Instructions for PerkinElmer® New Coronavirus Nucleic Acid Detection Kit v 9.0 For prescription use only. For in vitro diagnostic use only. For Emergency Use Authorization only. This manual is proprietary to PerkinElmer, Inc., and intended only for customer use in connection with the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose without the prior written consent of PerkinElmer. Follow the protocol included with the kit. Table of Contents Product Name ........................................................................... 3 Intended Use ............................................................................. 3 Principles of the Assay............................................................. 4 Kit Components and Packaging Specifications ...................... 5 Materials Required but Not Provided ....................................... 5 Storage & Handing Requirements............................................ 7 Warnings and Precautions ....................................................... 7 Instruments............................................................................... 9 Collection, Storage & Shipment of Specimens........................ 9 Assay Procedure .....................................................................11 Nucleic Acid Extraction and PCR Setup .................................11 Extraction and PCR setup on Pre-NAT II ................................11 Extraction and PCR setup on chemagic 360 ...........................12 Extraction from saliva sample on chemagic 360 ......................16 Amplification ..........................................................................16 Interpretation of Results..........................................................24 Kit Limitations..........................................................................28 Conditions of Authorization for the Laboratory......................30 Assay Performance .................................................................32 Limit of Detection ...................................................................32 Analytical Reactivity (Inclusivity) .............................................39 Analytical Specificity (Cross-reactivity) ....................................41 2 Interfering Substances Studies ...............................................43 Clinical Study .........................................................................44 Saliva Sample Stability Study .................................................51 FDA SARS-CoV-2 Reference Panel Testing ............................52 References ...............................................................................52 Appendix A: Specimen Pooling Implementation and Monitoring Guidelines .............................................................55 Appendix B: Verification Recommendations..........................58 Appendix C: Additional Label .................................................61 Product Name PerkinElmer® New Coronavirus Nucleic Acid Detection Kit Kit Contents 48 Tests Intended Use The PerkinElmer® New Coronavirus Nucleic Acid Detection Kit is a real-time RT-PCR in vitro diagnostic test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in human oropharyngeal swab and nasopharyngeal swab specimens collected by a healthcare provider (HCP), and anterior n asal swab specimens collected by an HCP or self-collected under the supervision of an HCP by any individual, including individuals without symptoms or other reasons to suspect COVID-19. This test is also for use with saliva samples collected using the SalivaSecure Saliva Collection Kit either by an HCP or self- collected under the supervision of an HCP in a healthcare setting from individuals suspected of COVID-19. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet requirements to perform high complexity tests. This test is also for the qualitative detection of nucleic acid from SARS-CoV-2 in pooled samples containing up to 5 individual upper respiratory swab specimens (i.e., oropharyngeal swab and nasopharyngeal swab specimens collected by an HCP and anterior nasal swab specimens collected by an HCP or self-collected under the supervision of an HCP) using individual vials containing transport media. Negative results from pooled testing should not be treated as definitive. If patient’s clinical signs and symptoms are inconsistent 3 with a negative result and results are necessary for patient management, then the patient should be considered for individual testing. Specimens included in pools with a positive or invalid result must be tested individually prior to reporting a result. Specimens with low viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing. Results are for the identification of SARS-CoV-2 RNA. SARS-CoV-2 RNA is generally detectable in upper respiratory specimens and saliva 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 bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all 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 treatment or other patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information. Negative results for SARS-CoV-2 RNA from saliva should be confirmed by testing of an alternative specimen type if clinically indicated. The PerkinElmer® New Coronavirus Nucleic Acid Detection 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 PerkinElmer® New Coronavirus Nucleic Acid Detection Kit is only for use under the Food and Drug Administration’s Emergency Use Authorization. Principles of the Assay The PerkinElmer® New Coronavirus Nucleic Acid Detection kit uses TaqMan- based real-time PCR technique to conduct in vitro reverse transcription of SARS-CoV-2 RNA, DNA amplification and fluorescence detection. The assay targets specific genomic regions of SARS-CoV-2: nucleocapsid (N) gene and Open Reading Frame 1ab (ORF1ab) gene. The TaqMan probes for the two amplicons are labeled with FAM and ROX fluorescent dyes respectively to generate target-specific signal. The assay includes an RNA internal control (IC, bacteriophage MS2) to monitor the processes from nucleic acid extraction to fluorescence detection. The IC probe is labeled with VIC fluorescent dye to differentiate its fluorescent signal from SARS-CoV-2 targets. The assay also uses a dUTP/UNG carryover prevention system to avoid 4 contamination of PCR products and subsequent false positive results. Kit Components and Packaging Specifications Catalog Number: 2019-nCoV-PCR-AUS (48 tests/kit) Component Specifications & Storage Name Loading Main Ingredients Conditions nCoV 950 2+ Reagent A μL ×1 tube Buffers, dNTPs, Mg -25 to -15°C nCoV 230 TE buffer, primers, Reagent B μL ×1 tube probes -25 to -15°C Taq DNA nCoV 170 ×1 tube polymerase, MMLV, -25 to -15°C Enzyme Mix μL RNasin, UNG nCoV Internal 1.4 TE buffer, Control mL ×1 tube bacteriophage MS2 -25 to -15°C SARS-CoV-2 RNA nCoV 1.4 ×2 fragments capsulated -25 to -15°C Positive mL tubes Control in bacteriophage nCoV 1.4 Negative ×2 mL TE buffer -25 to -15°C Control tubes Notes: 1) The reference materials and other components in the kit should be treated as potential sources of infection. 2) The use of this kit should be strictly in accordance with the nucleic acid amplification guidelines to operate in compliance with the requirements of the appropriate laboratories. 3) The components in different batches of the kit cannot be used interchangeably. Materials Required but Not Provided 1. RNA extraction reagents and instrument The PerkinElmer® Nucleic Acid Extraction Kits (KN0212) and PreNAT II (SY61) (software version 1.00.06). chemagic™ Viral DNA/RNA 300 Kit special H96 (CMG-1033, CMG-1033- S) and chemagic™ 360 (2024-0020) with chemagic™ Rod Head Set 96 (CMG-371) (chemagicTM MSM I software version 6.1.0.5). 2. PCR amplification instrument and software a. Applied Biosystems™ 7500 Real-Time PCR System: 4351104 with Laptop, 4351105 with desktop, software version 2.3. b. Applied Biosystems™ 7500 Fast Dx Real-Time PCR System: 4406984 with Laptop, 4406985 with desktop, software version 2.0.4. c. Applied Biosystems™ QuantStudioTM 3 Real-Time PCR System: 96-well block; REF: A28131, Design and Analysis Software v1.5.1. 5 d. Applied Biosystems™ QuantStudioTM 5 Real-Time PCR System: 384-well block: REF: A28135, Design and Analysis Software v1.5.1. e. Analytik Jena qTower3 / qTower3 G Real-Time PCR System: 844-00553- x, 844-00554-x, 844-00555-x, 844-00556-x, 844-00563-x, 844-00564-x, 844 00503-2, 844-00503-4, 844-00504-2 software version qPCRsoft 4.1. f. Analytik Jena qTower3 84 / qTower3 84 G Real-Time PCR System: 844- 00558-x, 844-00559-x, 844-00568-x, 844-00569-x, 844-00509-2, 844- 00509-4 software version qPCRsoft384 1.2. g. 96-well PCR plate: • 0.1