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SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (Test Code 39433)

Package Insert

For Emergency Use Only

For In-vitro Diagnostic Use - Rx Only

Intended Use

The Quest Diagnostics SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (“Quest SARS-CoV-2 rRT-PCR”) is a real-time

RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in upper and lower respiratory

specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates, and bronchoalveolar lavage)

collected from individuals suspected of COVID-19 by their healthcare provider.

This test is also for use with nasal swab specimens that are self-collected at home or in a healthcare setting by individuals

using an authorized home-collection kit when determined to be appropriate by a healthcare provider.

This test is for the qualitative detection of nucleic acid from the SARS-CoV-2 in pooled samples containing up to four of

the individual upper respiratory swab specimens (nasopharyngeal, mid-turbinate, anterior nares or oropharyngeal swabs)

that were collected in individual vials containing transport media from individuals suspected of COVID-19 by their

healthcare provider

. Negative results from pooled testing should not be treated as definitive. If patient’s clinical signs and

symptoms are inconsistent with a negative result or results are necessary for patient management, then the patient should

be considered for individual testing. Specimens included in pools with a positive, inconclusive, 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. The SARS-CoV-2 RNA is generally detectable in respiratory

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 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. Specimens that are self-collected will not be tested with an internal control to confirm that the

specimen was properly collected. Self-collected specimens from SARS-CoV-2 positive individuals may yield negative

results if the specimen was not collected properly.

Testing is limited to laboratories designated by Quest Diagnostics that are certified under the Clinical Laboratory

Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, and meet the requirements to perform high complexity tests.

Testing with the SARS-CoV-2 rRT-PCR test is intended for use by qualified and trained laboratory personnel specifically

instructed and trained in the techniques of real-time RT-PCR assays. The SARS-CoV-2 rRT-PCR test is only for use under

a Food and Drug Administration’s Emergency Use Authorization.

Summary and Explanation

An outbreak of pneumonia of unknown etiology in Wuhan City, Hubei Province, China was initially reported to WHO on

December 31, 2019. Chinese authorities identified a novel coronavirus (SARS-CoV-2), which has resulted in thousands of

confirmed human infections in multiple provinces throughout China and exported cases in several Southeast Asian

countries and more recently in Europe and the United States. Cases of severe illness and some deaths have been reported.

Summary of content (27 pages)

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SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (Test Code 39433) Package Insert For Emergency Use Only For In-vitro Diagnostic Use - Rx Only Intended Use The Quest Diagnostics SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR (“Quest SARS-CoV-2 rRT-PCR”) is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates, and bronchoalveolar lavage) collected from in
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The Quest Diagnostics SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR aids in the detection of SARS-CoV-2 RNA and diagnosis COVID-19 and is a real-time reverse transcription polymerase chain reaction test. The test’s primer and probe sets were designed to detect RNA from individuals suspected of COVID-19 by their healthcare provider.
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2019-nCoV_N1 Probe 2019-nCoV_N3 Forward Primer 2019-nCoV_N3 Reverse Primer 2019-nCoV_N3 Probe Reagent Preparation and Storage Primer and Probe 10µM stocks in TE Buffer Dilute Probes 100 µM stocks 1:10 in TE Buffer ex: 100 µL + 900 µL TE Buffer). Prepare aliquots in screw cap tubes. Dilute Primers 200 µM stocks 1:10 in TE Buffer ex: 50 µL + 950 µL TE Buffer). Prepare aliquots in screw cap tubes. Storage Store @ -60ºC to -90ºC Stability 1 year from date of preparation. Formulation sheet EFORM.129.
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DTT solution 500mM Add 100 µL of nuclease free water to one microtube containing DTT and mix with pipette tip. Add the entire 100µL DTT solution into 5mL of cold sterile 0.01 M PBS (pH 7.2) and mix briefly. Discard any unused reconstituted DTT SARS-CoV-2 PCR Mix Combine the ingredients in the amounts listed below. Dispense in 455 µL aliquots label as: NCOV PCR Mix Lot#/Prep: (preparation date. initials) Exp.
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3. 4. 5. 6. 7. 8. 9. 10. Authorization (EUA) for use in laboratories designated by Quest Diagnostics that are also certified under CLIA, and meet the requirements to perform high complexity tests.
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Is the specimen acceptable? • • • The specimen is within stability criteria. There is enough specimen in the collection tube. The patient name and second identifier match the test requisition.
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Procedure NOTE: For all procedures involving specimens, buttoned lab coats, gloves, and face protection are required minimum personal protective equipment. Report all accidents to your supervisor and in accordance with the Company’s policies and Procedures. When performing pooling, laboratories will monitor sample pooling in accordance with Attachment 1 – Protocol for Monitoring of Sample Pooling Testing Strategies.
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MagNA Pure 96 Nucleic Acid Extraction 1. 2. 3. 4. 5 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Refer to PROC.129.01298, Nucleic Acid Isolation on the MagNA Pure 96 Instrument for general instructions on using the MagNA Pure 96 Instrument All of the following steps are performed in the Specimen Preparation Area. Perform beginning of run maintenance on the MagNA Pure 96 instrument (as described in PROC.129.01298). In the Overview tab, select Enter Order, and select “External Lysis 450 µL ”.
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1. 2. 3. 4. SPECIMEN TRANSFER PROCEDURE for the Omega Method using the HAMILTON STAR (to be performed in Extraction Room) Total nucleic acids (DNA and RNA) are extracted from patient specimens and assay controls using the Mag-Bind Viral RNA Xpress Kit (Omega Bio-Tek) on the Hamilton MagEx STAR Autoload automated platform. Refer to PROC.129.01744- Hamilton MagEx STAR Autoload System Use and Maintenance for general instructions on using the Hamilton STAR.
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Omega Method using the Hamilton STAR Nucleic Acid Isolation 1. 2. 3. 4. 5. 6. 7. 8 9. 10. 11. Refer to PROC.129.01744, Hamilton MagEx STAR Autoload System Use and Maintenance for general instructions on using the Hamilton STAR. All of the following steps are performed in the Specimen Preparation Area. Perform daily maintenance on the Hamilton instrument.
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Setting Up Real-Time RT-PCR Reactions 1. 2. 3. 4 5. 6. 7. 8. 9 In the Reagent Room, thaw two vials of 2019-NCOV PCR MIX for every 96 samples (94 specimens + 2 controls) in the assay run. DO NOT thaw on a heat block or on the blowers of a biosafety hood. Add 325 µl of 4X TaqPath Enzyme for every 455 µl tube of NCOV PCR MIX. Vortex to mix and spin down. KEEP 96 WELL PLATE CHILLED ON A COLD BLOCK. To each well of the 96-well reaction plate, add 15 µL of 2019-NCOV PCR MIX as needed.
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Analyzing the Run Data, Exporting Results and Printing 2. Click on “Analysis”, and then click on “Analysis Settings”. For 2019-NCOV PCR MIX Manual Ct should be selected Set the threshold at 0.1 for NCOV1, automatic baseline Set the threshold at 0.01 for NCOV3, automatic baseline Set the threshold at 0.05 for IPC, automatic baseline 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Click the Analyze icon (฀) from the toolbar. Note: Wait approximately one minute for the analysis process to be completed.
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Interpretation of Results and Reporting Review patient results for unusual patterns, trends or distributions in patient results, such as an unusually high percentage of abnormal results, or unusually high percentage of non-reactive, or indeterminate, or reactive results. Computer aided tools should be used when available. Refer to SOP Quality Control Program and Molecular Infectious Diseases Department. Real-Time Group Results Review and Release Process.
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nCoV-N1 ND nCoV-N3 ND DET DET Only one of two SARSnCoV-2 targets are Detected ND ND Specimen Result Interpretation for Pooled Specimens IPC Interpretation Actions Within +/- 3 Ct of NOT DETECTED Report to public health authorities. Negative Control Repeat each constituent specimen in the pool Not Applicable (+/-) POOLED as a separate unpooled specimen. POSITIVE – DO NOT REPORT Repeat each constituent specimen in the pool Not Applicable (+/-) POOLED INCONCLUSIVE – as a separate unpooled specimen.
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Inhibitors or other types of interference may produce a false negative result. An interference study evaluating the effect of common cold medications was not performed. 7. Test performance can be affected because the epidemiology and clinical spectrum of infection caused by 2019-nCoV is not fully known.
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Conditions of Authorization for the Laboratory The Quest SARS-CoV-2 rRT-PCR test Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients and other authorized labeling are available on the FDA website: https://www.fda.
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• Authorized laboratories implementing pooling strategies for testing patient specimens must use the “Protocol for Monitoring of Specimen Pooling Testing Strategies” to evaluate the appropriateness of continuing to use such strategies based on the recommendations in the protocol.
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Performance Characteristics 1) Limit of Detection The Limit of Detection (LOD) is defined as the lowest SARS-CoV-2 RNA concentration that is successfully detected with a probability of 95% or greater. Sensitivity standards were prepared by serially diluting the SARS-CoV-2 viral RNA transcript containing an 1100 nucleotide region from the N gene in stabilizing buffer RNA Diluent P to the following concentrations: 2,580, 968, 363, 136, and 51 copies/mL.
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Human coronavirus HKU1 Human coronavirus NL63 Adenovirus (e.g. C1 Ad.
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8) Intra-assay Precision Intra-assay precision is defined as the reproducibility of a sample within an assay run and was evaluated by testing 3 replicates of 3 separate precision standards in a single run using the SARS-CoV-2 RNA Qualitative RT-PCR assay.
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in any of the replicates tested. One of the sputum specimens had invalid results for the internal control in all RTPCR reactions (replicates were out of range by about 1 cycle). The invalid sputum sample was noted to be highly mucopurulent in both the raw and pre-processed states, possibly causing the inhibitory result. Excluding the one invalid result, the specificity with presumed negative lower respiratory specimens was 100% (29/29, 95%CI 88.1100%).
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A specimen stability study was conducted to confirm that signal degradation at high temperatures would not occur during shipping. Contrived samples for this study were prepared by spiking a SARS-CoV-2 remnant positive patient sample into pooled remnant SARS-CoV-2 negative patient samples at concentrations targeting 2X LoD and 5-10X LoD. The remnant patient samples used for this study included upper respiratory swabs in two different transport media: VCM and sterile saline (0.9% NaCl).
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Each pool was tested and the percent of negative results for these pools was calculated. Of the 103 4-sample pools in the 1-3% prevalence group, 99.0% (102/103, 95%CI 94.9-99.8%) were negative, and 1/103 was inconclusive. Of the 107 4-sample pools in the 3-6% prevalence group, 99.1% (106/107, 95%CI 94.9-99.8%) were negative, and 1/107 was invalid. Of the 37 4-sample pools in the 6-10% prevalence group, 100% (37/37, 95%CI 90.6-100%) were negative. Overall in the study, 99.2% (245/247, 95% CI 97.1-99.
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In Silico Sensitivity in Population with Positivity Rate 1-10% (n = 3,091) Interval Number of Interval Number of # above Neg Inc Pos % Positive Percent [X*, 40] for samples [X*, 40] for samples both shifted Agreement* N1 with N1 Ct N3 with N3 Ct Thresholds values in values in the interval the interval 1-3% 820 [37.0, 40] 22 [37.45, 40] 0 0 0 22 798 100% (820/820) 95% CI 99.5-100% 3-6% 1,113 [38.3, 40] 7 [38.7, 40] 0 0 0 7 1106 100% (1,113/1,113) 95% CI 99.7-100% 6-10% 1,158 [37.65, 40] 17 [38.
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20) Omega Extraction Validation - Agreement with the MagnaPure Extraction Method (n = 168) Quest selected randomly 168 specimens that had been previously tested for SARS-CoV-2 RNA using the Quest Diagnostics SARS-CoV-2 Assay and the Magna Pure Method. Of the 168 specimens selected 78 were positive previously and 90 were negative previously. Specimens included a range of transport media types (VCM/UTM, UTMRT, PBS, Saline (0.9% NaCl) and Eswabs), and Ct values.
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Attachment 1 – Protocol for Monitoring of Specimen Pooling Testing Strategies Monitoring plan for use of pooling Laboratories should evaluate the appropriateness of the pooling and pool size using the FDA recommended monitoring procedure described below. Laboratories may also consider the sensitivity of pooled testing based on the assay’s Limit of Detection. Ongoing assessment of positivity rate during application of the initial selected n-sample pooling strategy: a.
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Table 1 Efficiency of pooling based on prevalence P, percent of positive subjects in the tested population nmaxefficiency (n corresponding to the maximal efficiency) 1% - 4% 6 5% - 6% 6 7% - 12% 6 13% - 25% 6 1% - 4% 5 5% - 6% 5 7% - 12% 5 13% - 25% 5 1% - 4% 4 5% - 6% 4 7% - 12% 4 13% - 25% 4 1% - 4% 3 5% - 6% 3 7% - 12% 3 13% - 25% 3 1% - 4% 2 5% - 6% 2 7% - 12% 2 13% - 25% 2 Efficiency (F) of n-sample pooling (a maximum increase in the number of tested patients