NHS Meningitides Real-TM,sacace -50t

Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 IVD For in Vitro Diagnostic Use For Professional Use Only NHS Meningitidis Real-TM Handbook Real time PCR kit for detection of Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae REF B25-50FRT 50 Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 NAME NHS Meningitidis Real-TM INTENDED USE Kit NHS Meningitidis Real-TM is a Real-Time test for the detection and differentiation of Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae in the biological materials. DNA is extracted from specimens, amplified using RT-amplification and detected using fluorescent reporter dye probes specific for N.meningitidis, H.influenzae, S.pneumoniae DNA and IC (Internal Control). PRINCIPLE OF ASSAY NHS Meningitidis Real-TM Test is based on three major processes: isolation of DNA from specimens, Real Time amplification of the DNA and NHS Meningitidis detection by the polymerase chain reaction (PCR) based on the amplification of pathogen genome specific region using specific primers and detection via fluorescent dyes. These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product. The real-time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run. NHS Meningitidis Real-TM PCR kit is a qualitative test which contain the Internal Control (IC). It must be used in the isolation procedure in order to control the process of each individual sample extraction and serves also to identify possible reaction inhibition. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 MATERIALS PROVIDED “Controls”  Negative Control C- *, 1,2 ml;  Internal Control (IC) **, 1,0 ml;  Pos DNA N. meningitidis C+, 0,1 ml;  Pos DNA H.influenzae C+, 0,1 ml  Pos DNA S.pneumoniae C+, 0,1 ml;  Pos IC C+, 0,1 ml;  DNA-buffer, 0,5 ml; “NHS Meningitidis Real-TM”: Real Time amplification  PCR-mix-1 N. meningitidis/IC, 0,6 ml;  PCR-mix-1 S. pneumoniae/ H. influenzae 0,6 ml;  PCR-mix-2, 2 x 0,3 ml;  TaqF Polymerase, 2 x 0,03 ml; Contains reagents for 55 reactions * must be used in the isolation procedure as Negative Control of Extraction. ** add 10 µl of Internal Control during the DNA purification procedure directly to the sample/lysis mixture MATERIALS REQUIRED BUT NOT PROVIDED  DNA extraction kit.  Tube racks.  Vortex mixer.  Desktop centrifuge with rotor for 1,5/2,0 ml tubes.  PCR Workstation.  Real Time Thermal cycler.  Disposable polypropylene microtubes for PCR.  Refrigerator for 2–8 °C, Deep-freezer for ≤ –16 °C.  Waste bin for used tips. STORAGE INSTRUCTIONS “Controls” must be stored at 2-8°C. “NHS Meningitidis Real-TM” must be stored at -20°C. The kit can be shipped at 2-8°C but should be stored at 2-8°C and -20°C immediately on receipt. QUALITY CONTROL In accordance with Sacace’s ISO 13485-Certified Quality Management System, each lot is tested against predetermined specifications to ensure consistent product quality. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 STABILITY NHS Meningitidis Real-TM test is stable up to the expiration date indicated on the kit label. The product will maintain performance through the control date printed on the label. Exposure to light, heat or humidity may affect the shelf life of some of the kit components and should be avoided. Repeated thawing and freezing of these reagents should be avoided, as this may reduce the sensitivity. WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only 1. Wear disposable gloves, laboratory coats and eye protection when handling specimens and reagents. Thoroughly wash hands afterward. 2. Do not pipette by mouth. 3. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. 4. Do not use a kit after its expiration date. 5. Dispose of all specimens and unused reagents in accordance with local regulations. 6. Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents. 7. Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0,5% sodium hypochlorite, or other suitable disinfectant. 8. Avoid contact of specimens and reagents with the skin, eyes and mucous membranes. If these solutions come into contact, rinse immediately with water and seek medical advice immediately. 9. Material Safety Data Sheets (MSDS) are available on request. 10. Use of this product should be limited to personnel trained in the techniques of DNA amplification. 11. PCR reactions are sensitive to contamination. Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in performing PCR in compliance with good laboratory practice. 12. Workflow in the laboratory must proceed in a uni-directional manner, beginning in the Extraction Area and moving to the PCR and Detection Area. Do not return samples, equipment and reagents in the area where you performed previous step. Some components of this kit contain sodium azide as a preservative. Do not use metal tubing for reagent transfer. Sampling of biological materials for PCR-analysis, transportation, and storage are described in details in the handbook of the manufacturer. It is recommended that this handbook is read before beginning of the work. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics. Use of this product should be limited to personnel trained in the techniques of DNA amplification (UNI EN ISO 18113-2:2012). Strict compliance with the user manual is required for optimal PCR results. Attention should be paid to expiration dates printed on the box and labels of all components. Do not use a kit after its expiration date. SAMPLE COLLECTION, STORAGE AND TRANSPORT NHS Meningitidis Real-TM can analyze DNA extracted from:  liquor (ready for extraction)- 0,1 ml; Specimens can be stored at +2-8°C for no longer than 12 hours, or frozen at -20°C to -80°C. Transportation of clinical specimens must comply with country, federal, state and local regulations for the transport of etiologic agents. DNA ISOLATION Any commercial RNA/DNA isolation kit, if IVD-CE validated for the specimen types indicated herein at the “SAMPLE COLLECTION, STORAGE AND TRANSPORT” paragraph, could be used. Sacace Biotechnologies recommends to use the following kits:  DNA-Sorb-B (Sacace, REF K-1-1/B/100);  DNA/RNA Prep (Sacace, REF K-2-9);  SaMag Bacterial DNA Extraction kit (Sacace, REF SM006); Please carry out the DNA extraction according to the manufacturer’s instructions. Add 10 μl of Internal Control during the DNA isolation procedure directly to the sample/lysis mixture. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 PROTOCOL: Total reaction volume is 25 µl, the volume of DNA sample is 10 µl. 1 Prepare required quantity of reaction tubes (2 tubes for each sample + Controls) 2 Prepare in a new sterile tube two Reaction Mixes.  The Reaction Mix S. pneumoniae/ H. influenzae: 10*(N+1) µl of PCR-mix-1 S. pneumoniae/ H. influenzae 5.0*(N+1) µl of PCR-mix-2 0.5*(N+1) µl of TaqF Polymerase  The Reaction Mix N. meningitidis/IC: 10*(N+1) µl of PCR-mix-1 N. meningitidis/IC 5.0*(N+1) µl of PCR-mix-2 0.5*(N+1) µl of TaqF Polymerase 3 Vortex the tube, then centrifuge shortly. Add 15 µl of prepared reaction mix into each appropriate tube. 4 Using tips with aerosol filter add 10 µl of DNA samples obtained at the stage of DNA isolation and mix carefully by pipetting. 5 Prepare for each panel 3 controls:  add 10 µl of DNA-buffer to the tube labeled Amplification Negative Control;  add 10 µl of Pos DNA S. pneumoniae C+ to the tube with PCR-mix-1 S. pneumoniae/ H. influenzae;  add 10 µl of Pos DNA H. influenzae C+ to the tube with PCR-mix-1 S. pneumoniae/ H. influenzae;  add 10 µl of Pos DNA N.meningitidis C+ to the tube with PCR-mix-1 N. meningitidis/ IC;  add 10 µl of Pos IC C+ to the tube with PCR-mix-1 N. meningitidis/ IC; Amplification Create a temperature profile on your Real-time instrument as follows: Rotor type instruments1 Plate type or modular instruments2 Stage Тemp, °С Time Fluorescence detection Cycle repeats Тemp, °С Time Fluorescence detection Cycle repeats Hold 95 15 min – 1 95 15 min – 1 Cycling 95 10 s – 45 95 10 s – 56 20 s 45 FAM(Green), JOE(Yellow) 56 30 s FAM, JOE/HEX/Cy3 72 10 s – 72 10 s – 1 For example Rotor-Gene™ 3000/6000/Q (Corbett Research, Qiagen) 2 For example, SaCycler-96™ (Sacace), CFX/iQ5™ (BioRad); Mx3005P™ (Agilent), ABI® 7300/7500/StepOne Real Time PCR (Applied Biosystems), SmartCycler® (Cepheid), LineGeneK® (Bioer) Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 INSTRUMENT SETTINGS Rotor-type instruments Channel Calibrate/Gain Optimisation… Threshold More Settings/ Outlier Removal Slope Correct FAM/Green from 5 Fl to 10 Fl 0.05 10 % On JOE/Yellow from 4 Fl to 8 Fl 0.05 5-10 % On Plate-type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline; otherwise, the threshold level should be raised. Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples. RESULTS ANALYSIS The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line. Put the threshold line at such level where curves of fluorescence are linear.  Streptococcus pneumoniae is detected on the FAM (Green) channel and Haemophilus influenzae on the JOE (Yellow)/HEX/Cy3 channel with PCR-mix-1 S. pneumoniae/ H. influenzae;  Internal Control (IC) is detected on the FAM (Green) channel and Neisseria meningitidis on the JOE (Yellow)/HEX/Cy3 channel with PCR-mix-1 N. meningitidis/ IC; The sample is considered to be positive if the value of Ct is different from zero (Ct < Boundary value) The sample is considered to be negative if the result is positive only on the channel Fam with PCR-mix-1 N. meningitidis/ IC and the Ct value is lower than boundary value. Ct boundary values Sample Channel Boundary Ct (Rotor-type) Boundary Ct (Plate-type) CS+ FAM/Green 38 40 C+N.meningitidis JOE/Yellow 38 40 C+S.pneumoniae FAM/Green 38 40 C+H.influenzae JOE/Yellow 38 40 C- FAM/Green 38 40 Clinical samples FAM/Green 38 40 JOE/Yellow 38 40 Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 PERFORMANCE Sensitivity Clinical material DNA extraction kit PCR kit Pathogen Analytical sensitivity, GE/ml* Cerebrospinal fluid DNA/RNAPrep NHS Meningitidis Real-TM Neisseria meningitidis 1х10 Haemophilus 3 influenzae Streptococcus pneumoniae * Genome equivalents (GE) of the microorganism per 1 ml of a clinical sample. Specificity The analytical specificity of NHS Meningitidis Real-TM PCR kit is ensured by selection of specific primers and probes as well as strict reaction conditions. The primers and probes were checked for possible homologies to all sequences deposited in gene banks by sequence comparison analysis. Specificity was evaluated by testing the following microorganism sand strains: Enterobacter aerogenes and E. cloacae; Enterococcus faecalis (GISK 29212); Escherichia coli (NCTC 9001) and E. coli (ATCC 25922); Haemophilus parainfluenzae and H. haemolyticus; Klebsiella oxytoca and K. pneumoniaе; Listeria monocytogenes; Moraxella catarrhalis; Neisseria cinereae, N. elongate, N. flavescens, N. gonorrhoeae, N. mucosa; N. sicca and N. subflava; Pantoea agglomerans; Proteus mirabilis; Pseudomonas aeruginosa (ATCC 27853); Salmonella enteritidis (GISK 1137) and S. typhi (Central Public Health Laboratory (London) 5715); Shigella flexneri 2a (GISK 1270) and S. sonnei (GISK 9090); Staphylococcus aureus (ATCC 25923) and S. saprophyticus (ATCC 15305), S. pneumoniae, S. agalactiae, S. milleri, S. mitis, S. mutans, S. pyogenes, S. salivarius, S. sanguis, S. suis and S. viridians; and Yersinia enterocolitica and Y. pseudotuberculosis. The analytical specificity was also confirmed by testing human DNA. Nonspecific results were not detected. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 TROUBLESHOOTING 1. Weak or absent signal of the IC (Fam (Green) channel) with PCR-mix-1 N. meningitidis/ IC: retesting of the sample is required.  The PCR was inhibited.  Make sure that you use a recommended DNA extraction method and follow the manufacturer’s instructions.  Re-centrifuge all the tubes before pipetting the extracted DNA for 2 min at maximum speed (12000-16000 g) and take carefully supernatant. Don’t disturb the pellet, sorbent inhibit reaction.  The reagents storage conditions didn’t comply with the instructions.  Check the storage conditions  The PCR conditions didn’t comply with the instructions.  Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol.  The IC was not added to the sample during the pipetting of reagents.  Make attention during the DNA extraction procedure. 2. Weak (Ct > boundary value) sample signal on the Fam/Joe (Yellow)/Cy3/HEX channel: retesting of the sample is required. 3. Joe (Yellow)/Cy3/HEX signal with Negative Control of extraction.  Contamination during DNA extraction procedure. All sample results are invalid.  Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol.  Use only filter tips during the extraction procedure. Change tips among tubes.  Repeat the RNA extraction with the new set of reagents. 4. Any signal with Negative PCR Control.  Contamination during PCR preparation procedure. All sample results are invalid.  Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents.  Pipette the Positive controls at the end.  Repeat the PCR preparation with the new set of reagents. Sacace™ NHS Meningitidis Real-TM VER 01.06.2021 KEY TO SYMBOLS USED * SaCycler™ is a registered trademark of Sacace Biotechnologies * CFX™ and iQ5™ are registered trademarks of Bio-Rad Laboratories * Rotor-Gene™ is a registered trademark of Qiagen * MX3005P® is a registered trademark of Agilent Technologies * ABI® is a registered trademark of Applied Biosystems * LineGeneK® is a registered trademark of Bioer * SmartCycler® is a registered trademark of Cepheid Sacace Biotechnologies Srl via Scalabrini, 44 – 22100 – Como – Italy Tel +390314892927 Fax +390314892926 mail: info@sacace.com web: www.sacace.com List Number Caution! Lot Number Contains sufficient for tests For in Vitro Diagnostic Use Version Store at NCA Negative Control of Amplification Manufacturer NCE Negative control of Extraction Consult instructions for use C+ Positive Control of Amplification Expiration Date IC Internal Control