Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit

Transcription

1 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE Diagenode DBDR-10-0-L096 Version 1 BD Date of issue: Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit Instructions for use Distributed by FOR USE WITH THE BD MAX TM SYSTEM Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

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3 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 1. INTRODUCTION GENERAL INFORMATION INTENDED USE PATHOGEN INFORMATION CONTENTS OF KIT MATERIAL MATERIAL REQUIRED (NOT PROVIDED) EQUIPMENT AND MATERIAL IN COMBINATION WITH DIAGENODE BORDETELLA PERTUSSIS AND PARAPERTUSSIS REAL-TIME PCR KIT (NOT PROVIDED) VALIDATION QUALITY CONTROL REAGENT STORAGE, HANDLING AND STABILITY SAMPLES COLLECTION, STORAGE AND TRANSPORT SAMPLE COLLECTION TYPE SAMPLE STORAGE SAMPLE TRANSPORT WARNINGS AND PRECAUTIONS PROTOCOL SAMPLE AND DIAGENODE CONTROL PREPARATION PROBE AND PRIMERS PREPARATION BD MAX TM SYSTEM AMPLIFICATION RESULTS TROUBLESHOOTING GUIDE PERFORMANCE EVALUATION ANALYTICAL SENSITIVITY PRECISION ANALYTICAL SPECIFICITY CLINICAL INVESTIGATION TEST LIMITATIONS QUALITY CONTROL REFERENCES EXPLANATION OF SYMBOLS NOTICE TO PURCHASER Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

4 Page 3 1. Introduction Real-time polymerase chain reaction, also called quantitative real-time polymerase chain reaction (qpcr), is a nucleic acid technology used to detect and quantify specific DNA sequences. This quantification can be measured in absolute or relative number of copies. Real-time PCR technology allows rapid, specific, and quantitative measurements of the presence of genes involved in infectious diseases, cancer, and genetic abnormalities. This powerful technology is used in Diagenode's kits for accurate pathogen detection for a number of diseases. One of the prominent attributes of qpcr technology is that amplified DNA is quantified as it accumulates in the reaction in "real-time" for each PCR amplification cycle, allowing for far more accurate measurements than with traditional end-point PCR. These real-time measurements are made possible by quantifying signals from fluorescent dyes that intercalate with the double-stranded DNA produced in each amplification cycle. Alternatively, one can use labeled DNA oligonucleotide probes with a dye (e.g. a TaqMan probe) that subsequently hybridize with the complementary DNA to allow real-time measurements during qpcr. The resulting fluorescent signals can be closely monitored and quantified using an instrument such as a thermal cycler adapted for qpcr. Figure 1: Technology using dual-labeled hydrolysis probes Figure 2: Specific hybridization of primers and probes to the target gene. Figure 3: Elongation and release of reporter dye by Taq polymerase.

5 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 2. General Information 2.1 Intended use The Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit as implemented on the BD MAX System is an automated in vitro diagnostic test for the direct qualitative detection of Bordetella pertussis in nasopharyngeal aspirates and throat swabs from patients with signs and symptoms of bacterial respiratory tract infection. The test utilizes real-time polymerase chain reaction (PCR) for the amplification of B. pertussis and B. parapertussis genes in the IS481 and IS1001 sequences respectively. The test utilizes the fluorogenic target-specific hybridization probes for the detection of the amplified DNA. The Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit is intended as an aid in the diagnosis of pertussis. This test allows also detecting Bordetella parapertussis in nasopharyngeal aspirates but is not to be used for diagnostic purposes. 2.2 Pathogen information Bordetella pertussis and parapertussis causes the disease commonly referred to as pertussis or whooping cough. Pertussis is a highly contagious respiratory disease resulting in million cases of disease each year. Thus, timely detection through PCR is essential in order to prevent the spread of disease and to reduce the risk of widespread outbreaks. PCR testing is ideal for prompt differential diagnosis of pertussis from other respiratory diseases such as atypical pneumonia, bronchitis, asthma, respiratory viruses or infections from mycobacterium. The disease most commonly affects infants and young children and can be fatal especially in infants less than 1 year of age: adults may also be affected. Older children and adults with waning natural or vaccine-induced immunity, in contrast present with a more diverse spectrum of symptoms that necessitate testing to establish a definitive diagnosis. 2.3 Contents of kit This kit is used with the BD MAX TM System. Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit: 96 PCR reactions (12.5 µl PCR volume), one box: I. Bordetella pertussis/parapertussis double-dye probe & primers (4 tubes) : Bordetella pertussis double-dye probe & primers (FAM, 520 nm) and Bordetella parapertussis double-dye probe & primers (Yellow dye, 553 nm): 65 µl each, red tube. II. Bordetella pertussis/parapertussis positive control: 1000 µl, green tube. III. Bordetella pertussis/parapertussis negative control (H2O (PCR grade)): 1000 µl, sky blue tube. IV. H2O (PCR grade): 1000 µl, sky blue tube. Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

6 Page 5 3. Material 3.1 Material required (Not provided) Pipettes (accurate between µl) Sterile pipette tips with filters Powder-free gloves Vortex mixer Lab coat RNase/DNase-free microcentrifuge tubes (1.5 and/or 2 ml) RNase/DNase free water Proteinase k solution 1 mg/ml (recommended buffer 20 mm Tris ph 8.3, 0.5% SDS) Heat block or water bath (55 C) Laminar flow hood Specimen container (nasopharyngeal aspirates) Vial of Liquid Amies medium with swab (ESwab*) *ESwab buffer : 3 g NaCl; 0.20 g KCl; 0.10 g CaCl2; 0.10 g MgCl2; 0.20 g KH2PO4; 1.15 g Na2HPO4; 1 g C2H3NaO2S in 1 l distilled water 3.2 Equipment and material in combination with Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit (Not provided) Real-time PCR instrument: BD MAX System BD MAX ExK TM -DNA-1 kit (Ref: ): - DNA Extraction tube - DNA Sample Buffer tube (SB1) - DNA Unitized Reagent Strip - Septum - Conical tube BD MAX DNA MMK (SPC) kit (Ref: ): - Master Mix (with Sample Process Control) tube - Primer and Probe Diluent tube BD MAX Microfluidic Cartridges (Ref : ) 4. Validation Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit has been validated on the parameters as described below: Collected samples PCR machine Master Mix Throat swabs and nasopharyngeal aspirates BD MAX TM System BD MAX TM DNA MMK (SPC) with DNA extraction/inhibition control

7 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 5. Quality Control Quality Control requirements must be performed in conformance with local, state and/or federal regulations or accreditation requirements and your laboratory s standard quality control procedures. Quality control procedures are intended to monitor reagent and assay performance. Control type Positive Negative Internal (SPC) For monitoring the following reagents and assay performance Substantial reagent failure including primer and probe integrity Reagent and/or environmental contamination Failure in extraction procedure and/or PCR procedure PCR inhibition in individual samples and Reagent failure or process error Test all assay controls and the internal control prior to running samples with each new kit lot to ensure all reagents and kit components are working properly. Good laboratory practice recommends including an internal control in each run. The internal control should be treated as a sample. Inclusion of one negative control and at least one positive control is recommended in each run performed. Failure of controls invalidates the run and results should not be reported. - If the positive control is not positive within the specified Ct range but the negative control is valid, repeat testing should be done starting from the original sample and using new aliquot of the positive control. If repeat results are still invalid, results should not be reported and testing should be repeated from a sample newly collected. - If the internal control is not positive within the specified Ct range or the negative control is invalid, repeat testing should be done starting from the original sample using a new internal control and a new negative control. If repeat results are still invalid, results should not be reported and a new sample should be collected and tested (see point 11. Troubleshooting Guide). 6. Reagent Storage, Handling and Stability Store all reagents (opened and unopened) at -20 C until the expiry date as defined below: Condition Storage temperature Expiry date Unopened reagent -20 C Expiry date stated on the label Always check the expiry date on the reagent labels. We recommend not exposing to more than 6 freeze-thaw cycles to avoid the degradation of compounds. Diagenode Bordetella pertussis and parapertussis Real-Time PCR kit is shipped frozen, should arrive frozen and should be stored frozen at -20 C after receipt. If the kit contents are not frozen, contact your local BD representative. Protect reagents from light. Caution Storage of the kit at room temperature can lead to the degradation of the primers/probes and positive control. This will lead to a decreased sensitivity and may result in a false negative outcome. Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

8 Page 7 7. Samples Collection, Storage and Transport 7.1 Sample Collection Type The PCR test is performed using throat swabs collected with ESwab or nasopharyngeal aspirates. 7.2 Sample Storage Respiratory samples must be stored at 2 to 8 C for up to 24 hours or frozen at -20 C or -70 C for storage longer than 24 hours. Throat swabs should be stored in Eswab Liquid Amies transport medium and aspirates in sterile nonbacteriostatic saline. The sensitivity of the PCR could be reduced through repeated freezing and thawing of respiratory samples; therefore, avoid freeze/thaw cycles. 7.3 Sample Transport Throat swabs must be transported in Eswab Liquid Amies transport medium and aspirates in sterile non-bacteriostatic saline. The samples must be transported following the local and national regulations for the transport of pathogen material. In order to avoid degradation of nucleic acid, we advise shipping at -20 C if transport time is more than 24 hours. In the laboratory, samples may be transported at room temperature. 8. Warnings and Precautions - If you receive a damaged parcel or thawed kits, please contact your local BD representative. Do not use the kit. General information - Specific lots of ExK TM DNA-1 kits produced (lot n and higher) have been tested using the Bordetella QC method, and have conformed to acceptance criteria. ExK TM DNA-1 lots to be distributed with the Diagenode Bordetella Assay kit will be tested with the Bordetella QC method by BD and released for this assay upon conformity. Therefore, it is a requirement to use ExK TM DNA-1 kits with lot numbers of or higher when testing with the Diagenode Bordetella Assay. Read all instruction before performing the experiment. Use of this product should be limited to personnel who have been trained in the techniques of realtime PCR. For in vitro diagnostic use.

9 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE Precautions Do not use the kit if the label that seals the outer box is broken. Do not use reagents if the protective box is open or torn upon arrival. Do not use reagents if the tube has been opened or damaged. Do not mix reagents from different kits and/or lots. Do not use expired reagents and/or materials. Do not mix caps between tubes or re-use caps as contamination may occur and compromise test results. Aerosol resistant tips must be used for all PCR mixtures. The PCR laboratory (safety cabinet, DNA/ RNA extraction platform, bench coat, etc.) must be cleaned after each PCR experiment with appropriate solutions. Additional controls may be tested according to guidelines or requirements of local, state, provincial and/or federal regulations or accrediting organizations. In cases where other PCR tests are conducted in the same general area of the laboratory, care must be taken to ensure that the Diagenode Respiratory Bordetella pertussis and parapertussis Real-Time PCR kit, BD MAX ExK TM -DNA-1 kit, BD MAX MMK (SPC) any additional reagents required for testing, and the BD MAX System are not contaminated. Gloves must be changed before manipulating reagents and cartridges. Discard the kit with a suspected contamination. Always handle specimens as if they are infectious and in accordance with safe laboratory procedures such as those described in local legislation documentation. Wear protective clothing and disposable gloves while handling kit reagents. Wash and disinfect hands thoroughly after performing the test. The kit should not be used by anyone displaying symptoms of the disease being detected by the kit. Do not pipette by mouth. Do not ingest any components from the kit. Do not smoke, drink, or eat in areas where specimens or kit reagents are being handled. The experiments should not be carried out during pregnancy due to risks to the unborn baby. Dispose of unused reagents and waste in accordance with country, federal, provincial, state and local regulations. Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

10 Page 9 9. Protocol Collect specimen and label appropriately (see point 7.1 Samples Collection Type) 9.1 Sample and Diagenode control preparation Sample pretreatment For swabs: Pipette 750 µl of specimen into BD Sample Buffer Tube (SB1). Close each tube with septum cap and vortex. For aspirates: Pipette 375 μl of Proteinase K solution and 375 μl of specimen into a sterile microcentrifuge tube. (If specimen volume is less than 375 μl, use PBS to adjust the volume). Vortex 10 seconds. Incubate 1h at 55 C Pipette 750 µl of the specimen with Proteinase K mixture into BD Sample Buffer Tube (SB1). Close each tube with septum cap and vortex Diagenode negative and positive control preparation (optional) To validate the run and for result interpretation, perform a positive and negative control: For the positive control: Pipette 10 µl of Diagenode positive control and 740 µl of RNase/DNase free water into BD Sample Buffer Tube (SB1). Close each tube with septum cap and vortex, and un-cap the tube prior to use. For the negative control: Pipette 10 µl of Diagenode negative control and 740 µl of RNase/DNase free water into BD Sample Buffer Tube (SB1). Close each tube with septum cap and vortex and un-cap the tube prior to use. 9.2 Probe and primers preparation PCR Mix (per strip/reaction): 1. Count the total number of samples and/or controls to be tested in one run. 2. Shortly before running the assay, prepare the following complete primers/probes mix by multiplying each component volume by the total number of samples, plus 20% overage accounting for pipetting errors (see table below). Diagenode Bordetella pertussis/parapertussis primers and probes (red tube) 2.5 µl BD Primers and Probe Diluent (included in BD MAX TM MMK (SPC) ) 2 µl H2O (PCR grade) (Sky blue tube) 8 µl Final volume for one strip: 12.5µL

11 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE Example of calculations for several strips/ reactions: Number of percentage error Primers/Probe Primers/Probe H 2 O (PCR grade) Total samples of pipetting (µl) diluent (µl) (µl) (µl) 3 20% 9 7,2 28, % 12 9,6 38, % 15 12,0 48, % 18 14,4 57, % 21 16,8 67, % 24 19,2 76, % 27 21,6 86, % 30 24,0 96, % 33 26,4 105, % 36 28,8 115, % 39 31,2 124, % 42 33,6 134, % 45 36,0 144, % 48 38,4 153, % 51 40,8 163, % 54 43,2 172, % 57 45,6 182, % 60 48,0 192, % 63 50,4 201, % 66 52,8 211, % 69 55,2 220, % 72 57,6 230, BD MAX TM System amplification Note: Refer to the BD MAX System User s Manual for detailed instructions Creating test Diagenode Bordetella Note: If you already have created the Diagenode Bordetella test, you can skip step and go directly to On the «Run» screen of the BD MAX TM System, select the «Test Editor» tab. 2. Click the «Create Test» button. 3. In the «Test Name» window, name your test: Diagenode Bordetella. 4. In the «Extraction Type» drop down menu, choose ExK TM DNA-1 5. In the «Master Mix Format» drop down menu, choose DNA MMK 6. In «Channel Settings», set «Gains» and «Threshold» as follow : Channel 475/ / / / /715 Gain Threshold Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

12 Page In «GuardRail», select «Default» 8. In «Test Details»,enter the PCR profile*: New Step New Step Cycle 1 Cycle 45 Type Profile type Time (s) Hold Temp ( C) 98.0 Detect Type Profile type Time (s) Temperature Temp ( C) Detect *The validity indicator provides a real-time indication that the step being created passes the system s validity checking. A green dot and the words VALID STEP indicate the parameters for the step are valid. A red dot and the words INVALID STEP indicate that some aspect of the step is not valid. A prompt indicates what aspect of the step is failing the validity check, and what must be done to correct the step. 9. Click the «Save Test» button BD MAX TM Rack set up Please refer to BD MAX User s Manual for additional information. 1. Load the BD MAX racks with the appropriate number of DNA Unitized Reagent Strips (URS). Gently tap each URS onto a hard surface to ensure that the liquids are at the bottom. 2. Snap the DNA Extraction tube(s) and DNA MMK (SPC) Tube(s) into their corresponding positions in the URS (see figure 1). 3. Snap the empty conical tube in position Pipette 12.5 μl of complete primers/probes Mix (prepared in section 9.2) into the conical tube in position 3 (Ready DNA tube) of the DNA URS. Make sure there are no bubbles and the liquid is at the bottom of the tube.

13 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE Instrument set up 1. Select the <Work List> tab on the <Run> screen of the BD MAX software. 2. In the Assay drop down menu, select Diagenode Bordetella (if not already created see Section 9.3.1). 3. Enter the Sample Buffer Tube barcode corresponding to each specimen/patient or control using the barcode scanner or manual entry. Start with Position 1 of Rack A. 4. Place the septum-capped Sample Buffer tube into its corresponding position in the BD MAX TM Rack(s). 5. Enter the specimen/patient identification information into the work list, using either the barcode scanner or manual entry. Continue until all Sample Buffer tubes are entered. Ensure that the specimen/patient ID and the Sample Buffer tubes are accurately matched. 6. Place the rack into the BD MAX TM System (Rack A is positioned on the left side of the BD MAX System and Rack B on the right side). 7. Load the BD MAX TM PCR cartridge(s). 8. Close the BD MAX System door. 9. Click Start Run 10. Results 1. In main menu, click the Results button. 2. Double click on your run in the list and click the new tab Run.. 3. In the print tab, select: Result Protocol Details PCR 4. Click on create report and then click the Print Report button. Results interpretation with BD MAX TM software 2.74 or 2.70: 1. For a run to be valid a. No BD MAX TM System failures. b. (optional) Negative control has Ct value of -1 for all channels except 680/715. c. (optional) Positive control has Ct value of 30.0 ± 3.3 for channels 475/520, and 530/ If valid, interpret the specimen results following the chart below: a. A Ct value of 0 indicates that there was no Ct value calculated. That sample s curve must be reviewed manually with threshold line adjustment to generate a recalculated Ct value. b. A Ct value of -1 indicates that no amplification occurred. c. Any other Ct value must be interpreted in correlation with PCR curves ( PCR Analysis tab) and according to the following table: Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

14 Ct 475/520 B. p Ct 530/565 B.pp Ct 680/715 SPC Results Interpretation Page Bordetella pertussis/parapertussis* not detected. ANY -1 ANY Bordetella pertussis detected. -1 ANY ANY Bordetella parapertussis detected.* ANY ANY ANY Both Bordetella pertussis/parapertussis* detected > 36 Or -1 Inhibitory Specimens** No diagnosis can be established. * The Bordetella parapertussis s detection has not been validated. Do not use this result for diagnostic purposes. **For Inhibitory specimens: Repeat test with the original specimen stored at 2 to 8 C by preparing a new Sample Preparation Reagent tube from specimen. Alternatively, test a newly collected specimen. 11. Troubleshooting Guide Refer to the troubleshooting section of the BD MAX TM System User s Manual for additional information. Description Possible Causes Corrective Actions PCR inhibited by exogenous or endogenous Repeat sample testing. substances Target and internal control do not amplify. Target amplifies, but the internal control does not amplify. Spike in all amplification curves at the same point. Very low fluorescence across all samples, including internal control Liquid handling problem Target out competed internal control for resources during reaction Power surge Primer or probe degradation Instrument issue Check URS and Microfluidic Cartridge to determine where liquid handling problem occurred and re-run sample. If problem persists, contact your local BD representative Verify that the sample has early amplification. Verify that the system is connected to the UPS (Uninterruptible Power Supply). Check kit expiration date and verify kit was stored correctly. Refer to BD MAX User s Manual. For other trouble shootings/questions, please contact your local BD representative or the Diagenode customer support.

15 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 12. Performance Evaluation 12.1 Analytical sensitivity The analytical detection limit in consideration of the purification (LOD) was assessed for Bordetella pertussis in Eswab and nasopharyngeal aspiration (NPA) matrixes and for Bordetella parapertussis in Eswab. To determine the analytical sensitivity in consideration of the purification of the Diagenode B. pertussis and parapertussis Real-Time PCR Kit, dilution series of quantified inactivated B. pertussis and parapertussis pathogens have been set up in Eswab medium and in a pool of negative nasopharyngeal aspirations. Samples were analyzed on the BD MAX TM Instrument in combination with the Diagenode B. pertussis and parapertussis Real-Time PCR Kit. Testing was carried out on different days on 24 replicates. The results were determined by a probit analysis. Analytical sensitivity (LOD) as defined as the lowest concentration at which 95% of replicates are tested positive are presented in the table below. Matrix LOD 95% (copies/ml) Eswab 184 B. pertussis NPA 504 B. parapertussis Eswab Precision The precision data of the Diagenode Bordetella pertussis and parapertussis Real-Time PCR Kit have been collected by means of the BD MAX TM System and allow the determination of the intra-run variability (variability of multiple results of samples of the same concentration within one experiment) and the inter-run variability (variability of multiple results of the assay generated by four different runs carried out on three different days) of the Diagenode Bordetella pertussis and parapertussis Real-Time PCR Kit on the BD MAX TM System. The data obtained were used to determine the standard deviation and the coefficient of variation for the specific pathogen. Precision data of the Diagenode Bordetella pertussis and parapertussis Real-Time PCR Kit run on the BD MAX TM System have been collected using ESwab and nasopharyngeal aspiration (NPA) matrix spiked with B. pertussis (Bp) and B. parapertussis (Bpp) inactivated pathogens. Testing was performed with 24 replicates. The precision data were calculated on the basis of Ct values of the amplification curves as following: intra-assay variation = mean CV of each individual run; inter-assay variation = CV of the mean Ct value of each individual run. Results are presented in the table below. Test Matrix Pathogen concentration (c/ml) Mean Ct Standard deviation Coefficient variation (%) Intra-assay Bp Eswab Eswab Inter-assay Bp Eswab Eswab Intra-assay Bpp Eswab Inter-assay Bpp Eswab Intra-assay Bp NPA NPA Inter-assay Bp NPA NPA Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com of

16 Page Analytical specificity The specificity of the Diagenode Bordetella pertussis and parapertussis Real-Time PCR Kit is first and foremost ensured by the selection of the primers and probes, as well as the selection of stringent reaction conditions. The primers and probes were checked for possible homologies to all in gene banks published sequences by sequence comparison analysis. This analysis shows the well-known crossreaction between B. pertussis and B.holmesii (100% homology) and B. pertussis and B. bronchiseptica (73% homology). To determine the experimental specificity of the Diagenode Bordetella pertussis and parapertussis Real- Time PCR Kit, the pathogens listed in the following table have been tested for cross-reactivity. The specificity study confirms the cross-reaction between B. pertussis and B. holmesii. However, in the specificity study, B. pertussis doesn t recognise B. bronchiseptica. This may be due to ATCC strains that were used in this study. Indeed, the B. bronchiseptica strain does not contain the IS481 insertion that is targeted by Diagenode B. pertussis probe and primers and is therefore not recognised, while the ATCC strain for B. holmesii contains the insertion and is detected. For all the other strains, the level of crossreaction was < %. Pathogens Bordetella bronchiseptica Bordetella holmesii Bordetella hinzii Bordetella avium Bordetella trematum Legionella pneumophila Mycoplasma pneumoniae Chlamydophila pneumoniae Streptococcus pneumoniae Klebsiella pneumoniae Streptococcus pyogenes Enterococcus faecalis Staphylococcus aureus Neisseria meningitidis Pseudomonas aeruginosa Enterobacter aerogenes Heamophilus influenzae Escherichia coli

17 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 12.4 Clinical investigation Performance characteristics of the Bordetella pertussis and parapertussis Real-Time PCR kit were established during a retrospective study at one reference laboratory in comparison to a validated homebrew PCR method. A total of 307 samples (199 negative samples and 108 Bordetella pertussis positive samples) were retrospectively tested with the Diagenode Bordetella pertussis and parapertussis Real Time PCR kit on the BD Max TM. Samples consisted in throat swabs and in nasopharyngeal aspirations with a solution of proteinase K added. Samples were initially extracted on the easymag and analyzed with the reference home-brew real-time PCR method in combination with the icycler IQ Real Time Detection system (BioRad) and the ABI 2x mastermix. 750 µl of sample was added on the BD Max TM Sample Buffer Tube (SB1). Diagnostic sensitivity, diagnostic specificity and accuracy were calculated for swabs and nasopharyngeal aspirations (NPA). Results are summarized in the following tables: NPA Bordetella pertussis and parapertussis Real-Time PCR kit on BD Max TM System Swab Bordetella pertussis and parapertussis Real-Time PCR kit on BD Max TM System Positive Negative Positive Negative Reference method Positive Negative Reference method Positive Negative Diagnostic sensitivity (lower Bound 95%CI): 97% (87%) 92% (83%) Diagnostic specificity (lower Bound 95%CI): 100% (96%) 98% (94%) Accuracy: Κ = 0.98 (almost perfect agreement) Κ = 0.91 (almost perfect agreement) 11 samples (7 positive Bordetella pertussis and 4 negative Bordetella pertussis) were detected positive for Bordetella parapertussis with the Bordetella pertussis and parapertussis Real-Time PCR kit. The infectious status of these samples for Bordetella parapertussis was unknown. Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

18 Page Test Limitations All reagents must exclusively be used for in vitro diagnostics. A strict compliance with the user manual is required for optimal results. This test has been validated for the qualitative detection of Bordetella pertussis in nasopharyngeal aspirates and throat swabs collected using ESwab. This test has been validated for use on the BD MAX TM System using software version 2.70 and This test shows a cross-reaction between B. pertussis and B.holmesii and between B. pertussis and B. bronchiseptica. Negative results do not preclude infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. The detection of viral nucleic acid is dependent upon proper specimen collection, handling, transportation, storage and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false negative values resulting from improperly collected, transported or handled samples. There is a risk of false negative values due to the presence of sequence variants in the viral targets of the assay, procedural errors, amplification inhibitors in samples, or inadequate number of organisms for amplification. False negative results may occur due to loss of nucleic acid. The inhibition control has been added to the test to aid in the identification of samples that contain inhibitors to PCR amplification. The control does not indicate whether or not nucleic acid has been lost due to inadequate collection, transport or storage of samples. There is a risk of false positive values resulting from cross-contamination by target organism, their nucleic acids or amplified product, or from non-specific signal in the assay. 14. Quality Control Diagenode has obtained ISO 9001 and ISO certifications for design, manufacture and sale of IVD devices using Nucleic Acid technology for infectious diseases. Diagenode Bordetella pertussis and parapertussis Real-Time PCR is tested against predetermined specifications to ensure the product quality. 15. References Kate E. Templeton, Sitha A. Scheltinga, Anneke van der Zee, Bram M. W. Diederen, Anna M. Kruijssen, Herman Goossens, Ed Kuijper, and Eric C. J. Claas (2003) Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii for Clinical Diagnosis. Journal of clinical microbiology, p

19 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE 16. Explanation of Symbols Catalogue number Batch code Contains sufficient for <n> tests Upper limit of temperature Use by yyyy-mm In vitro diagnostic medical device Consult instructions for use Control Negative control Positive control Keep away from sunlight Manufacturer Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

20 Page 19 This product is optimized for use in the polymerase chain reaction (PCR) covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffmann-La Roche ltd. (Roche). No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers, when used in conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Center Drive, Foster City, California or at Roche Molecular Systems, Inc, 1145 Atlantic Avenue, Alameda, California BD MAX TM System is a trademark of Becton, Dickinson and Company.

21 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

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23 DIAGENODE Bordetella pertussis and parapertussis INSTRUCTIONS FOR USE MA-Bor-BD-v1_28_05_13 Diagenode s.a. Avenue de l hôpital,1 Tour GIGA, 3rd Floor 4000 Liège Belgium Tel: Fax: info@diagenodediagnostics.com Europe Diagenode sa / CHU - Tour GIGA - B34 3 rd Floor // Avenue de l Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) // Mail: info@diagenode.com

24 Page 23 Diagenode sa CHU, Tour GIGA, 3rd floor Avenue de l hôpital, Liège - BELGIUM Tel info@diagenodediagnostics.com