GMDN TERM

SARS-CoV-2 nucleic acid IVD, kit, nucleic acid technique (NAT)

INTENDED USE

Wollerau Pharma COVID-19 PCR Testing Kit is a fluorescence PCR kit and is intended for the qualitative detection of COVID-19 nucleic acid in human throat swabs, sputum, and bronchoalveolar lavage fluid (BALF) samples. Positive results are indicative of the presence of COVID-19 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out a bacterial infection or co-infection with other viruses. 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.

SUMMARY

Coronavirus (CoV) belongs to the Coronaviridae family and is divided into three types: α, β and γ. Alpha and beta are only pathogenic to mammals and gamma mainly cause bird infections. CoV is mainly transmitted through direct contact with secretions or aerosols and droplets. There is also evidence that it can be transmitted through the fecal-oral route. The COVID-19 novel coronavirus was discovered in 2019 in Wuhan, China with viral pneumonia cases and clinical manifestations were fever, fatigue, cough, and other symptoms. These can rapidly develop into severe pneumonia, respiratory failure, septic shock, multiple organ failure and severe acid-base metabolism disorders, etc.

TEST PRINCIPLE

Wollerau Pharma COVID-19 PCR Testing Kit is an In Vitro diagnostic test based on PCR technology, developed for specific detection of SARS-CoV-2 viral RNA. The probe system is based on the standard TaqMan® hydrolysis probe system Technology. The COVID-19 specific ORF1ab probe and N prob are labelled with the Cy5 and FAM fluorophore respectively. A dU-UNG enzyme system is included in the kit to prevent contamination and false-positive results. A set of internal control primers and probe are included in the design to avoid false-negative results due to sampling failure

STORAGE INSTRUCTIONS

• Store at -20°C with a shelf life of 6 months.
• Repeated freezing and thawing shall not exceed 5 times.
• Refer to packaging label for expiration date.
• Protect fluorogenic primer/probe mix from light.

CONTENTS OF THE KIT

REQUIRED EQUIPMENT AND CONSUMABLES (NOT PROVIDED)

• A PCR instrument and software. The kit is designed to be compatible with all RT-PCR devices including Applied Biosystems® 7500 Real-Time PCR Systemand Roche® LightCycler 480 II.
• PCR hood
• Benchtop microcentrifuge
• Vortex mixer
• Adjustable micropipettes (2 or 10 μl, 200 μl and 1000 μl)
• Adjustable multichannel micropipettes (5-50 μl)
• Racks for 1.5 mL microcentrifuge tubes
• 1.5 mL microcentrifuge tubes (DNase/RNase free)
• Aerosol barrier pipette tips with filters
• Disposable gloves
• 10% bleach (1:10 dilution of commercial 5.25-6.0% hypochlorite bleach)
• DNAZap™ (Ambion, Catalogue no: AM9890), or equivalent • RNAse Away™ (Fisher Scientific, Catalogue no: 11580095) or equivalent
• 0.2 mL PCR reaction plates.

NUCLEIC ACID EXTRACTION

The kit can be used with automatic and manual nucleic acid extraction kits and instruments, supplied by Wollerau Pharma or other manufacturers. We recommend the QIAamp Viral RNA Mini Kit. Please consult the relevant Instruction For Use (IFU) and Materials Safety Data Sheet (MSDS), available from the manufacturer, before using your chosen IVD extraction kit/ system.

SAMPLE REQUIREMENTS

• Follow specimen collection device manufacturer instructions for proper collection methods.
• Specimens for virus detection should reach the laboratory as soon as possible after collection. Correct handling of specimens during transportation is essential. Specimens that can be delivered promptly to the laboratory can be stored and shipped at 2-8°C.
• When there is likely to be a delay in specimens reaching the laboratory, the use of viral transport medium is strongly recommended.
• Specimens may be frozen to - 20°C or ideally -70°C and shipped on dry ice if further delays are expected (see Table 2). It is important to avoid repeated freezingand thawing of specimens.
• Ensure that adequate standard operating procedures (SOPs) are in use and that staff are trained for appropriate specimen collection, storage, packaging, andtransport.
• All specimens collected for laboratory investigations should be regarded as potentially infectious.
• Ensure that health care workers who collect specimens adhere rigorously to infection prevention and control guidelines.
• Specific WHO interim guidance has been published.

TEST PROCEDURE

NOTE: For all procedures involving specimens, buttoned lab coats, gloves, and face protection are required minimum personal protective equipment. Report allaccidents to your supervisor and in accordance with the your institution’s policies and procedures.

1. RNA Extraction from Samples

• Please refer to the instructions of the extraction Kit for the specific operations.
• Samples should be tested as soon as possible to avoid degradation.
• Quality control requirements must be performed in conformance with local, state and federal regulations or accreditation requirements and the user's laboratory'sstandard quality control procedures.

2. PCR setup

• PCR A should be fully dissolved before use. Ensure PCR A and PCR B are concentrated at the bottom of the tubes.
• For each reaction, prepare reagents according to the following table:

• Add 4 μL of extracted nucleic acid (from patient specimens and controls) to the appropriate well with PCR A and PCR B reaction mixtures.. Briefly centrifuge the plate to collect the reactions at the bottom of the wells and to eliminate any air bubbles.Regent Volume / μLPCR A 15PCR B 1Sample(Positive/Negtive Control) 4Total 20

3. PCR Amplification

• Select FAM for N detection, Cy5 for ORF1ab detection, and HEX/VIC for internal control gene detection.
• Place the PCR tube into a fluorescence PCR instrument for amplification.

Applied Biosystems® 7500 Settings:

• Select Quantitation-standard Curve for the experiment type.
• Choose TaqMan®Reagents in the Experiment Properties window.
• In Plate Setup window, define the targets as:

• FAM channel（Reporter：FAM，Quencher：None),
• Cy5 channel（Reporter：Cy5，Quencher：None),
• HEX or VIC channel（Reporter：VIC，Quencher：None).

• For passive reference, select None.
• Negative, positive, and unknown samples should be set as NTC, PTC, and Unknown respectively.
• In the Run Method window, thermal cycles are set according to the table on the right.

LightCycler® 480 Settings:

• After opening the software, select “NewExperiment", click to enter the toolbar.
• Select the appropriate Detection Format, click“Customise"
• Check the desired filter combination; set theprogram according to the parameters in the table onthe right.
• Click "Subset Editor" to proceed with samplearrangement, click "Sample Editor", set the samplename, type. After setting, save the document
• Click “Start Run” to run the program.

QUALITY CONTROL CRITERIA

Before interpreting sample results, it is necessary to verify the success of the run. If the following criteria are not satisfied, then testing needs to be repeated:

• Negative quality control: Ct not detected in FAM and Cy5, or Ct>40
• Positive quality control: FAM, Cy5 show s-shaped curves and Ct≤34
• Unknown samples：FAM, Cy5 Ct≤40

Quality control requirements must be performed in line with applicable regulations.

Always include a Negative Control and a Positive Control in each run.

Always include an Extraction Control in each sample extraction batch and in subsequent runs.

INTERPRETATION

• SARS-CoV-2 Negative: FAM and Cy5 have no Ct value or Ct>40.
• SARS-CoV-2Positive: FAM and Cy5 show S-shaped curves, and Ct ≤40
• Presumptive SARS-CoV-2 Positive: If FAM or Cy5 shows a typical S-shaped curve

For instrument specific guidance on correctly assigning Ct values follow manufacture instructions. Please manually inspect amplification curves for all samplesassigned a Ct value to verify the positive amplification.

LIMITATIONS

• For use only by personnel trained in the techniques of rRT-PCR and in vitro diagnostic procedures. A thorough understanding of the instructions for use is necessary for successful use of the product. Reliable results will only be obtained by using precise laboratory techniques and accurately following these instructions for use and instructions from manufacturers of any kits, consumables and instruments.
• Samples must be collected, transported, and stored using appropriate procedures and conditions. Improper collection, transport, or storage of specimens may hinder the ability of the assay to detect the target sequences.
• Extraction and amplification of nucleic acid from clinical samples must be performed according to the specified methods listed in this procedure. Other extraction approaches and processing systems have not been evaluated.
• A false-negative result may occur if a specimen is improperly collected, transported or handled. False-negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms are present in the specimen.
• A false-positive result may arise from cross-contamination during specimen handling or preparation, or between patient samples.
• The impacts of vaccines, antiviral therapeutics, antibiotics, chemotherapeutic or immunosuppressant drugs have not been evaluated. The kit cannot rule outdiseases caused by other bacterial or viral pathogens.
• Negative results do not preclude infection, and should not be the sole basis of a patient management decision.
• Results from the kit should be used as an adjunct to clinical observations and other information available to the physician.

PERFORMANCE

Evaluation was conducted with contrived samples, including 5 positive contrived samples and 12 negative references. The resulted coincidence rate was 100%. The references were tested 10 times repeatedly. The Ct value variation coefficient (CV) was ≤5%.

Limit of detection: The Limit of detection (LoD) was defined as the lowest concentration of analyte that could be reliably detected at least 95% of the time. First a tentative LoD was detected by testing five limiting dilutions, prepared by spiking quantified SARS-CoV-2 whole viral RNA into oropharyngeal matrix negative for SARSCoV-2. The LoD was confirmed by testing single replicates of the 20 extraction eluates as 1.0×103 copies/mL.

Cross-reactivity: Cross-reactivity was evaluated by in silico analysis and by wet testing whole organisms and purified nucleic acids from pathogens potentially found in upper respiratory specimens. The kit did not cross-react with common respiratory pathogens and other pathogens with the same or similar infection symptoms as the site of infection, including NL63, HKU1, 229E, OC43, Influenza A, Influenza B, Respiratory Syncytial, Adenovirus, Mycoplasma Pneumonia, and Bacillus Pertussis.

Interference: The following interferon reagents did not have significant effect on the test results: Interferon (α-2b≤ 180 μg/mL), Hydroxymezoline hydrochloridespray(≤150 μg /mL), Budesonide nasal spray(≤256 g /mL), Erythromycin(≤50 mg/mL), Roxithromycin (≤7.5mg/mL), Azithromycin (≤100mg/mL).

DISPOSAL

Dispose of hazardous or biologically contaminated materials according to the practices of your institution

SAFETY PRECAUTIONS

• Handle all specimens as if infectious using safe laboratory procedures.
• Site- and activity-specific risk assessments should be performed to determine if enhanced biosafety precautions are warranted based onsituational needs.
• Refer to the following CDC guidelines: Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons UnderInvestigation for Coronavirus Disease 2019 (https://www.cdc.gov/coronavirus/2019- nCoV/lab/guidelines-clinical-specimens.html)
• Do not eat, drink, smoke, apply cosmetics, or handle contact lenses where reagents or human specimens are being handled.
• Do not pipette by mouth.
• Safety Data Sheets are available upon request.
• Manipulation of potentially infected specimens should be performed in a certified Class II BSC in a BSL-2 facility or higher. This includes aliquotingand/or diluting specimens and nucleic acid extraction procedures involving potentially infected specimens.
• Use appropriate personal protective equipment including but not limited to disposable gloves, laboratory coat/gown, and eye protection whenhandling specimens, reagents, pipettes, and other equipment.

TECHNICAL PRECAUTIONS

• Do not use expired reagents.
• Avoid contamination of clinical specimens or reagents with previously amplified PCR products. Assay workflow should entail separate workingareas for specimen preparation, reaction set-up, and amplification/detection. Wear disposable gloves and change them before entering aseparate working area.
• Maintain dedicated supplies and equipment for each of the separate working areas.
• Use a clean lab coat and clean disposable gloves for reaction set-up.
• Change gloves between samples and whenever contamination is suspected
• Always use DNase/RNase-free aerosol barrier pipette tips. Use a fresh DNase/RNase-free aerosol barrier pipette tip for each reagent orspecimen transfer.
• Avoid DNase/RNase contamination of the specimens. Decontaminate work surfaces, pipettes, and centrifuges with 10% bleach, DNAZapTMPCR DNA Degradation Solutions, and/or RNase AWAYTM Decontamination Reagent. Remove residual bleach with 70% ethanol.
• Avoid contamination by keeping reagent and reaction tubes capped or covered as much as possible.
• Do not open sealed PCR plates or PCR tubes after amplification to avoid contamination with amplified PCR products.
• Keep extracted RNA on cold block or on ice during reaction set-up.
• Keep rRT-PCR master mix on cold block or on ice during reaction set-up.