Fungiplex Universal RUO Real- Time PCR Kit

Transcription

1 Instructions for Use Fungiplex Universal RUO Real- Time PCR Kit Research Use Only Kit for broad identification of fungal species directly from samples without culture For General Purpose Use (In the United States, Japan and Taiwan: For Research Use Only (RUO)) This product has no declared clinical intended purpose and is not for clinical diagnostic use. Any clinical diagnostic use is at the user's own risk and responsibility. CARE products are designed to support our worldwide customers with highquality consumables, accessories and dedicated kits. Language: en Revision A (April 2018)

2 1 Product description Fungiplex Universal RUO Real-Time PCR Kit components 4 2 Inspection, storage and stability Inspection on arrival Storage temperature Suitable storage area Stability 5 3 Risk and safety information General precautions Biological risks Limitations of the procedure 7 4 Materials required but not provided General equipment Real-time PCR instrumentation 9 5 Specimen collection and DNA extraction Recommended extraction protocols used throughout verification 10 6 Test procedure PCR set-up 11 7 Thermal cycler conditions ABI Rotor-Gene Q CFX QuantStudio LightCycler 480 II 18 8 Interpretation of results Internal control Positive and negative controls Unknown / patient samples 20 9 Performance characteristics Disposal Explanation of symbols Manufacturer 24 Page 2 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

3 1 Product description The Fungiplex Universal RUO Real-Time PCR Kit (referred to as Fungiplex Universal RUO PCR Kit) is designed for research use only, to rapidly detect the presence of fungal DNA from samples without culture. It detects a broad range of fungal species including those with low prevalence, and those that are not detected by diagnostic biomarkers, such as from the Zygomycetes or Cryptococcus genera, see Table 1 for more details. Table 1 Genera detected by the Fungiplex Universal RUO Real-Time PCR Kit Apophysomyces Fusarium Rhizomucor Aspergillus Histoplasma Rhizopus Candida Lichtheimia Saccharomyces Cladosporium Microsporum Saksenaea Coccidioides Mucor Scedosporium Cryptococcus Paecilomyces Syncephalastrum Cunninghamella Penicillium Talaromyces Exophiala Pneumocystis Trichosporon The Fungiplex Universal RUO PCR Kit has been designed to run on the same conditions as Bruker s Fungiplex range of real-time PCR assays. Fungiplex Universal RUO PCR Kit The assay is designed to be used across common laboratory PCR equipment and can process up to 50 samples from post-extraction to result in less than 2 hours. The use of universal primers and multiple probes on the same fluorophore (Cy5) results in the detection of a broad range of fungal genera. The rapid, reliable detection provided by this test has the potential to improve the understanding of fungal disease, from a research perspective, while quality controlled manufacture ensures that the user can have full confidence in assay quality and reproducibility. References 1. Ala-Houhala M et al., Clin. Microbiol. Infect., 2017; Drgona L et al., Eur J. Clin. Microbiol. Infect. Dis., 2014; 33: Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 3 of 24

4 1.1 Fungiplex Universal RUO Real-Time PCR Kit components Reagents are provided in quantities that will allow 50 tests to be performed. Fungiplex Universal Real-Time PCR Kit Reagent Volume per tube No. of tubes Cap color Reactions per tube PCR Mix (50) 750 µl 1 Orange 50 Fungiplex Universal Mix 60 µl 1 Purple 50 PCR Water 800 µl 1 White 50 Fungiplex Universal Control Kit Reagent Volume per tube No. of tubes Cap color Internal control 135 µl 2 Red Fungiplex Universal Positive Control 180 µl 2 Blue Negative Control 240 µl 2 White Ordering Information Product Part Number Fungiplex Universal RUO Real-Time PCR Kit Page 4 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

5 2 Inspection, storage and stability 2.1 Inspection on arrival Check the Fungiplex Universal RUO Real-Time PCR Kit on arrival. If the packaging is damaged the kit must not be used. Additionally, all reagents contained within the Fungiplex Universal RUO Real-Time PCR Kit are shipped frozen and should arrive frozen. If reagents are not frozen upon receipt or if the tubes have been compromised during shipment, contact Bruker Daltonik GmbH for assistance (see section 12 for details). 2.2 Storage temperature 25 C 15 C The Fungiplex Universal RUO Real-Time PCR Kit is shipped on dry ice and should be stored within this advised temperature range on receipt. 2.3 Suitable storage area The internal reagent box named Fungiplex Universal Real- Time PCR Kit should be stored in an amplicon free laboratory. The internal reagent box named Fungiplex Universal Control Kit, which includes an internal control and positive and negative assay controls, should be stored in the area of the facility where the DNA template / sample template is added. 2.4 Stability The performance of the Fungiplex Universal RUO Real-Time PCR Kit has shown no adverse effects when used over 10 freeze-thaw cycles. All reagents contained within the Fungiplex Universal RUO Real-Time PCR Kit have been validated for use within the expiration date stated on the kit label and must be used within this time period. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 5 of 24

6 3 Risk and safety information There are no hazardous substances included in the manufacture of the Fungiplex Universal RUO Real- Time PCR Kit. The composition of all kit reagents poses no specific risk to the user or to their property. Additional chemicals and materials may be required for procedures described in these Instructions for Use. Carefully read any warnings, instructions, or Safety Data Sheets provided by the supplier and follow general safety regulations when handling chemicals, biohazards, or other materials. 3.1 General precautions This Fungiplex Universal RUO PCR Assay is for Research Use Only. This test is verified only on the PCR platforms listed herein. Test procedure should be performed as outlined within these Instructions for Use. Any deviation from the outlined protocols may result in assay failure or generate erroneous results. This test is for use with extracted DNA. Standard precautions and institutional guidelines should be followed when handling all samples. Low concentrations of DNA can be unstable if stored for long periods. Bruker advises that sample storage time should be minimized before testing. Do not use reagents after their stated expiry date. Do not mix reagents from different tubes or kits, even if they are from the same lot. Do not substitute reagents from different manufacturers. Take care to preserve the purity of kit reagents. Avoid contamination from positive controls and specimens by following good laboratory practice and organising and segregating workflow appropriately. Ensure all additional required consumables are DNase/RNase free. Wear protective clothing and disposable gloves when handling kit reagents. Do not use kit reagents if the label seal is broken upon receipt. Do not eat, drink or smoke in areas where specimens or kit reagents are being handled. Dispose of unused reagents and waste in accordance with country, state and local regulations. 3.2 Biological risks The Fungiplex Universal RUO Real-Time PCR Kit involves working with potentially dangerous and transmissible biological material. Personnel working with these systems are responsible for reading and following all necessary health and safety precautions. It is very important to wear appropriate personal protective equipment at all times; a lab coat, protective gloves and safety glasses as a minimum. Page 6 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

7 3.3 Limitations of the procedure This assay is a qualitative test and does not provide quantitative data on any detected targets. False positive results may arise from external contamination of original sample or contamination during PCR set-up. Positive results do not rule out co-infection with additional organisms that are not detected via this test. Extraction efficiency may impact results and could result in false negative outcomes. Preliminary data indicates that stability of extracted DNA may be affected by freezing and storage. It is therefore recommended that samples are processed as quickly as possible after collection. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 7 of 24

8 4 Materials required but not provided 4.1 General equipment Equipment Recommended nucleic acid extraction platform Verified real-time thermal cycler instrument Class II hood / PCR station Vortex mixer Mini centrifuge Centrifuge capable of microplate processing (recommended) Fridge (2 to 8 C) and freezers (-25 C to -15 C) Micropipettes for volumes of 1 to 1000 µl Reagents Extraction reagents Positive extraction control (recommended, e.g. previously characterized positive sample) Negative extraction control (recommended, e.g. nuclease free water) Consumables Appropriate DNase/RNase free plasticware for PCR preparation DNase/RNase free pipette tips Disposable gloves, powderless Page 8 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

9 4.2 Real-time PCR instrumentation The Fungiplex Universal RUO Real-Time PCR Kit has been validated across multiple real-time PCR instruments. The instruments and a list of consumables used throughout internal verification studies are advised below in Table 2. Table 2 Real-time PCR instrumentation specific details Manufacturer Applied Biosystems Real-time PCR platform model Software version ABI 7500 Fast 2.3 Bio-Rad CFX Applied Biosystems Roche Qiagen QuantStudio 5 LightCycler 480 II Rotor-Gene Q 5plex HRM Q-Series Verified consumables MicroAmp Fast Optical 96-Well MicroAmp Optical 8-Cap Strips LightCycler 480 Multiwell Plate 96 (white) MicroAmp Optical 8-Cap Strips Qiagen Strip Tubes and Caps, 0.1 ml Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 9 of 24

10 5 Specimen collection and DNA extraction The quality of the extracted DNA is inherently linked to the sensitivity achieved by real- time PCR assays and therefore samples must be collected and stored appropriately following the guidelines below: Samples should be extracted following the recommendations for extracting DNA from Aspergillus fumigatus published by the European Aspergillus PCR Initiative 3,4 (EAPCRI). Good laboratory practice recommends including at least one positive and negative extraction control per analysis. Extracted samples should be stored between -80 C and -20 C for long term storage. Note Low concentrations of DNA can be unstable if stored for long periods. References 3. White PL et al., J. Clin.Microbiol., 2010; 48: White PL et al., J. Clin.Microbiol., 2011; 49:, Recommended extraction protocols used throughout verification The protocol validated for the extraction of DNA is referenced in Table 3. Bruker advises each user to perform an internal validation on their specific platform / procedure. Table 3 Extraction platform Extraction protocol validated Extraction kit used Extraction input volume Extraction elution volume Qiagen EZ1 EZ1 DSP Virus Kit 400 µl 60 µl Page 10 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

11 6 Test procedure 6.1 PCR set-up ATTENTION Bruker recommends the use of a clean room (PCR suite) to set up all PCR reactions with addition of template in a suitable area out with the clean room facility. To avoid any risk of contamination, users should follow good laboratory practice at all times and segregate workflow appropriately. DNase/RNase free plasticware should be used. Before beginning PCR set- up, the user has the responsibility to assign samples to the appropriate wells within the appropriate plasticware; this information should be recorded for traceability purposes. Within an amplicon free area, remove the Fungiplex Universal Real- Time PCR Kit from the freezer and allow the reagents to thaw. Briefly vortex and centrifuge the reagents. Prepare a PCR reaction mastermix using the reagents and volumes detailed in Table 4. Volumes are given per reaction well and should be multiplied by the number of reaction wells, taking into account the number of samples, positive and negative controls in the analysis. The prepared PCR reaction mastermix should be thoroughly mixed and centrifuged for 10 seconds. Table 4 Preparation volumes for the PCR reaction mastermix in an amplicon free area Assay kit reagents Volume PCR Mix (50) 10.0 µl Fungiplex Universal Mix 1.0 µl PCR Water 3.0 µl Total volume (per well) 14.0 µl Example: For 8 samples, 1 positive control and 1 negative control, the final volume of the PCR reaction mastermix will be = 140 µl. Before dispensing into reaction wells, transfer the PCR reaction mastermix to the template/sample addition area for inclusion of the internal control. Remove the Fungiplex Universal Control Kit from the freezer and allow the reagents to thaw. Briefly vortex and centrifuge the reagents. Internal control material should be added to the PCR reaction mastermix. The internal control is added at 1 µl per reaction well (see Table 5). Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 11 of 24

12 Table 5 Final PCR reaction mastermix volume with inclusion of internal control Mastermix reagents + internal control PCR reaction mastermix (from Table 4) Volume 14 µl Internal control material 1 µl Total volume (per well) 15 µl Example: For 10 reaction wells (8 samples, 1 positive control and 1 negative control) 10 µl of the internal control material must be added to the bulk mastermix. For 10 reaction wells this will provide a final PCR reaction mastermix volume of 150 µl. Dispense 15 µl of PCR reaction mastermix into the required wells of the appropriate plasticware. For each sample, add 5 µl of extracted sample material to the PCR reaction mastermix (see Table 6). For each positive control, add 5 µl of Fungiplex Universal Positive Control to the dispensed PCR reaction mastermix. At least one positive control reaction should be included to provide details on reaction efficiency. For each negative control, add 5 µl of Negative Control to the dispensed PCR reaction mastermix. At least one negative control reaction should be included to provide details on contamination. Table 6 Dispensed volumes per reaction well of PCR reaction mastermix PCR reaction well composition Volume PCR reaction mastermix + internal control 15 µl Sample/control material 5 µl Final volume (per well) 20 µl The plasticware should then be sealed and transferred to a verified thermal cycler for amplification. Ensure the samples are centrifuged briefly before placing in the thermal cycler. Please refer to the instrument manual for instructions on setting up an amplification run. Amplification should be carried out according to the instrument- specific parameters in section 7. Page 12 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

13 7 Thermal cycler conditions 7.1 ABI 7500 Refer to the manufacturer's instruction manual for information on how to operate the real-time PCR instrument and perform data analysis. This instrument collects all dyes as default see Table 7 for the dyes used with the Fungiplex Universal RUO Real-Time PCR Kit. Table 7 Detector channels used in the ABI 7500 instrument Targets Reporter Dye Channel FAM Cy5 Quencher None None Target of Fungiplex Universal RUO PCR Assay Configure the ABI 7500 with the following settings: Instrument Type: 7500 Fast (96 wells) Experiment type: Quantitation Standard Curve Reagents used: TaqMan Reagents Ramp speed: Standard Reaction volume: 20 µl Passive reference dye: None Internal Control (IAC) See Table 8 for the thermal profile settings. Data should be collected for all 45 cycles. Table 8 PCR parameters for the Fungiplex Universal RUO PCR Assay Fungal target Step Temperature ( C) Time Number of cycles Data collection Activation min 1 Off Denaturation 95 5 s Off 45 Annealing / extension s On To analyze the data, ensure the passive reference dye is switched to None and in Options deselect the box for Threshold Auto for each target. The baseline should continue to be calculated automatically. Use the information in Table 9 to set the thresholds for each detector channel in the Analysis settings window. Table 9 ABI 7500 Threshold values for the Fungiplex Universal RUO PCR Assay Channel FAM Cy5 Target(s) Internal control Fungal target Threshold ,000 Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 13 of 24

14 It is important to visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and cannot be attributed to background noise recorded above the defined thresholds. Page 14 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

15 7.2 Rotor-Gene Q Refer to the manufacturer's instruction manual for information on how to operate the real-time PCR instrument and perform data analysis. Select the appropriate fluorophore within each channel and assign to the relevant target, as shown in Table 10. Table 10 Detector channels used in the Rotor-Gene Q instrument Reporter Dye Channel GREEN RED Reporter dye FAM Cy5 Target of Fungiplex Universal RUO PCR Assay Internal control Fungal target Configure the Rotor-Gene Q Series / Q-Rex software with the following settings: Select 72-well rotor Reaction volume: 20 µl Auto Gain should be used for the selected dye channels only. For Rotor- Gene Q set to implement before first acquisition on the default well, for Q- Rex use the default / Auto- gain method. See Table 11 for the thermal profile settings. Data should be collected for all 45 cycles. Table 11 PCR parameters for the Fungiplex Universal RUO PCR Assay Step Temperature ( C) Time Number of cycles Data collection Activation min 1 Off Denaturation 95 5 s Annealing / extension To analyze data, apply the following settings: Rotor-Gene Q Series Off s 45 On Normalization: select Dynamic Tube and Slope Correct. Analyze all cycles and enter the threshold and outlier removal values for each channel as detailed in Table 12. Table 12 Rotor-Gene Q Series threshold values for the Fungiplex Universal RUO PCR Assay Channel GREEN RED Target(s) Internal control Fungal target Threshold Outlier removal 5% 9% It is important to visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and cannot be attributed to background noise recorded above the defined thresholds. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 15 of 24

16 7.3 CFX96 Refer to the manufacturer's instruction manual for information on how to operate the real-time PCR instrument and perform data analysis. This instrument collects all dyes as default see Table 13 for the dyes used with the Fungiplex Universal RUO Real-Time PCR Kit. Table 13 Detector channels used in the CFX96 Fluorophore FAM Cy5 Target detected with Fungiplex Universal RUO PCR Assay Internal control Fungal target Configure the CFX96 with the following settings: Sample volume: 20 µl Scan Mode: All channels Plate Type: BR clear See Table 14 for the thermal profile settings. Data should be collected for all 45 cycles. Table 14 PCR parameters for the Fungiplex Universal RUO PCR Assay Step Temperature ( C) Time Number of cycles Data collection Activation min 1 Off Denaturation 95 5 s Off 45 Annealing / extension s On To analyse the data, under Baseline Setting select the following: Baseline Subtracted Curve Fit Apply fluorescent drift correction To set the threshold values for each detector channel navigate to Baseline threshold > Single threshold > User defined and enter the values from Table 15. Baseline Cycles should remain autocalculated. Table 15 CFX96 Threshold values for the Fungiplex Universal RUO PCR Assay Channel FAM Cy5 Target(s) Internal control Fungal target Threshold It is important to visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and cannot be attributed to background noise recorded above the defined thresholds. Page 16 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

17 7.4 QuantStudio 5 Refer to the manufacturer's instruction manual for information on how to operate the real-time PCR instrument and perform data analysis. This instrument collects all dyes as default see Table 16 for the dyes used with the Fungiplex Universal RUO Real-Time PCR Kit. Table 16 Detector channels used in the QuantStudio 5 instrument Reporter Dye Channel FAM Cy5 Quencher None None Target detected with Fungiplex Universal RUO PCR Assay Internal control Fungal target Configure the QuantStudio 5 with the following settings: Sample volume: 20 µl Standard mode (with ramp rates of 1.6 C / s) See Table 17 for the thermal profile settings. Data should be collected for all 45 cycles. Table 17 PCR parameters for the Fungiplex Universal RUO PCR Assay Step Temperature ( C) Time Number of cycles Data collection Activation min 1 Off Denaturation 95 5 s Off 45 Annealing / extension s On To analyze the data, ensure the passive reference dye is switched to None and deselect the box for Threshold: Auto for each target. The baseline should continue to be calculated automatically. Use the information in Table 18 to set the threshold values for each detector channel in the Analysis settings window. Table 18 QuantStudio 5 Threshold values for the Fungiplex Universal RUO PCR Assay Channel FAM Cy5 Target(s) Internal control Fungal target Threshold ,500 It is important to visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and cannot be attributed to background noise recorded above the defined thresholds. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 17 of 24

18 7.5 LightCycler 480 II Refer to the manufacturer's instruction manual for information on how to operate the real-time PCR instrument and perform data analysis. Configure the LightCycler 480 II with the following settings: Set up detection format to run the Fungiplex Universal RUO Real-Time PCR Kit by navigating to Settings > Set detection format and name new file (suggested file name: Fungiplex Universal RUO PCR Assay) o o Tick and label FAM Tick and label Cy5 All dyes should be set up to have a Melt Factor of 1, a Quant Factor of 10 and a Maximum Integration Time of 2 seconds. Save the detection format. Reaction Volume: 20 µl. Table 19 details the targets detected by the corresponding Filter Combinations. Set these dyes as the reporter dyes. Table 19 Detector channels used in the LightCycler 480 II instrument Filter comb Target detected with Fungiplex Universal RUO PCR Assay Internal control Fungal target Use the detection format set up above (suggested settings name: Fungiplex Universal RUO PCR Assay) and use the LightCycler 480 II with the following settings: Reaction Volume: 20 µl See Table 20 for the thermal profile settings. Data should be collected for all 45 cycles. Table 20 PCR parameters for the Fungiplex Universal RUO PCR Assay Step Temperature ( C) Time Number of cycles Data collection Activation min 1 None Denaturation 95 5 s None 45 Annealing / extension s Acquire Analyze data with Absolute Quantification / Second Derivative Max settings. There is no requirement for a color compensation file with this Fungiplex Universal RUO PCR Assay. The use of thresholds is not applicable see Table 21. Table 21 LightCycler 480 II threshold values for the Fungiplex Universal RUO PCR Assay Channel FAM Cy5 Target(s) Internal control Fungal target Threshold 2 nd derivative auto-calculated 2 nd derivative auto-calculated Page 18 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

19 In 2nd derivative auto- calculated analysis mode, positive wells (red) and uncertain wells (blue) determined by the software are to be considered positive and negative wells (green) should be considered negative. It is important to visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and matches the automatic scoring of the software. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 19 of 24

20 8 Interpretation of results 8.1 Internal control Detection of the internal control is not required with a positive result. In instances where the internal control has failed but the sample has been reported as positive for a fungal target, the positive result should be considered valid. In instances where the sample is reported as negative for all targets and the internal control is negative, the user should repeat the assay using the same extract. If the internal control is then reported as positive, it is likely that the original result was due to a handling error / PCR inhibition and any new results should be reported. If the internal control is still reported as negative after repeat testing, the sample should be re-tested, starting from the extraction step. 8.2 Positive and negative controls At least one positive and negative control provided in the control kit should be included in each run. Each negative or positive control should be prepared and tested in the same manner as any sample under investigation. A negative control that yields a positive test result is indicative of a sample handling or contamination problem. The assay run should be repeated using a fresh aliquot of negative control material, ensuring that the work area and equipment are properly decontaminated and extreme care is taken during PCR set-up. A positive control that yields a negative result is indicative of reagent failure or sample handling error. Ensure all reagents have been stored correctly and, where applicable, are within expiry date before repeating the assay run taking extreme care during PCR set-up. Expected results for the positive control are provided in Table 22. Table 22 Expected positive control results Fungiplex Universal RUO Positive Control Expected result Fungal target positive 8.3 Unknown / patient samples Always visually inspect the amplification plots for each sample to ensure that the results recorded are due to true amplification and cannot be attributed to background noise recorded above the defined thresholds. Table 23 summarizes the result interpretation with the Fungiplex Universal RUO Real-Time PCR Kit. Table 23 Results achieved with Fungiplex Universal RUO Real-Time PCR Kit Channel Reported target C t value Result Interpretation Green/FAM Internal control >10 Internal control PASS N/A or <10 Internal control FAIL Red/Cy5 Fungal target <45 Positive result N/A Negative result Page 20 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

21 9 Performance characteristics The performance characteristics of the Fungiplex Universal RUO Real-Time PCR Kit are highlighted in Table 24. This details the analytical performance with known quantities of material analyzed at the defined Limit of Detection (LOD) and above. Table 24 Analytical performance characteristics of the Fungiplex Universal RUO Real-Time PCR Kit per instrument Plasmid samples CFX QuantStudio LightCycler 480 II ABI7500 Assay sensitivity (%) Assay specificity (%) Rotor-Gene Q-Series The defined limits of detection are quoted per instrument in Table 25. Additional fungal species are theoretically detected but no claims are made on performance. Table 25 LODs of Fungal Targets tested using the Fungiplex Universal RUO Real-Time PCR Kit Target CFX QuantStudio LightCycler 480 II LOD Concentration in input copies of DNA ABI7500 Apophomyces elegans 50 ipc 50 ipc 100 ipc 50 ipc 50 ipc Aspergillus chevalieri 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Aspergillus flavus 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Aspergillus fumigatus 50 ipc 50 ipc 200 ipc 50 ipc 50 ipc Aspergillus glaucus 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Aspergillus lentulus 50 ipc 50 ipc 200 ipc 50 ipc 50 ipc Aspergillus nidulans 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Aspergillus niger 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Aspergillus ustus 50 ipc 50 ipc - 50 ipc 50 ipc Aspergillus versicolour 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida albicans 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Rotor-Gene Q-Series Candida dubliniensis 50 ipc 50 ipc 200 ipc 200 ipc 200 ipc Candida famata 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida glabrata 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida guilliermondii 50 ipc 50 ipc 200 ipc 50 ipc 200 ipc Candida inconspicua 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida kefyr 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida krusei 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida lusitaniae 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 21 of 24

22 Target CFX QuantStudio LightCycler 480 II ABI7500 Rotor-Gene Q-Series Candida parapsilosis 200 ipc 200 ipc 50 ipc 200 ipc 200 ipc Candida tropicalis 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Candida viswanthii 50 ipc 50 ipc 200 ipc 200 ipc 50 ipc Cladosporium spp. 200 ipc 50 ipc 200 ipc 200 ipc 50 ipc Coccidioides immitis 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Cryptococcus neoformans 50 ipc 50 ipc 100 ipc 50 ipc 50 ipc Cunninghamella elegans 400 ipc 400 ipc 400 ipc 50 ipc 400 ipc Exophiala dermatitidis 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Fusarium chlamydosporum 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Fusarium dimerum 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Fusarium oxysporon 50 ipc 50 ipc 200 ipc 200 ipc 200 ipc Fusarium prolificans 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Fusarium solani 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Histoplasma capsulatum 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Lichtheimia corymbifera 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Microsporum canis 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Mucor circinelloides 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Paecilomyces lilacinus 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Pneumocystis spp. 300 ipc 200 ipc 200 ipc 200 ipc 200 ipc Rhizomucor pusillus 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Rhizopus oryzae 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Saccharomyces cerevisiae 200 ipc 50 ipc 200 ipc 50 ipc 200 ipc Sakenaea vasiformis 50 ipc 50 ipc 100 ipc 50 ipc 50 ipc Scedosporium apiospermum 200 ipc 50 ipc 200 ipc 50 ipc 50 ipc Scedosporium prolificans 200 ipc 50 ipc 200 ipc 200 ipc 50 ipc Syncephalastrum racemosum 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Talaromyces marneffei 50 ipc 50 ipc 50 ipc 50 ipc 50 ipc Trichosporon spp. 100 ipc 200 ipc 200 ipc 200 ipc 100 ipc Page 22 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A

23 10 Disposal General disposal Dispose of any unused reagents, waste and shipping materials in accordance with country, state and local regulations. Dry ice disposal Dry ice contains CO 2 and must be handled with gloves in a well ventilated area. Dispose of any dry ice in accordance with country, state and local regulations. Packaging disposal Please dispose of all packaging in accordance with local recycling regulations. The above instructions for disposal apply to EU member states. Correct disposal of this product will help conserve natural resources and ensure that it is recycled in a manner that prevents potential negative consequences for the environment and human health. For more information, please contact your local waste disposal service. Revision A Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Page 23 of 24

24 11 Explanation of symbols The following symbols may be present on the Fungiplex Universal RUO Real-Time PCR Kit labels or within these Instructions for Use. Symbol Definition Symbol Definition Use by date YYYY-MM-DD Contains sufficient for <n> tests Lot number Product reference number Manufacturer Upper and lower storage temperature limitation 12 Manufacturer Bruker Daltonik GmbH Fahrenheitstraße Bremen Germany Support Support.Molecular-Testing@bruker.com Phone: Sales Information For general information please contact: ms.sales.bdal@bruker.com For ordering information please contact: care@bruker.com Phone: Web: Descriptions and specifications supersede all previous information and are subject to change without notice. Copyright 2018 Bruker Daltonik GmbH ( ) Page 24 of 24 Fungiplex Universal RUO Real-Time PCR Kit Instructions for Use Revision A