Manual FTD BCE. Quantitative assay for in vitro diagnostics

Transcription

1 Manual 32 reactions (catalog no. FTD ) 64 reactions (catalog no. FTD ) Quantitative assay for in vitro diagnostics For use with the ABI 7500, ABI 7500 Fast, ViiA 7, Bio-Rad CFX96, LightCycler 480, Rotor-Gene 3000/6000/Q and SmartCycler FTD , FTD Fast Track Diagnostics Luxembourg S.à.r.l.; 29, rue Henri Koch; L-4354 Esch-sur-Alzette; Luxembourg FTD _64 MANUAL v2 2017_02 EN

2 Table of Contents 1. IDENTIFICATION OF THE MANUFACTURER IDENTIFICATION OF THE PRODUCT INTENDED USE PATHOGEN INFORMATION CONTENTS PRECAUTIONS AND WARNINGS SAFETY INFORMATION HANDLING REQUIREMENTS SAFE WASTE DISPOSAL STORAGE AND STABILITY CONDITIONS PRINCIPLE OF THE METHOD ADDITIONALLY REQUIRED EQUIPMENT SAMPLES PROCEDURE PRELIMINARY EXTRACTION PROCEDURE USING THE EASYMAG MAIN PCR SETUP PROCEDURE PROGRAMMING OF THE THERMOCYCLER ASSAY VALIDATION SETUP ON THE ABI QUANTIFICATION INTERPRETATION OF RESULTS TROUBLESHOOTING VALIDATION LEGEND OF SYMBOLS FTD _64 MANUAL v2 2017_02 EN - 2 -

3 1. Identification of the manufacturer Fast Track Diagnostics Luxembourg S.à.r.l. 29, rue Henri Koch L-4354 Esch-sur-Alzette Tel.: Fax: Identification of the product Category: Multiplex Real-time PCR for detection and quantification of human polyomavirus 1, human cytomegalovirus and Epstein-Barr virus including internal control. Reference: FTD Test for 32 reactions. FTD Test for 64 reactions. Reagents in the kits are sufficient for 32 or 64 reactions. These kit sizes allow maximal flexibility from 1 to 27 patients in FTD and 1 to 59 patients in FTD According PCR run amounts are shown in Table 1. Table 1: Minimum and maximum patient amounts and according run amounts possible for FTD and FTD FTD minimum maximum amount patients 1 27 amount runs 5 1 FTD minimum maximum amount patients 1 59 amount runs 10 1 Indication: For in vitro diagnostics. 3. Intended use is an in vitro test for the quantitative detection of viral nucleic acid as an aid to the evaluation of infections with human polyomavirus 1, human cytomegalovirus and Epstein-Barr virus. FTD _64 MANUAL v2 2017_02 EN - 3 -

4 4. Pathogen information Human polyomavirus 1 (BKV) consists of dsdna and is a member of the polyomavirus family. The BK virus rarely causes disease since many people who are infected with this virus are asymptomatic. If symptoms do appear, they tend to be mild: respiratory infection or fever. These are known as primary BK infections. The virus then disseminates to the kidneys and urinary tract where it persists for the life of the individual. It is thought that up to 80% of the population contains a latent form of this virus, which remains latent until the body undergoes some form of immunosuppression. Typically, this is in the setting of kidney transplantation or multi-organ transplantation. Presentation in these immunocompromised individuals is much more severe. Clinical manifestations include renal dysfunction (seen by a progressive rise in serum creatinine), and an abnormal urinalysis revealing renal tubular cells and inflammatory cells. Human cytomegalovirus (HCMV) is also known as human herpesvirus 5 (HHV-5). The virus carries its genetic information on dsdna. Transmission may occur in utero, perinatal or postnatal. Congenital cytomegalovirus infection is the leading infectious cause of mental retardation, cerebral palsy and hearing loss. HCMV infection is typically unnoticed in healthy people, but can be lifethreatening for the immunocompromised, such as HIV-infected persons, organ transplant recipients, or newborns. Common complications include pneumonitis, retinitis and colitis. The virus is generally passed from infected people to others through direct contact with body fluids, such as urine, saliva, breast milk or sexual contact. It can also be spread through transplanted organs and blood transfusions. Epstein-Barr virus (EBV), also called human herpesvirus 4, is a member of the herpesvirus family. The virus is composed of a double stranded, linear DNA genome enclosed by a protein capsid. It is best known as the cause of infectious mononucleosis (glandular fever), an acute but self-limiting disease affecting children and young adults. It occurs worldwide, and most people become infected with EBV at some point in their life. Symptoms of infectious mononucleosis are fever, sore throat, and swollen lymph glands. Sometimes, a swollen spleen or liver involvement may develop. There is no specific treatment, other than treatment of the symptoms. No antiviral drugs or vaccines are available. It is also associated with particular forms of cancer, such as Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and central nervous system lymphomas associated with HIV. FTD _64 MANUAL v2 2017_02 EN - 4 -

5 5. Contents Table 2: Table of contents: PP= primer and probe, IC= internal control, QS= quantification standard, NC= negative control, IU= international units, BKV= human polyomavirus 1, HCMV= human cytomegalovirus, EBV= Epstein-Barr virus, MCMV= murine cytomegalovirus Contents FTD FTD BCE PP Primer/Probe mix BKV, HCMV, EBV & MCMV (IC) 1 x 48 µl 2 x 48 µl BCE QS1 BCE QS2 BCE QS3 BCE QS4 Quantification standard including (plasmid): BKV 8x10 3 IU/mL, HCMV 4x10 3 IU/mL and EBV 4x10 3 IU/mL Quantification standard including (plasmid): BKV 8x10 4 IU/mL, HCMV 4x10 4 IU/mL and EBV 4x10 4 IU/mL Quantification standard including (plasmid): BKV 8x10 5 IU/mL, HCMV 4x10 5 IU/mL and EBV 4x10 5 IU/mL Quantification standard including (plasmid): BKV 8x10 6 IU/mL, HCMV 4x10 6 IU/mL and EBV 4x10 6 IU/mL 1 x 150 µl 2 x 150 µl 1 x 150 µl 2 x 150 µl 1 x 150 µl 2 x 150 µl 1 x 150 µl 2 x 150 µl NC Negative control 1 x 2000 µl 1 x 4000 µl IC Internal control 1 x 128 µl 2 x 128 µl Enzyme 25x RT-PCR Enzyme mix (Fast-track mastermix) 1 x 32 µl 2 x 32 µl Buffer 2x RT-PCR Buffer (Fast-track mastermix) 1 x 400 µl 2 x 400 µl Each vial contains additional volume for pipetting inaccuracy. The box itself, the cover of the box and each vial are labeled with a lot number. FTD _64 MANUAL v2 2017_02 EN - 5 -

6 6. Precautions and warnings 6.1 Safety information Warning notice: the negative control contains lysis buffer. Hazardous pictogram: Signal word: Warning Hazardous statements: H315: Causes skin irritation. H317: May cause an allergic skin reaction. H319: Causes serious eye irritation. Precautionary statements: Prevention: P280: Wear protective gloves/protective clothing/eye protection/face protection. 6.2 Handling requirements Use of this product should be limited to personnel trained in the techniques of PCR. This product should be used in accordance with Good Laboratory Practice. Take the normal precautions required for handling all laboratory reagents. Do not mix reagents from different lots. Do not use the product after its expiration date. 6.3 Safe waste disposal Dispose of unused reagents and waste in accordance with country, state or local regulations. FTD _64 MANUAL v2 2017_02 EN - 6 -

7 7. Storage and stability conditions The components of the FTD product should be stored in the original packaging at 20 C and are stable until the expiration date stated on the label. The product is shipped in frozen packages which should ensure a transport temperature under +10 C (satisfactory, according to stability studies). The reagents within a kit are suitable for 32 or 64 reactions. Freeze the product immediately after usage. More than 9x thawing and freezing of the reagents per tube should be avoided, as this may reduce assay sensitivity. We recommend aliquoting the reagents according to your needs after the first thawing. For stability performance data, please refer to 8. Principle of the method Viral DNA is amplified simultaneously in the same tube by polymerase chain reaction. The presence of specific pathogen sequences in the reaction is detected by an increase in fluorescence observed from the relevant dual-labeled probes, and is reported as a cycle threshold value (Ct) by the Real-time thermocycler. The assay uses murine cytomegalovirus (MCMV) as an extraction control- the internal control (IC) - which is introduced by the laboratory into each sample and also the negative control at the lysis buffer stage of the extraction process. FTD _64 MANUAL v2 2017_02 EN - 7 -

8 9. Additionally required equipment FTD kits are suited for use with the Applied Biosystems 7500/7500Fast (Thermo Fisher Scientific), CFX96 (BIO-RAD), LightCycler 480 (Roche) and Rotor-Gene 3000, 6000, Q (Qiagen) and SmartCycler (Cepheid; in combination with Life Science software 2.0d). The assay has been fully validated on an Applied Biosystems 7500 with Fast-track mastermix and with the NucliSENS easymag (biomérieux). If you want to use different extraction methods, please firstly check their compatibility with FTD. For using the SmartCycler we recommend the FTD smartmix. Disposable powder-free gloves Pipettes (adjustable) Sterile pipette tips with filters Vortex mixer Desktop centrifuge For the ABI 7500, CFX96 and LightCycler 480, 96 well PCR plates and plate sealers are recommended. For the usage of the Rotor-Gene 3000/6000/ Q and SmartCycler use appropriate tubes and caps. Sample rack The validation file of Fast-track mastermix and a detailed compatibility list are available under Samples The quantitative validation of was done with extracted nucleic acid from human EDTA or Citrate plasma with NucliSENS easymag and ABI In clinical performance evaluation studies the test was also successfully qualitatively used with human urine, EDTA blood, plasma and serum. In case human urine, EDTA blood or serum are intended to use for quantitative analysis, the procedure has to be validated by the user. Attention: The blood samples should be shipped cooled (+2 C to +8 C) and the separated plasma/ serum deep frozen (-20 C). Samples of heparinised patients must not be used as heparin is a PCR interfering substance. FTD _64 MANUAL v2 2017_02 EN - 8 -

9 11. Procedure 11.1 Preliminary extraction procedure using the easymag If you want to use different extraction methods, please firstly check their compatibility at Extraction of specimens and negative control with the easymag : 1. Thaw the negative control (NC, white cap) and the internal control (IC, dark blue cap). Before use, the reagents have to be thawed completely, mixed (by short vortexing) and spun down briefly. 2. Extract your samples and the NC. We recommend a starting volume for the extraction of 200 µl and an elution volume of 55 µl. If you use different in- and output volumes you can find the formula for the calculation in chapter 15, Figure 2. Note: It is well recognised that sensitivity is increased if a larger volume of clinical material is extracted into a small volume of eluate. This is particularly important with samples where pathogen load is expected to be low, such as CSF (and others). Where extreme sensitivity is required, such as certain clinical situations, we recommend an input volume up to 1mL. However, we have to mention that, depending of the method used, the extraction of such volume could be not completely efficient and, therefore, could prevent accurate quantification. Please follow manufacturer`s extraction kit recommendations. 3. Add 2 µl internal control (IC, blue cap) directly to the lysis buffer of each extraction. Never add the internal control directly to the sample unless they are in lysis buffer. Adding the internal control to each of the samples and to the negative control is a very important step to see if the nucleic acid isolation has been successful and to check for possible PCR inhibition. 4. Do not extract quantification standards as they are plasmids and will be inhibited. 5. Make sure to refreeze the left over volumes of NC and IC right after usage. FTD _64 MANUAL v2 2017_02 EN - 9 -

10 11.2 Main PCR setup procedure Preparation of PCR with Fast track mastermix: 1. Thaw reagents for the reaction: BCE PP, the quantification standards (QS1-4) and 2x RT-PCR buffer (Fast-track mastermix, light blue cap) of Fast-track mastermix. The quantification standards and the extracted NC have to be included in each run. Before use, the reagents have to be thawed completely, mixed (by short vortexing) and spun down briefly. The quantification standards need to be thawed at room temperature for minutes and vortexed thoroughly right before use. Make sure to keep 25x RT-PCR enzyme (Fast-track mastermix, orange cap) of Fast-track mastermix in a freezer or on a cooling block at all times. 2. Pipette the required amount of 2x RT-PCR buffer in a 1.5ml tube. Do not immerge the whole tip into the liquid when pipetting 2x RT-PCR buffer to avoid waste of material and to obtain accurate volumes. Pipetting must be done very slowly to prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing. 3. Add according amount (see Table 3) of BCE PP to 2x RT-PCR buffer. Take care to change the tips after each pipetting step. 4. Pipette the required amount (see Table 3) of 25x RT-PCR enzyme to BCE PP with 2x RT-PCR buffer (reaction mix). Do not immerge the whole tip into the liquid when pipetting 25x RT-PCR enzyme to avoid waste of material and to obtain accurate volumes. Pipetting must be done very slowly to prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing. Take care to change the tips after each pipetting step. Vortex the complete master mix briefly and spin it down. If you use the SmartCycler please add per reaction 4µl of FTD smartmix to reaction mix. 5. Make sure to refreeze the remaining volumes of PP, QS1-4 and 2x RT- PCR buffer (Fast-track mastermix) after usage. FTD _64 MANUAL v2 2017_02 EN

11 Table 3: Shown are the amounts of reagents that are needed for 1, 15, 32 and 64 wells. FTD : The PPmix is sufficient for 32 reactions (+ pipetting inaccuracy). A minimum of 1 patient up to maximum 27 patients plus QS1-3 and NC is possible. FTD : The PPmix is sufficient for 64 reactions (+ pipetting inaccuracy). A minimum of 1 patient up to maximum 59 patients plus QS1-3 and NC is possible. Number of reactions FTD /64 Buffer 12.5 µl µl 400 µl 800 µl PPmix 1.5 µl 22.5 µl 48 µl 96 µl Enzyme 1 µl 15 µl 32 µl 64 µl Total 15 µl 225 µl 480 µl 960 µl Preparation of a 96 well plate for the ABI 7500: All our tests are validated on ABI 7500, Bio-Rad CFX96, LightCycler 480 and RotorGene. If you intend to use ABI 7500, Bio-Rad CFX96 or the LightCycler 480 you must use appropriate plates and adhesive films. For RotorGene and SmartCycler use adequate tubes and caps. If you intend to run our tests on a different cycler, please firstly refer to: Preparation of a 96 well plate for ABI Take a 96 well plate which is compatible with the ABI Pipette 15 µl of the reaction mix in the wells. 3. Add 10 µl of the extracted samples, the extracted negative control and the quantification standards (which are not extracted; thaw at room temperature for minutes and vortex thoroughly right before use). Each run must include a negative control and the quantification standards. 4. Mix briefly by pipetting up and down. 5. Close the plate with the ABI optical adhesive film. 6. Slightly vortex the plate and centrifuge briefly afterward. 7. Put the plate in the ABI Figure 1 (12 patients + QS1-4 + NC) shows an example for location of samples and controls on an ABI 7500 plate. FTD _64 MANUAL v2 2017_02 EN

12 Figure 1: Schematic presentation of an example for location of samples and controls on a 96 well plate for the ABI Rows A-H; columns 1-12= layout of the 96 well plate S1; S2; S3;, S12= reaction mix and samples 1-12 QS1-4= reaction mix and quantification standards (C1-4) NC= reaction mix and extracted negative control (C5) Yellow background= reaction mix with BCE PP (A1-12, C1-5) FTD _64 MANUAL v2 2017_02 EN

13 12. Programming of the thermocycler Pay particular attention to the settings for the detectors: Table 4: Settings for the detectors. PP mix Pathogen Dye Detection wavelength (nm) * Epstein-Barr virus green 520 BCE human cytomegalovirus yellow 550 murine cytomegalovirus (IC) orange 610 human polyomavirus 1 red 670 *The mentioned detection wavelengths are from the ABI They can be slightly different on other machines. NEW! Fast-track mastermix PCR programme 2: 50 C for 15 minutes hold 94 C for 1 minute hold 40 cycles of: 94 C for 8 seconds 60 C for 1 minute FTDliquid kits and FTlyo kits usage: To use FTlyo and FTDliquid kits on one plate or FTlyo kits alone, use the PCR programme 2. To use only FTDliquid kits, FTD is recommending the optimized PCR programme 2, as all validations are done with this program. But be aware, that you still can use the former PCR programme 1 as well. Fast-track mastermix PCR programme 1: 42 C for 15 minutes hold 94 C for 3 minutes hold 40 cycles of: 94 C for 8 seconds 60 C for 34 seconds Detailed information on programming of the thermocyclers is provided in the instruction manuals of the cyclers which can be downloaded from our homepage FTD _64 MANUAL v2 2017_02 EN

14 IMPORTANT NOTES: If you use the ABI 7500, it is necessary to change the setting for the passive reference dye. (By default, the ROX dye is selected). After the step specifying the detectors and task for each well, click finish and the software will create the plate document. Click on a well, or click-drag, to select replicate wells. Enter the sample name and change the passive reference to none. If you use the ABI 7500 Fast, do NOT use the fast programme. If you use the RotorGene turn off auto-gain optimization and set gains for yellow, orange, red and green channels on 5. If you use the LightCycler 480, it is necessary for you to perform one FTD color compensation run before you start using FTD tests. FTD advises to run a new FTD color compensation annually and after each maintenance of your device. The reagents for FTD color compensation are supplied for free by FTD. Use Abs Quant/Fit Points for analysis of the run. FTD recommends the usage of transparent 96well plates. If you use the SmartCycler, please be aware that it is currently validated in combination with the Cepheid, Life Science software 2.0d only and with the FTD smartmix (FTD). If you want to use different enzymes please refer to FTD. FTD _64 MANUAL v2 2017_02 EN

15 13. Assay validation Set a threshold as follows: 1. All negative controls should be below the threshold. If there is a potential contamination (appearance of a curve in the negative control or a cluster of curves in specimens at high Ct for example above 36), results obtained are not interpretable and the whole run (including extraction) has to be repeated. 2. All the quantification standards (QS1, QS2, QS3 and QS4) must show a positive (i.e. exponential) amplification trace. After setting up a standard curve following parameters should be observed: Slope: -3.9 to -2.9 R 2 : 0.97 to 1.00 Eff: % For detailed information check chapter 15 Quantification. 3. Check the component trace before accepting the exponential trace as real. Contact the equipment manufacturer or FTD for advice (support@fasttrackdiagnostics.com). 4. All internal controls must show a positive (i.e. exponential) amplification trace. The internal control must fall below a Ct of 33. If the internal control is above CT 33, this points to a purification problem or a strong positive sample that can inhibit the IC. FTD _64 MANUAL v2 2017_02 EN

16 14. Setup on the ABI Open your experiment 2. On the drop down menu on the left choose Analysis [A] and Amplification Plot [B]. 3. Modify the Graph Type [C] as you prefer to Linear or Log and the Color [D] to Target. FTD _64 MANUAL v2 2017_02 EN

17 4. In the top right corner of your screen choose Analysis settings [E]. 5. A new window opens: Analysis settings; highlight all targets of all tests [F]. 6. Unclick Use Default Settings, Automatic Threshold and Automatic Baseline [G] and Apply Analysis Settings [H]. FTD _64 MANUAL v2 2017_02 EN

18 7. For advanced analysis, you can also change your settings for each target in the options window. Here you can modify the threshold and baseline for every single parameter [I]. 8. Check the positive controls, negative controls and internal controls first. They have to follow the specifications mentioned in point 13 (Assay validation). 9. If all controls meet the specified ranges, check your samples for positive traces. 10. Your Ct results for all color channels will be displayed on the View Well Table window. FTD _64 MANUAL v2 2017_02 EN

19 15. Quantification FTD has decided to follow decision 2002/364/EC on common technical specifications for in vitro diagnostic medical devices: quantification standards have to be provided that allow the quantification of pathogens in International Units per milliliter (IU/mL) if available. The standards for absolute quantification that are included in FTD kits are correlated with World Health Organisation standards. These standards are calibrated following extensive international studies and play an important role for the traceability and comparability of results between laboratories. Although FTD understands that certain customers, in particular clinician`s, still prefer copies per ml. FTD gives conversion factors for BKV to convert obtained IU/mL to copies/ml (Table 5). Pay attention to the settings for the quantities for QS1-QS4 of : The quantitative results obtained with are reported in International Units (IU/ml) for HCMV, EBV and BKV. Our quantities (IU/ml) for HCMV, EBV and BKV are determined with the 1 st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques [NIBSC code: 09/162 (Version 3.0, Dated 30/11/2010)], the 1 st WHO International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques [NIBSC code: 09/260 (Version 2.0, Dated 12/01/2012)] and the 1 st WHO International Standard for BKV DNA for Nucleic Acid Amplification Techniques [NIBSC code: 14/212 (Version 1.0, Dated 07/01/2016)] using, Applied Biosystems 7500 and the NucliSENS easymag and plasma as matrix. The given quantities and conversion factor are specific for the mentioned platforms and for an extraction of 200µl plasma and an elution volume of 55µl. If extraction and/or elution volumes are differing to the recommended volumes please use the formula for taking the dilution factor in account (Figure 2). Table 5 shows the values of the quantification standards QS1 QS4 for BKV, EBV and HCMV determined with the mentioned specification. Table 6 shows according slope, R 2 and efficiency ranges which need to be reached for a correct quantification. FTD _64 MANUAL v2 2017_02 EN

20 Result of Quantity calculated with FTD QS in IU/ml or copies/ml Extraction volume recommended by FTD (µl) x Actually used Elution volume (µl) x = Elution volume recommended by FTD (µl) x Actually used Extraction volume (µl) Result for Quantity (IU/ml or copies/ml) 1 x 10 3 x 200µl x 55µl 55µl x 400µl = 5 x 10 2 Figure 2: Calculation including specific dilution factor. Table 5: Overview of quantities of BKV, HCMV and EBV within the FTD quantification standards (QS1-QS4). The quantities are given in IU/ml for BKV, EBV and HCMV. BKV= human polyomavirus 1, HCMV= human cytomegalovirus, EBV= Epstein-Barr virus, IU= international units, CF= conversion factor CF QS IU/mL QS1 4 x 10 3 HCMV 0.4 QS2 4 x 10 4 QS3 4 x 10 5 QS4 4 x 10 6 QS1 4 x 10 3 EBV 0.4 QS2 4 x 10 4 QS3 4 x 10 5 QS4 4 x 10 6 QS1 8 x 10 3 BKV 4.0 QS2 8 x 10 4 QS3 8 x 10 5 (e.g. conversion for BKV: 1000 cop/ml x 4.0= 4000 IU/mL) QS4 8 x 10 6 Table 6: Overview of slope, R 2 and efficiency ranges. specification range R to 1.00 slope -3.9 to -2.9 efficiency % FTD _64 MANUAL v2 2017_02 EN

21 1. In the drop down menu on the left choose Setup (J.). 2. Choose Assign Targets and Samples (K.). 3. Select the well containing one QS (L.) and start to specify the Task (M.): you need to choose S for standard. 4. Next to the Task block you can fill in the Quantity for each pathogen included in the test. Check in table 3 for the exact values of concentration (N.). 5. Continue to set up the concentrations for all quantification standards (QS1-QS4). FTD _64 MANUAL v2 2017_02 EN

22 6. For the internal control you do not need to change any settings. 7. In the drop down menu on the left choose Analysis (O.). 8. Select Standard Curve (P.) and mark the wells containing QS1 to QS4. 9. Choose your Targets (Q.) and review your obtained standard curve (R.). FTD _64 MANUAL v2 2017_02 EN

23 Figure 3-6 show amplification plots and according standard curves for BKV, HCMV and EBV you should obtain. Figure 3: Amplification plot for QS1-4. (red= BKV, green= EBV, purple= HCMV) Figure 4: Standard curve for BKV with QS1-4. FTD _64 MANUAL v2 2017_02 EN

24 Figure 5: Standard curve for EBV with QS1-4. Figure 6: Standard curve for HCMV with QS Interpretation of results The QS1-QS4 and any positive samples will show an exponential fluorescence trace. Any specimen displaying an exponential trace is considered as positive. For example, if a sample shows an exponential fluorescence trace at a wavelength of 550 (yellow channel) it contains human cytomegalovirus DNA. FTD _64 MANUAL v2 2017_02 EN

25 17. Troubleshooting No signal with quantification standards Incorrect programming of the temperature profile of the thermocycler Compare the temperature profile to the manual. Incorrect configuration of the PCR reaction Check your work steps by means of the pipetting scheme and repeat the PCR if necessary. Check calibration of pipettes. Incorrect handling of the quantification standards Inadequate or no vortexing and thawing at room temperature The storage conditions for one or more product components did not comply with the instructions or the FTD kit has expired. Please, check the storage conditions and the expiration date (see the product label) of the reagents and use a new test, if necessary. Weak or no signal of the internal control The PCR conditions do not comply with the protocol. Check the PCR conditions and repeat the PCR with correct settings if necessary. The PCR was inhibited or no / too little internal control was added during the extraction. Make sure that your extraction method is compatible with FTD kits. A strong positive signal of a pathogen can occasionally inhibit the fluorescence of an internal control. Signals within the negative control A contamination occurred during preparation of the PCR or during extraction Repeat the PCR with new reagents in replicates. We recommend pipetting the positive controls last. Make sure that work space and instruments are decontaminated at regular intervals. If you have any further questions or if you encounter problems, please contact support@fast-trackdiagnostics.com. FTD _64 MANUAL v2 2017_02 EN

26 18. Validation For detailed validation data such as sensitivity, specificity, clinical studies and external quality panel results, please refer to the related validation file at: Legend of symbols FTD _64 MANUAL v2 2017_02 EN