP20874/2021 | 100% (3/3) |
| Iota; B.1.526 | 0810619CFHI | USA/NY-WADSWORTH-21025952-01/2021 Isolate 1 | 100% (3/3) |
| B.1 | 0810621CFHI | USA/NY-Wadsworth-103677-01/2020 | 100% (3/3) |
| B.1.595 | 0810622CFHI | USA/NY-Wadsworth-33126-01/2020 | 100% (3/3) |
| Zeta; P2 | 0810618CFHI | USA/NY-Wadsworth-21006055-01/2021 | 100% (3/3) |
| Omicron; BA.2.3 | 0810643CFHI | USA/MD-HP24556/2022 | 100% (3/3) |
In silico analysis was performed to determine inclusivity (reactivity) of the TaqPath COVID-19 Diagnostic PCR Kit primer/probe sequences with all known strains/isolates of SARS-CoV-2 from GISAID and GenBank databases from March 2020 to April 2024. Mismatch and melting temperature analyses were performed and genomes with 100% identity and/or melting temperature (Tm) greater than the annealing temperature were considered reactive. Analysis indicated that >99% of the sequences are reactive based on 100% homology or Tm greater than annealing temperature to at least two of the three assay gene targets of the TaqPath COVID-19 Diagnostic PCR Kit. Evaluation of assay components that do not match 100% to target sequences indicates that most primer or probe mismatches are unlikely to affect assay function.
Sequences from major SARS-CoV-2 variants that were evaluated and found to be reactive with the assay include but are not limited to Alpha, Beta, Delta, Epsilon, Eta, Gamma, Iota, Kappa, Lambda, Mu, Omicron, Theta, and Zeta. Specific Omicron pango lineages evaluated and found to be reactive with the assay include but are not limited to BA.2.86, XBB.1.9.1, XBB.1.9.2, XBB.2.3, XBB.1.16, XBB.1.5, CH.1.1, JN.1, and KP.2.
5. Assay Reportable Range:
Not applicable; this is a qualitative assay.
6. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a) Quality Controls
Please refer to Instrument Description Information (Section C.4) above for assay controls.
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# b) Specimen Stability Studies
The stability of SARS-CoV-2 targets was evaluated using nasopharyngeal (NP) swabs collected in viral transport media (VTM). Positive contrived samples were prepared by spiking inactivated SARS-CoV-2 virus into pooled NP swab clinical matrix collected in VTM. Samples were tested fresh (T0) to establish a baseline and then aliquoted and stored at: 6 hours at ambient temperature $(15^{\circ}\mathrm{C}$ to $30^{\circ}\mathrm{C})$ , 6 days refrigerated $(2^{\circ}\mathrm{C}$ to $8^{\circ}\mathrm{C})$ , 35 days frozen $(-30^{\circ}\mathrm{C}$ to $-10^{\circ}\mathrm{C})$ , and 35 days frozen at $\leq -70^{\circ}\mathrm{C}$ . To establish stability, samples from each condition were withdrawn and tested at intermediate timepoints including the final stated timepoint and one time point beyond the final stability claim. The results of the specimen stability study support the claims listed in Table 13.
Table 13: Stability of Swab Specimens in Viral Transport Media
| Storage Temperature | Stability |
| --- | --- |
| Ambient: 15°C to 30°C | 4 hours |
| Refrigerated: 2°C to 8°C | 3 days (72 hours) |
| Frozen: -30°C to -10°C | 30 days; stable up to 1 freeze-thaw cycles |
| Frozen: ≤ -70°C | 30 days; stable up to 3 freeze-thaw cycles |
# c) Fresh vs. Frozen Studies
A fresh vs. frozen study was performed to demonstrate equivalency between fresh and frozen SARS-CoV-2 nasopharyngeal (NP) swabs in viral transport media (VTM). Positive contrived samples were prepared by spiking inactivated SARS-CoV-2 virus in negative pooled NP swab matrix at a concentration of 0x, 1.5x and 5x LoD. Samples were tested at baseline and subsequently after 1x and 2x freeze-thaw cycles when stored at $-10^{\circ}\mathrm{C}$ to $-30^{\circ}\mathrm{C}$ , and after 1x, 2x, 3x and 4x freeze-thaw cycles when stored at $\leq -70^{\circ}\mathrm{C}$ . Ten replicates were tested for samples prepared at 0x and 5x LoD and 40 replicates were tested for samples at 1.5x LoD. All samples were negative at 0x LoD and all samples spiked at 1.5x and 5x LoD were positive. The results demonstrated that freeze-thaw up to two cycles when stored at $-10^{\circ}\mathrm{C}$ to $-30^{\circ}\mathrm{C}$ , and freeze-thaw up to four cycles when stored at $\leq -70^{\circ}\mathrm{C}$ , does not impact the performance of the TaqPath COVID-19 Diagnostic PCR Kit.
# d) Transport Media Comparison
The following six different transport media were evaluated for compatibility with the TaqPath COVID-19 Diagnostic PCR Kit:
- Remel MicroTest M4RT Multi-Microbe Media
- Remel MicroTest M5 Multi-Microbe Media
- Remel MicroTest M6 Multi-Microbe Media
- Copan Universal Transport Medium (UTM-RT)
- Bartels FlexTrans Transport Medium
BD Universal Viral Transport Medium
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Comparable qualitative and quantitative results were obtained with all the six transport media types in the study.
# 7. Detection Limit:
The Limit of Detection (LoD) of the TaqPath COVID-19 Diagnostic PCR Kit was determined by serially diluting gamma-irradiated SARS-CoV-2 virus (USA-WA1/2020) into pooled negative nasopharyngeal (NP) swab matrix. Samples were randomized and blinded prior to testing on two PCR instruments: QuantStudio 5 Dx RT-PCR instrument and 7500 Fast Dx RT-PCR instrument. LoD is defined as the lowest concentration of SARS-CoV-2 RNA that can be detected at a rate of at least $95\%$ .
A preliminary LoD was determined by testing five replicates of three-fold dilutions of quantified SARS-CoV-2 inactivated virus. Subsequently, the LoD was confirmed by testing 20 replicates to demonstrate a $\geq 95\%$ detection rate at the LoD.
The study established the LoD of the TaqPath COVID-19 Diagnostic PCR Kit to be 50 genomic copy equivalent (GCE)/mL when used with both, the QuantStudio 5 Dx RT-PCR instrument and the 7500 Fast Dx RT-PCR instrument. Table 14 below summarizes the results for each SARS-CoV-2 target, including mean cycle threshold (Ct), standard deviation (SD), COVID-19 positivity, and $\%$ assay target hit rate.
Table 14: Summary of LoD Results and Mean Ct Values for SARS-CoV-2 for the TaqPath COVID-19 Diagnostic PCR Kit
| Virus Concentration (GCE/mL) | COVID-19 Positivity (#Detected/#Tested) | Assay Target Hit Rate (%)[1] (#Detected/#Tested) | | | | Mean Ct (SD)[2] | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | ORF1ab | N gene | S gene | MS2 | ORF1ab | N gene | S gene | MS2 |
| 7500 Fast Dx RT-PCR Instrument | | | | | | | | | |
| 150 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 32.21 (0.45) | 32.70 (0.32) | 32.29 (0.54) | 25.53 (0.22) |
| 50 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 80% (16/20) | 100% (20/20) | 33.74 (0.84) | 34.20 (0.87) | 33.91 (0.65) | 25.45 (0.25) |
| 17 | 70% (14/20) | 80% (16/20) | 85% (17/20) | 45% (9/20) | 100% (20/20) | 34.70 (0.78) | 35.10 (0.91) | 35.21 (1.22) | 25.33 (0.24) |
| 0 | 0% (0/20) | 0% (0/20) | 0% (0/20) | 0% (0/20) | 100% (20/20) | N/A | N/A | N/A | 25.21 (0.20) |
| QuantStudio 5 Dx RT-PCR Instrument | | | | | | | | | |
| 150 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 32.92 (0.55) | 33.00 (0.38) | 32.76 (0.59) | 26.05 (0.17) |
| 50 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 34.24 (0.95) | 34.82 (0.84) | 34.15 (1.02) | 26.10 (0.14) |
| 17 | 85% (17/20) | 75% (15/20) | 85% (17/20) | 70% (14/20) | 100% (20/20) | 35.00 (0.54) | 36.06 (0.85) | 35.17 (0.82) | 26.10 (0.26) |
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| 0 | 0% (0/20) | 0% (0/20) | 0% (0/20) | 0% (0/20) | 100% (20/20) | N/A | N/A | N/A | 25.77 (0.20) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
[1]Assay target hit rate is calculated by the number of assay targets that are called positive divided by the number of replicates tested. Based on the calling rules of the assay, COVID-19 positive is called if two or more of the three assay targets (Orf1ab, N gene, S gene) are called positive. Samples can have different combinations of positive targets, thus contributing to a less than 100% hit rate on the assay target level but 100% hit rate on the COVID-19 positivity call.
[2]For concentration levels that did not produce a positive result, no Ct statistics is displayed and it is denoted as 'N/A'.
# LoD Determination with WHO International Standard for SARS-CoV-2 RNA
The LoD of the TaqPath COVID-19 Diagnostic PCR Kit was determined using contrived positive specimens that were created by spiking the reconstituted WHO International Standard for SARS-CoV-2 RNA (NIBSC code: 20/146) in pooled negative nasopharyngeal (NP) swab matrix in viral transport media (VTM). The study was conducted in two phases. In Phase I, a preliminary LoD was determined by testing serial dilutions of contrived positive samples at nine concentrations. In Phase II, the LoD was confirmed by testing 20 replicates at three concentrations based around the preliminary LoD.
The LoD of the TaqPath COVID-19 Diagnostic PCR Kit using the WHO International Standard for SARS-CoV-2 RNA (NIBSC code: 20/146) was 150 IU/mL for the 7500 Fast Dx RT-PCR instrument and 50 IU/mL for the QuantStudio 5 Dx RT-PCR instrument. The LoD results for each SARS-CoV-2 target, including mean Ct, standard deviation (SD), % COVID-19 positivity, and % assay target hit rate are summarized in Table 15 below.
Table 15: Summary of LoD Results and Mean Ct Values for SARS-CoV-2 for the TaqPath COVID-19 Diagnostic PCR Kit Using WHO International Standard
| Concentration (IU/mL) | COVID-19 Positivity (#Detected/#Tested) | Assay Target Hit Rate (%)[1] (#Detected/#Tested) | | | | Mean Ct (SD)[2] | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | ORF1ab | N gene | S gene | MS2 | ORF1ab | N gene | S gene | MS2 |
| 7500 Fast Dx RT-PCR Instrument | | | | | | | | | |
| 150 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 32.36 (2.40) | 33.75 (0.57) | 32.66 (0.49) | 24.99 (0.20) |
| 50 | 90% (18/20) | 90% (18/20) | 75% (15/20) | 95% (19/20) | 100% (20/20) | 34.49 (0.73) | 35.58 (0.69) | 34.30 (0.84) | 25.01 (0.16) |
| 17 | 50% (10/20) | 55% (11/20) | 50% (10/20) | 55% (11/20) | 100% (20/20) | 35.01 (0.76) | 35.80 (0.70) | 35.26 (1.06) | 25.18 (0.21) |
| 0 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) | N/A | N/A | N/A | 25.31 (0.04) |
| QuantStudio 5 Dx RT-PCR Instrument | | | | | | | | | |
| 150 | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 100% (20/20) | 33.52 (0.59) | 34.27 (0.61) | 33.52 (0.70) | 25.22 (0.18) |
| 50 | 95% (19/20) | 90% (18/20) | 80% (16/20) | 90% (18/20) | 100% (20/20) | 35.09 (0.83) | 35.64 (0.71) | 34.91 (0.94) | 25.44 (0.16) |
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K233453 - Page 21 of 25
| 17 | 50% (10/20) | 50% (10/20) | 45% (9/20) | 50% (10/20) | 100% (20/20) | 35.33 (0.93) | 36.64 (0.35) | 35.73 (0.55) | 25.60 (0.12) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | 0% (0/3) | 0% (0/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) | N/A | N/A | N/A | 25.46 (0.03) |
[1]Assay target hit rate is calculated by the number of assay targets that are called positive divided by the number of replicates tested. Based on the calling rules of the assay, COVID-19 positive is called if two or more of the three assay targets (Orf1ab, N gene, S gene) are called positive. Samples can have different combinations of positive targets, thus contributing to a less than 100% hit rate on the assay target level but 100% hit rate on the COVID-19 positivity call.
[2]For concentration levels that did not produce a positive result, no Ct statistics is displayed and it is denoted as 'N/A'.
# 8. Assay Cut-Off:
Once a TaqPath COVID-19 Diagnostic PCR Kit test run is completed, the data from the RT-PCR instrument's data collection software is imported into COVID-19 Interpretive Software IVD Edition for analysis and interpretation. The COVID-19 Interpretive Software calculates the interpretive results for each sample and control based on the cycle threshold (Ct) value obtained for each of the targets. The following Ct cutoff values for the assay targets is used during result interpretations.
Table 16: Ct Cutoff Values for Assay Targets in TaqPath COVID-19 Diagnostic PCR Kit
| Sample or Control | Target | Ct Cutoff |
| --- | --- | --- |
| Positive Control | MS2 | Valid Ct values are >37 |
| | Viral targets | Valid Ct values are ≤37 |
| Negative Control | MS2 | Valid Ct values are ≤32 |
| | Viral targets | Valid Ct values are >37 |
| Clinical samples | MS2 | Valid Ct values are ≤32[1] |
| | Viral targets | Positive Ct values are ≤37 |
[1]If any of the viral targets is positive, the Ct for MS2 can be >32.
# 9. Carry-Over:
Carry-over/cross-contamination rate of the TaqPath COVID-19 Diagnostic PCR Kit was evaluated by testing contrived SARS-CoV-2 high-positive (formulated at approximately 1×10⁵ plaque forming units (PFU)/mL in pooled negative nasopharyngeal swab matrix) and SARS-CoV-2 negative samples in a checkerboard pattern. Ten runs using two KingFisher Flex purification instruments, two operators, and one of each real time PCR instruments—7500 Fast Dx RT-PCR instrument and QuantStudio 5 Dx RT-PCR instrument, were performed. Each run tested 47 replicates of contrived SARS-CoV-2 high positive samples and 47 replicates of SARS-CoV-2 negative samples.
Out of a total of 470 negative samples tested, four false positive SARS-CoV-2 results were obtained. Two false positive results were obtained on 7500 Fast Dx RT-PCR instrument and two false positive results on QuantStudio 5 Dx RT-PCR instrument. The total carry-over/cross-contamination rate of the TaqPath COVID-19 Diagnostic PCR Kit was determined to be 0.85% (4/470).
{21}
B Comparison Studies:
1. Method Comparison with Predicate Device:
Refer to the Clinical Studies Section of this document.
2. Matrix Comparison:
Not applicable.
C Clinical Studies:
The clinical performance of the TaqPath COVID-19 Diagnostic PCR Kit was established in a multi-site prospective study with three external testing sites evaluating nasopharyngeal (NP) and anterior nasal (AN) swab specimens in BD universal viral transport (UVT) medium from individuals with signs and symptoms of respiratory tract infection. One NP swab and one AN swab was collected from each participant enrolled in the all-comer prospective study from 11 geographically diverse locations in the US. The swab collection order was alternated between the NP and the AN swab. Testing of the TaqPath COVID-19 Diagnostic PCR Kit was conducted on the 7500 Fast Dx RT-PCR instrument and QuantStudio 5 Dx RT-PCR instrument. A composite comparator approach, using three highly sensitive FDA cleared/authorized SARS-CoV-2 molecular assays was used to evaluate the performance of the TaqPath COVID-19 Diagnostic PCR Kit. The composite comparator result was defined as the concordant results from two comparator assays. In case of discordance between the initial two comparator assays, the sample was tested by a third assay and the result of the third test determined the composite comparator result.
From April to August 2023, a total of 1,076 subjects were enrolled in the prospective clinical study. After exclusion of samples due to withdrawal from the study, not meeting the inclusion criteria or incomplete consent forms, shipment delays at collection sites or specimen leakage during transit, a total of 1,055 NP swab specimens and 1,052 AN swab specimens were tested in the study. Of the 1,055 NP swab specimens tested, two NP swab specimens with inconclusive/invalid results on the 7500 Fast Dx and QuantStudio 5 Dx RT-PCR instruments were excluded from data analysis, resulting in 1,053 evaluable NP swab specimens on each instrument. Of the 1,052 AN swab specimens, three had inconclusive result on the 7500 Fast Dx Real-Time PCR instrument and were excluded from data analysis, resulting in 1,049 evaluable AN swab specimens on the 7500 Fast Dx RT-PCR instrument and 1,052 evaluable AN swab specimens on the QuantStudio 5 Dx RT-PCR instrument. Specimens evaluated using the TaqPath COVID-19 Diagnostic PCR Kit were tested either fresh (tested within 72 hours of collection when stored at $2^{\circ}\mathrm{C} - 8^{\circ}\mathrm{C}$) or frozen (stored at $\leq -70^{\circ}\mathrm{C}$ and tested within 30 days of collection).
a) Clinical Performance of TaqPath COVID-19 Diagnostic PCR Kit for Nasopharyngeal Swabs
Out of 1,053 NP swab specimens evaluated, 89 were positive and 964 were negative according to the composite comparator method. On the 7500 Fast Dx RT-PCR instrument, the TaqPath COVID-19 Diagnostic PCR Kit had a positive percent agreement (PPA) of $98.9\%$ (88/89) and a negative
K233453 - Page 22 of 25
{22}
percent agreement (NPA) of 98.4% (949/964). On the QuantStudio 5 Dx RT-PCR instrument, the TaqPath COVID-19 Diagnostic PCR Kit had a PPA of 98.9% (88/89) and a NPA of 98.7% (951/964). Performance is summarized in the tables below.
Table 17: Clinical Performance Estimates of the TaqPath COVID-19 Diagnostic PCR Kit on 7500 Fast Dx RT-PCR Instrument with NP Swabs Specimens
| TaqPath COVID-19 Diagnostic PCR Kit on 7500 Fast Dx RT-PCR Instrument | Composite Comparator | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive | 88 | 15 | 103 |
| Negative | 1 | 949 | 950 |
| Total | 89 | 964 | 1,053 |
| | Percent Agreement | | 95% CI[1] |
| Positive Percent Agreement (PPA) | 88/89 | 98.9% | 93.9%-99.8% |
| Negative Percent Agreement (NPA) | 949/964 | 98.4% | 97.4%-99.1% |
[1] Wilson score
Table 18: Clinical Performance Estimates of the TaqPath COVID-19 Diagnostic PCR Kit on QuantStudio 5 Dx RT-PCR Instrument with NP Swabs Specimens
| TaqPath COVID-19 Diagnostic PCR Kit on QuantStudio 5 Dx RT-PCR Instrument | Composite Comparator | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive | 88 | 13 | 101 |
| Negative | 1 | 951 | 952 |
| Total | 89 | 964 | 1,053 |
| | Percent Agreement | | 95% CI[1] |
| Positive Percent Agreement (PPA) | 88/89 | 98.9% | 93.9%-99.8% |
| Negative Percent Agreement (NPA) | 951/964 | 98.7% | 97.7%-99.2% |
[1]Wilson score
## b) Clinical Performance of TaqPath COVID-19 Diagnostic PCR Kit for Anterior Nasal Swabs
Out of 1,049 AN swab specimens evaluated on the 7500 Fast Dx RT-PCR instrument, 85 were positive and 964 were negative according to the composite comparator method. On the 7500 Fast Dx RT-PCR instrument, the TaqPath COVID-19 Diagnostic PCR Kit had a PPA of 98.8% (84/85) and a NPA of 97.8% (943/964). Out of 1,052 AN swab specimens evaluated on the QuantStudio 5 Dx RT-PCR instrument, 85 were positive and 967 were negative according to the composite comparator method. On the QuantStudio 5 Dx RT-PCR instrument, the TaqPath COVID-19 Diagnostic PCR Kit had a PPA of 98.8% (84/85) and a NPA of 98.0% (948/967). Performance is summarized the tables below.
Table 19: Clinical Performance Estimates of the TaqPath COVID-19 Diagnostic PCR Kit on 7500 Fast Dx RT-PCR Instrument with AN Swabs Specimens
| TaqPath COVID-19 Diagnostic PCR Kit on | Composite Comparator |
| --- | --- |
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K233453 - Page 24 of 25
| 7500 Fast Dx RT-PCR Instrument | Positive | Negative | Total |
| --- | --- | --- | --- |
| Positive | 84 | 21 | 105 |
| Negative | 1 | 943 | 944 |
| Total | 85 | 964 | 1,049 |
| | Percent Agreement | | 95% CI[1] |
| Positive Percent Agreement (PPA) | 84/85 | 98.8% | 93.6%–99.8% |
| Negative Percent Agreement (NPA) | 943/964 | 97.8% | 96.7%–98.6% |
[1]Wilson score
Table 20: Clinical Performance Estimates of the TaqPath COVID-19 Diagnostic PCR Kit on QuantStudio 5 Dx RT-PCR Instrument with AN Swabs Specimens
| TaqPath COVID-19 Diagnostic PCR Kit on QuantStudio 5 Dx RT-PCR Instrument | Composite Comparator | | |
| --- | --- | --- | --- |
| | Positive | Negative | Total |
| Positive | 84 | 19 | 103 |
| Negative | 1 | 948 | 949 |
| Total | 85 | 967 | 1,052 |
| | Percent Agreement | | 95% CI[1] |
| Positive Percent Agreement (PPA) | 84/85 | 98.8% | 93.6%–99.8% |
| Negative Percent Agreement (NPA) | 948/967 | 98.0% | 97.0%–98.7% |
[1]Wilson score
Other Clinical Supportive Data:
Not applicable
## D Clinical Cut-Off:
Not applicable
## E Expected Values/Reference Range:
Samples were collected from 11 geographically diverse collection sites across the US. The SARS-CoV-2 positivity rates, as determined by the TaqPath COVID-19 Diagnostic PCR Kit during the clinical study, per collection site and overall, by specimen type, are shown in the table below.
Table 21: SARS-CoV-2 Positivity Rates by Collection Site Based on the TaqPath COVID-19 Diagnostic PCR Kit
| Collection Site | QuantStudio 5 Dx RT-PCR Instrument | | 7500 Fast Dx RT-PCR Instrument | |
| --- | --- | --- | --- | --- |
| | NP Positivity Rate | AN Positivity Rate | NP Positivity Rate | AN Positivity Rate |
| D&H, Florida | 28.2% (61/216) | 29.0% (63/217) | 28.2% (61/216) | 30.1% (65/216) |
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| KUR-B, New York | 3.9% (3/76) | 3.9% (3/77) | 3.9% (3/77) | 3.9% (3/77) |
| --- | --- | --- | --- | --- |
| KUR-E, South Carolina | 12.7% (13/102) | 12.9% (13/101) | 13.9% (14/101) | 11.9% (12/101) |
| KUR-M, Texas | 2.5% (2/81) | 2.5% (2/81) | 2.5% (2/81) | 2.5% (2/81) |
| KUR_MB, California | 7.7% (1/13) | 7.7% (1/13) | 7.7% (1/13) | 7.7% (1/13) |
| KUR-P, South Carolina | 5.8% (3/52) | 5.8% (3/52) | 5.8% (3/52) | 5.8% (3/52) |
| KUR-R, California | 0.0% (0/2) | 0.0% (0/2) | 0.0% (0/2) | 0.0% (0/2) |
| WR-C, Tennessee | 3.3% (2/60) | 1.7% (1/59) | 3.3% (2/60) | 1.7% (1/58) |
| WR-L, Florida | 6.7% (8/120) | 5.0% (6/119) | 6.7% (8/120) | 5.1% (6/118) |
| WR-S, California | 2.9% (7/240) | 2.9% (7/240) | 3.3% (8/240) | 2.9% (7/240) |
| WR-V, Nevada | 1.1% (1/91) | 4.4% (4/91) | 1.1% (1/91) | 5.5% (5/91) |
| Overall | 9.6% (101/1053) | 9.8% (103/1052) | 9.8% (103/1053) | 10.0% (105/1049) |
F Other Supportive Instrument Performance Characteristics Data:
Not applicable
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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