general information laboratory name sponsor mako …

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MAKO Medical Center TaqPath™ COVID-19 Combo Kit by ThermoFisher Validation New Assay Validation

TaqPath™ COVID-19 Combo Kit by ThermoFisher Validation

GENERAL INFORMATION

Laboratory Name Sponsor Mako Medical 1 Industry Drive Henderson, NC 27537

Contact Person Matthew Tugwell Technical Supervisor Assay Name TaqPath™ COVID-19 Detect SARS-CoV-2 Assay Type Qualitative Detection Methodology RT-PCR Specimen Types nasopharyngeal swabs, E swabs, 0.45%

saline and flock swabs. Clinical Purpose Detection of SARS-CoV-2 infection Laboratory Medical Director Dr. Chad Rund

PURPOSE

The purpose of this validation is to validate TaqPath™ COVID-19 Combo Kit by ThermoFisher on the QuantStudio™ 12k Flex PCR. TaqPath™ COVID-19 Combo Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid form SARS-CoV-2 in Nasopharyngeal swabs. The TaqPath™ COVID-19 Combo Kit is for use only under Emergency Use Authorization (EUA). This testing is a high complexity test. Testing results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in nasopharyngeal swabs, E swabs, 0.45% saline with flock swabs during the acute phase of the 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 bacterial infection or c0-infection with other viruses. The agent detected may not be the definite cause of disease. All positive results are required to report to the appropriate public health authorities. Negative results do not impede SARS-CoV-2 infection and should not be used as the single bases for patient management decisions. Negative results must be combined with clinical observations, patient history and epidemiological information.

The specific nucleic acid sequences from the genome of the SARS-CoV-2 that will be measured in this validation are N gene, S gene, ORF1ab, and MS2.

INTENDED USE:

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SARS-CoV-2 assay is a real-time RT-RCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal swabs, E-Swabs, 0.45% saline and flock swabs, from individuals suspected of COVID-19. Testing is limited to Mako Medical Genetic Laboratory and is Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C 263 a certified high-complexity laboratory.

Results are for the identification and detection of SARS-C0V-2 RNA. The SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase of the infection. Positive results are indicative of active infection with SARS-CoV-2 but do not rule out bacterial infection of co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories with 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 management decisions. Negative results must be combined with clinical observations, patient history and epidemiological information.

The assay is intended for use by CLIA certified high-complexity laboratories with experience in developing molecular diagnostics and is only for use under the Food and Drug Administration’s Emergency Use Authorization.

TEST PRINCIPLE:

The assay is real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in Nasopharyngeal swabs, Eswabs, and Flock swabs in 0.45% saline.

VALIDATION INCLUDES:

1. Repeatability (Intra-assay Precision) 2. Accuracy 3. Limit of Detection 4. Cross-reactivity 5. Interface testing

All samples used in this validation will be nasopharyngeal swabs, E swabs, 0.45% saline and flock swabs.

Repeatability:

The Technologist will be using positive DNA control that is undiluted in a serial dilution

10,000 2500 Repeated 3x 1000 250 100 25 10

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Extraction:

The Technologist will be using positive DNA control that is spiked with water in a serial dilution.

10,000 7,500 2,500 X3 1000 750 250 100 75 25 10 >100 good

LOD and Accuracy information will come from the process listed above. Cross reactivity will be done with positive and negative controls. The results will be measured to see if cross reactivity is present.

Interface testing will be conducted by Mako LIS department in Apollo.

PROCEDURE:

1. Testing – Follow the instructions in the test kit for TaqPath™ COVID-19 Combo Kit by ThermoFisher.

2. Patient samples should be prepared under a laminar airflow hood of biological safety cabinet.

3. Expectable samples for testing include nasopharyngeal swabs, E swabs, 0.45% saline and flock swabs.

4. Extract RNA from patient sample using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit and the KingFisher™

5. Perform RT-PCR using the QuantStudio™ 12k Flex PCR 6. Analyze the data 7. Review the controls 8. Review the patient results and interpretation

CONTROLS

Controls used in this validation are included in the TaqPath™ COVID-19 assay kit known as COVID-19 Control Kit. There are three levels of controls. Controls are running with every plate during testing.

CONTROL USED TO MONITOR ASSAYS Positive Control RT-PCR reaction setup and

reagent integrity All three SARS-CoV-2 assays

MS2 Phage Control RNA extraction MS2 Negative Control Cross-contamination during

RNA extraction and reaction setup

All three Sars-CoV-2 assays MS2 assay

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SPECIMEN REJECTION CRITERIA

Specimen sent to the lab other than nasopharyngeal swabs, E swabs, 0.45% saline and flock swabs.

EQUIPMENT

• QuantStudio™ 12k Flex PCR system • TaqPath™ COVID-19 Kit by ThermoFisher • KingFisher™ Express-96 Magnetic Particle Processor

REAGENTS/CONSUMABLES

• Nasopharyngeal swabs • E-Swab (Copan Diagnostics Inc. cat. # 480C) • 0.45% Saline cat # CN4510 • Sterile tubes cat # 62.612.016 • Flock swabs cat. #25-3606-U • TaqPath™ COVID-19 Combo kit (cat. # A47813)

RESULTS INTERPERATION TABLE

Results will be released as:

Pathogen Detected

No Pathogen Detected

PRODUCT MANUFACTURING

Please note that Under the Emergency Authorization (EUA) most if the 21 CFR 820 Quality System Regulation (QSR) requirements can be waived for the duration of the EUA. FDA expects that the high complexity laboratories performing these tests follow comparable practices as much as possible and may consider previous compliance history when determining whether to waive

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certain QSR requirements for a specific product. Please note adverse events, per 21 CFR part 803, must be reported to authorized devices.

DATA ANALYSIS

1. Repeatability (Intra-assay Precision) 2. Accuracy 3. Limit of Detection -Describe the specific material used in study, (live or inactivated viral

stocks, viral RNA, or in vitro transcripts) and the LOD with appropriate units for our assay.

4. Cross-reactivity – Describe in abbreviated form our cross-reactivity study and list the microorganisms tested, including whether this was performed either in in silico or wet testing. Organisms recommended for testing are listed in table. Samples used in cross-reactivity are: Results Positive A Positive B Negative Viruses Adenovirus + Detected Not Detected Not Detected Coronavirus 229E + Detected Not Detected Not Detected Coronavirus HKU1 Not Detected + Detected Not Detected Coronavirus NL63 + Detected Not Detected Not Detected Coronavirus OC43 Not Detected + Detected Not Detected Human Metapneumovirus A+B

+ Detected Not Detected Not Detected

Influenza A H1 + Detected Not Detected Not Detected Influenza H1N1 pdm09 + Detected Not Detected Not Detected Influenza A H3 Not Detected + Detected Not Detected Influenza B Not Detected + Detected Not Detected Parainfluenza Virus 1 + Detected Not Detected Not Detected Parainfluenza Virus 2 Not Detected + Detected Not Detected Parainfluenza Virus 3 + Detected Not Detected Not Detected Parainfluenza Virus 4 Not Detected + Detected Not Detected Respiratory Syncytial Virus A+B

Not Detected + Detected Not Detected

Rhinovirus/Enterovirus + Detected Not Detected Not Detected Bacteria Bordetella Pertussis Not Detected + Detected Not Detected Chlamydophila Pneumoniae Not Detected + Detected Not Detected Mycoplasma Pneumoniae + Detected Not Detected Not Detected

Samples used in testing were supplied from QI A stat-Dx RP Control Panel M360. 5. Interface testing

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LIM Program Values: MS2 <27,000 N Gene <35,701 ORF1ab <33,512 S Gene <36,760

6. Include our analytical sensitivity indicated that there was 100% detection greater than 250 copies of all SARS-CoV-2 strains.

DAY 1 – Linearity

N Gene ORF1ab S Gene Copies Ct Ct Ct 10000 24.428 21.957 22.461 10000 24.477 22.359 22.264 10000 24.477 22.418 21.789

2500 26.295 24.397 23.067 2500 26.464 24.959 23.128 2500 26.329 24.741 23.913 1000 27.673 25.787 25.553 1000 27.502 25.403 25.718 1000 27.565 25.672 25.484

250 29.468 27.747 26.599 250 29.584 27.793 26.746 250 29.616 27.698 25.653 100 31.288 29.159 27.616 100 31.154 29.518 28.147 100 31.302 29.311 28.748

25 34.472 31.196 28.949 25 33.596 32.665 30.328 25 33.244 32.246 30.732 10 34.989 34.371 33.262 10 34.888 33.948 30.175 10 34.852 36.826 31.503

R² = 0.9554

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27

29

31

33

35

37

39

41

50 500 5000

Ct

Copies

N Gene (Water Extraction) Linearity

R² = 0.9624

20222426283032343638

5 50 500 5000

Ct

Count

ORF1ab qPCR Assay Linearity

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See forms attached

Linearity Study is acceptable with 95% accuracy.

DAY 2 – Precision and Extraction

N gene ORF1ab S Gene Copies Ct Ct Ct 10000 28.421 25.81 26.025 10000 28.255 25.326 25.962

7500 28.933 26.191 26.456 7500 29.341 26.676 26.976 5000 29.657 27.178 27.723 5000 29.714 26.896 27.27 2500 30.988 29.182 28.836 2500 30.848 27.917 28.805 1000 32.313 29.099 29.291 1000 32.263 28.765 29.5

750 31.998 28.513 29.267 750 32.782 29.391 29.809 500 33.322 29.961 30.133 500 33.56 29.926 30.575 250 34.925 30.539 31.088 250 34.494 30.93 31.653 100 39.384 25.912 35.777 100 38.076 32.211 36.577

75 38.539 33.418 34.996 75 38.459 31.283 36.054

R² = 0.9471

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2224

262830

3234

5 50 500 5000

Ct

Count

N Gene qPCR Assay Linearity

R² = 0.9554

252729313335373941

50 500 5000

Ct

Copies

N Gene (Water Extraction) Linearity

R² = 0.6149

25262728293031323334

50 500 5000

Ct

Copies

ORF1ab (Water Extraction) Linearity

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Precision and extraction are acceptable with one outlier in ORF1ab water extraction.

Day 3 – LOD 20 runs

E-Swab E-Swab E-Swab Saline Saline Saline UTM UTM UTM Replicate N Gene ORF1ab S Gene N Gene ORF1ab S Gene N Gene ORF1ab S Gene 250 Copies rep01 36.113 19.958 35.562 32.673 28.571 30.897 33.045 33.096 33.636 250 Copies rep02 UND 28.865 UND 32.691 31.934 31.949 34.758 30.935 30.796 250 Copies rep03 32.709 26.163 30.87 32.584 28.216 33.123 32.528 32.472 30.221 250 Copies rep04 33.577 31.987 6.847 32.735 28.967 32.067 32.016 31.437 31.705 250 Copies rep05 36.499 30.804 32.11 32.295 28.849 32.028 32.395 31.249 32.227 250 Copies rep06 35.406 27.625 36.707 33.06 29.488 32.116 32.175 33.61 32.973 250 Copies rep07 33.13 32.61 UND 33.139 27.422 32.698 33.107 24.969 31.965 250 Copies rep08 34.224 27.944 UND 33.543 27.997 36.852 33.871 33.002 15.224 250 Copies rep09 34.225 30.681 31.329 32.705 26.638 32.743 34.359 27.447 32.823 250 Copies rep10 32.684 30.703 28.498 33.284 27.735 31.969 33.124 26.077 30.34 250 Copies rep11 34.853 26.146 UND 31.881 27.242 31.252 33.089 30.355 31.876 250 Copies rep12 33.925 29.842 33.104 31.936 27.017 31.253 34.465 32.282 UND 250 Copies rep13 33.805 29.998 26.335 31.328 27.381 30.317 33.18 28.165 30.704

R² = 0.9125

2527293133353739

50 500 5000

Ct

Copies

S gene (Water Extraction) Linearity

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250 Copies rep14 34.45 30.387 34.407 31.761 28.038 31.347 31.405 31.778 30.927 250 Copies rep15 34.326 27.844 UND 31.733 28.027 32.375 32.756 34.452 32.875 250 Copies rep16 32.811 30.225 29.683 31.635 26.279 31.503 32.947 24.402 35.772 250 Copies rep17 UND 25.011 35.169 32.96 24.211 31.494 34.929 32.975 UND 250 Copies rep18 33.35 28.536 UND 32.799 27.385 32.709 32.876 31.964 34.687 250 Copies rep19 34.012 28.508 33.879 31.633 28.676 31.048 33.176 32.728 26.601 250 Copies rep20 32.759 28.994 UND 31.383 26.445 30.398 *39.195 UND 29.367

*Internal Control Failed Testing Performance

E Swab 19/20 =95% Saline 20/20 = 100% UTM 19/20 = 95%

Day 4 – Method Comparison – Medium Comparison

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Assay Specific:

MS2 E-swab Saline Viral Transport Copies Rep CT CT CT

>10,000 1 23.19 24.091 23.206 2 24.023 23.741 23.514

>10,000 1 24.002 23.606 24.042 2 24.336 23.557 23.738

>10,000 1 23.733 23.521 23.853 2 23.725 23.32 23.857

>10,000 1 23.641 23.271 23.952 2 23.291 23.286 23.555

>10,000 1 23.296 23.809 23.597 2 24.047 23.439 24.08

>10,000 1 24.011 23.568 23.903 2 23.955 23.711 24.142

>10,000 1 23.718 23.423 23.606 2 23.872 23.238 23.622

>10,000 1 23.589 23.142 24.186 2 23.481 23.246 23.452

>10,000 1 23.975 23.151 23.607

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2 23.879 23.44 23.559

>10,000 1 24.114 23.18 23.612 2 24.042 23.415 24.055

>10,000 1 23.637 22.786 24.484 2 23.863 22.828 24.322

>10,000 1 23.185 23.133 24.423 2 23.088 23.225 24.004

>10,000 1 24.509 23.097 23.658 2 24.684 23.558 23.465

N gene E-swab Saline Viral Transport Copies Rep CT CT CT

10,000 1 29.381 28.331 28.357 2 30.145 28.181 28.691

7,500 1 30.504 29.339 30.192 2 30.534 29.458 29.83

5,000 1 33.493 30.341 31.39 2 32.675 30.089 31.115

2,500 1 31.351 29.231 30.153 2 31.201 28.51 29.655

1,000 1 32.353 30.501 31.336 2 33.649 30.764 32.018

750 1 35.614 33.37 33.006 2 35.561 32.731 34.189

500 1 Undetermined 36.745 38.017 2 38.048 35.835 36.475

250 1 35.296 33.378 34.024 2 35.701 34.113 33.851

100 1 Undetermined Undetermined Undetermined 2 Undetermined 36.929 Undetermined


50 1 Undetermined Undetermined Undetermined 2 Undetermined Undetermined Undetermined



ORF1ab E-swab Saline Viral Transport

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Copies Rep CT CT CT

10,000 1 27.583 26.63 26.398 2 28.205 26.828 27.248

7,500 1 27.233 27.563 29.187 2 27.274 27.945 28.412

5,000 1 31.377 28.099 30.269 2 22.795 28.806 30.31

2,500 1 30.246 27.852 26.289 2 29.367 26.86 27.884

1,000 1 30.986 28.287 29.738 2 30.861 28.795 29.813

750 1 32.354 30.71 31.179 2 31.408 30.1 30.652

500 1 Undetermined 33.338 34.032 2 35.607 32.212 37.293

250 1 32.598 31.245 33.007 2 33.512 31.591 26.469

100 1 25.923 32.163 Undetermined 2 Undetermined Undetermined Undetermined

75 1 Undetermined Undetermined 23.74 2 29.48 33.54 Undetermined

50 1 28.104 Undetermined Undetermined 2 34.527 33.959 Undetermined

25 1 23.515 Undetermined Undetermined 2 36.991 Undetermined Undetermined

10 1 27.969 32.483 26.61 2 34.268 Undetermined Undetermined

S gene E-swab Saline Viral Transport Copies Rep CT CT CT

10,000 1 28.147 25.751 26.345 2 28.373 24.639 26.665

7,500 1 28.788 27.067 29.171 2 27.856 27.035 28.558

5,000 1 31.348 28.653 30.591 2 26.903 28.425 30.454

2,500 1 30.063 27.221 29.659 2 29.731 25.353 29.381

1,000 1 30.593 28.653 29.551 2 31.728 29.23 29.873

750 1 Undetermined 31.385 31.539

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2 32.705 30.665 32.417

500 1 Undetermined 33.195 Undetermined 2 Undetermined 33.77 33.954

250 1 34.096 32.016 32.662 2 36.76 32.055 33.196






Medium Graphs:

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Positive Controls:

Sample Name

Target Name Ct

PosA MS2 24.671 PosA N gene Undetermined PosA ORF1ab Undetermined PosA S gene Undetermined PosB MS2 24.614 PosB N gene Undetermined PosB ORF1ab Undetermined PosB S gene Undetermined

Day 5 – Sample Comparison with Sonic Lab

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Sample Target CT Result 323924450 MS2 22.116 Pos 323924450 N gene 24.643 323924450 ORF1ab 22.551 323924450 S gene 23.015 323924460 MS2 21.148 Pos 323924460 N gene 27.442 323924460 ORF1ab 25.751 323924460 S gene 26.072 323964337 MS2 23.126 Neg 323964337 N gene UND 323964337 ORF1ab UND 323964337 S gene UND 323964341 MS2 20.73 Pos 323964341 N gene 17.678 323964341 ORF1ab 15.364 323964341 S gene 15.804 323964345 MS2 22.102 Inconclusive 323964345 N gene UND 323964345 ORF1ab 29.761 323964345 S gene UND 323964349 MS2 20.682 Neg 323964349 N gene UND 323964349 ORF1ab UND 323964349 S gene UND 323964353 MS2 21.95 Neg 323964353 N gene UND 323964353 ORF1ab UND 323964353 S gene UND 323964357 MS2 22.221 Pos 323964357 N gene 29.968 323964357 ORF1ab 27.046 323964357 S gene 27.534 323964576 MS2 20.691 Inconclusive 323964576 N gene UND 323964576 ORF1ab 31.245 323964576 S gene UND 323964577 MS2 17.69 Pos 323964577 N gene 11.788 323964577 ORF1ab 9.906 323964577 S gene 10.431

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323971641 MS2 22.905 Neg 323971641 N gene UND 323971641 ORF1ab UND 323971641 S gene UND 323971645 MS2 21.067 Neg 323971645 N gene UND 323971645 ORF1ab UND 323971645 S gene UND 323971649 MS2 22.805 Neg 323971649 N gene UND 323971649 ORF1ab UND 323971649 S gene UND 323971653 MS2 22.907 Neg 323971653 N gene UND 323971653 ORF1ab UND 323971653 S gene UND 323971657 MS2 23.111 Neg 323971657 N gene UND 323971657 ORF1ab UND 323971657 S gene UND 323971661 MS2 20.725 Neg 323971661 N gene UND 323971661 ORF1ab 37.176 323971661 S gene UND 323971665 MS2 22.686 Neg 323971665 N gene UND 323971665 ORF1ab UND 323971665 S gene UND 323971669 MS2 21.241 Neg 323971669 N gene UND 323971669 ORF1ab UND 323971669 S gene UND 323971873 MS2 20.826 Neg 323971873 N gene UND 323971873 ORF1ab UND 323971873 S gene UND 323971877 MS2 22.22 Neg 323971877 N gene UND 323971877 ORF1ab UND 323971877 S gene UND 323971881 MS2 22.852 Neg

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323971881 N gene UND 323971881 ORF1ab UND 323971881 S gene UND 323971885 MS2 22.069 Neg 323971885 N gene UND 323971885 ORF1ab UND 323971885 S gene UND 323971889 MS2 22.948 Neg 323971889 N gene UND 323971889 ORF1ab UND 323971889 S gene UND 323972558 MS2 22.007 Pos 323972558 N gene 18.353 323972558 ORF1ab 16.08 323972558 S gene 16.525 323972566 MS2 22.686 Neg 323972566 N gene UND 323972566 ORF1ab UND 323972566 S gene UND 323972570 MS2 22.481 Neg 323972570 N gene UND 323972570 ORF1ab UND 323972570 S gene UND 323972646 MS2 22.751 Neg 323972646 N gene UND 323972646 ORF1ab UND 323972646 S gene UND 323972872 MS2 22.519 Neg 323972872 N gene UND 323972872 ORF1ab UND 323972872 S gene UND 323972880 MS2 22.921 Neg 323972880 N gene UND 323972880 ORF1ab UND 323972880 S gene UND 323972884 MS2 23.036 Neg 323972884 N gene UND 323972884 ORF1ab UND 323972884 S gene UND 323972911 MS2 21.434 Pos 323972911 N gene 17.2

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323972911 ORF1ab 15.201 323972911 S gene 15.525 323972939 MS2 20.963 Pos 323972939 N gene 33.192 323972939 ORF1ab 30.828 323972939 S gene 31.642 323973077 MS2 22.448 Inconclusive 323973077 N gene UND 323973077 ORF1ab 31.912 323973077 S gene UND 323973205 MS2 21.963 Pos 323973205 N gene 22.942 323973205 ORF1ab 20.701 323973205 S gene 21.079 324052807 MS2 21.303 Pos 324052807 N gene 21.495 324052807 ORF1ab 18.953 324052807 S gene 19.144 324094355 MS2 21.209 Pos 324094355 N gene 24.649 324094355 ORF1ab 22.078 324094355 S gene 22.17 324125642 MS2 22.665 Inconclusive 324125642 N gene UND 324125642 ORF1ab 29.822 324125642 S gene UND 324246821 MS2 20.618 Inconclusive 324246821 N gene UND 324246821 ORF1ab 24.318 324246821 S gene UND 324246850 MS2 22.611 Neg 324246850 N gene UND 324246850 ORF1ab UND 324246850 S gene UND 324309735 MS2 19.595 Pos 324309735 N gene 13.916 324309735 ORF1ab 12.1 324309735 S gene 12.452 324327676 MS2 22.005 Pos 324327676 N gene 27.295 324327676 ORF1ab 24.682

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324327676 S gene 25.376 324327680 MS2 21.451 Pos 324327680 N gene 32.067 324327680 ORF1ab 29.672 324327680 S gene 31.061 324335755 MS2 20.739 Pos 324335755 N gene 16.737 324335755 ORF1ab 14.295 324335755 S gene 14.241 324335768 MS2 17.192 Pos 324335768 N gene 10.932 324335768 ORF1ab 9.104 324335768 S gene 10.448 324348657 MS2 20.783 Pos 324348657 N gene 28.461 324348657 ORF1ab 26.163 324348657 S gene 26.969 324370600 MS2 21.413 Pos 324370600 N gene 16.939 324370600 ORF1ab 14.151 324370600 S gene 14.093 324375696 MS2 20.992 Pos 324375696 N gene 24.838 324375696 ORF1ab 22.345 324375696 S gene 22.844 324375698 MS2 20.684 Pos 324375698 N gene 23.153 324375698 ORF1ab 20.723 324375698 S gene 20.764 324380638 MS2 21.089 Pos 324380638 N gene 29.662 324380638 ORF1ab 27.023 324380638 S gene 27.914 324383480 MS2 21.117 Pos 324383480 N gene 19.153 324383480 ORF1ab 19.95 324383480 S gene 20.244 324383750 MS2 20.982 Pos 324383750 N gene 25.599 324383750 ORF1ab 23.104 324383750 S gene 23.903

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324393745 MS2 20.727 Neg 324393745 N gene UND 324393745 ORF1ab UND 324393745 S gene UND 324393749 MS2 22.729 Neg 324393749 N gene UND 324393749 ORF1ab UND 324393749 S gene UND 324411015 MS2 20.903 Pos 324411015 N gene 23.949 324411015 ORF1ab 21.444 324411015 S gene 21.707 324411832 MS2 20.915 Pos 324411832 N gene 20.169 324411832 ORF1ab 17.951 324411832 S gene 18.352 324411976 MS2 20.951 Pos 324411976 N gene 21.047 324411976 ORF1ab 18.683 324411976 S gene 18.89 324416706 MS2 20.551 Pos 324416706 N gene 22.986 324416706 ORF1ab 20.725 324416706 S gene 21.02 324429251 MS2 21.208 Inconclusive 324429251 N gene UND 324429251 ORF1ab 33.722 324429251 S gene UND 324430839 MS2 21.454 Pos 324430839 N gene 16.939 324430839 ORF1ab 15.06 324430839 S gene 15.316 324432602 MS2 22.059 Pos 324432602 N gene 17.629 324432602 ORF1ab 14.465 324432602 S gene 15.853 NC MS2 21.291 Expected NC N gene UND NC ORF1ab UND NC S gene UND Pos MS2 UND Expected

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Pos N gene 30.294 Pos ORF1ab 28.093 Pos S gene 28.169 NTC MS2 UND Expected NTC N gene UND NTC ORF1ab UND NTC S gene UND

Validation was 100% recovery for COVID-19.

Ten samples were sent to North Carolina State Virology Laboratory for testing. Mako sent 5 positive patients and 5 negative patients for COVID-19 testing comparability testing. The following are the results recovered by the State Lab:

All ten samples were 100% comparable. Please see printed results below. Patient information has been blackened out, but the accession number will identify patient results.

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Interpretation for Positive results on Mako Medical Reports

Interpretation for Not Detected results on Mako Medical Reports

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ACCEPTANCE CRITERIA/CLINICAL EVALUATION

The validation was successful. All testing was found acceptable and samples were reproducible. There was not cross-reactivity found.

1. Unmet Need addressed by the product a. This section will be completed by FDA

2. Approved/cleared Alternative products a. Currently no methods for detection of SARS-CoV-2 have been approved/cleared

by FDA. 3. Benefits and Risks

a. This section will be filled out by FDA 4. Fact Sheet for Health care providers and patients

a. https://www.fda.gov/emergency-preparedness-and-response/mcm-issues/coronavirus-disease-2019-covid-19#fastfacts

5. Standing Operation Procedure is attached with this Validation on COVID-19. 6. Record Keeping and reporting information to the FDA: The laboratory will track adverse

events and report to FDA under 21 CFR Part 803. Ap website is available to report on adverse events, and this website is referenced in the fact sheet for Health Care providers. The laboratory will maintain will information the performance of the test, and report to FDA any suspected change in associated with the EUA and ensure these records are maintained until notified by FDA. Such records will be made available to FDA for inspection upon request.

DEVIATION:

REFERENCES:

ThermoFisher TaqPath™ COVID-19 Combo Kit Cat # A47813 and A47814

MMQCI Maine Molecular Quality Controls. QIAstat-Dx RP Control Panel M360

RESPONSIBILITIES:

Performing the validation: Matthew Tugwell and Lauren Moon

Validation Author: Kristen Harding and Matthew Tugwell

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Approving the Validation outline: Steve Hoover and Medical Director

Conduct Validation study: Matthew Tugwell

Reviewing the results: Steve Hoover and Medical Director

Conduct LIS Validation study: Matthew Tugwell and Megan Ziller

Approving Study results: Steve Hoover and Medical Director

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