HHV 6 Quant Real-TM (NEW) ,sacace -100t

Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 IVD For in Vitro Diagnostic Use HHV6 Real-TM Quant Handbook Real Time PCR kit for quantitative detection of Human Herpes Virus 6 REF V10-100FRT 100 Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 NAME HHV6 Real-TM Quant INTENDED USE kit HHV6 Real-TM Quant is an in vitro Real Time amplification test for quantitative detection of Human Herpes Virus 6 in the biological materials. DNA is extracted from samples, amplified using real time amplification with fluorescent reporter dye probes specific for pol-gene of HHV6 and Internal Control (IC). Test contains an IC (-globine gene) which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition. PRINCIPLE OF ASSAY kit HHV6 Real-TM Quant is based on two major processes: isolation of DNA from specimens and Real Time amplification. Amplification results of HHV6 DNA are detected on the Joe/HEX/Yellow and -globine gene used as Internal Control is detected on the Fam/Green channel. If the sample is not correctly prepared or well stored (insufficient quantity < 2,0 x 104 genomes/sample) the Internal Control will not be detected or it comes very low. Kit contains quantitative standards for simultaneous detection of DNA HHV6 and -globine gene in one tube which allows to avoid labouring procedure of leukocytes extraction from blood and their calculation. Calculate the concentration of HHV6 DNA in standard quantity of cells, for example in 106 human cells using the following formula: Copies HHV6 DNA in 106 cells* = copies HHV6 DNA in reaction/copies human DNA in reaction x 2*106 *106 cells contains 2*106 human genomes Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 MATERIALS PROVIDED “HHV6 Real-TM Quant”: Real Time amplification  PCR-mix-1 “HHV6/Glob”, 2 x 0,6 ml  PCR- buffer-FRT, 2 x 0,3 ml  TaqF Polymerase, 2 x 0,03 ml  Negative Control, 1,2 ml;*  Pos HHV6 & Human DNA C+, 2 x 0,2 ml;**  DNA-buffer (C-), 0,5 ml  Quantitation Standard HHV6 & Glob: o QS1, (concentration 104 copies/sample) 0,2 ml, o QS2, (concentration 102 copies/sample) 0,2 ml, Contains reagents for 120 test. * must be used during the sample preparation procedure as Negative Control: add 100 µl of C– (Negative Control) to the tube labeled Cneg; **add 90 µl of C– (Negative Control) and 10 µl of Pos HHV6 & Human DNA C+ to the tube labeled Cpos MATERIALS REQUIRED BUT NOT PROVIDED  Real Time Thermal cycler  Reaction tubes  Workstation  Pipettes (adjustable)  Sterile pipette tips with filters  Vortex mixer  Freezer, refrigerator STORAGE INSTRUCTIONS HHV6 Real-TM Quant must be stored at – 20°C. The kits can be shipped at 2-8°C but should be stored at 2-8°C and -20°C immediately on receipt. STABILITY HHV6 Real-TM Quant 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. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 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. WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following:  Use sterile pipette tips with aerosol barriers and use new tip for every procedure.  Store extracted positive material (samples, controls and amplicons) away from all other reagents and add it to the reaction mix in a separate area.  Thaw all components thoroughly at room temperature before starting an assay.  When thawed, mix the components and centrifuge briefly.  Use disposable gloves, laboratory coats and eye protection when handling specimens and reagents. Thoroughly wash hands afterwards.  Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas.  Do not use a kit after its expiration date.  Dispose of all specimens and unused reagents in accordance with local authorities’ regulations.  Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices.  Clean and disinfect all sample or reagent spills using a disinfectant such as 0.5% sodium hypochlorite, or other suitable disinfectant.  Avoid sample or reagent contact with the skin, eyes, and mucous membranes. If skin, eyes, or mucous membranes come into contact, rinse immediately with water and seek medical advice immediately.  Material Safety Data Sheets (MSDS) are available on request.  Use of this product should be limited to personnel trained in the techniques of DNA amplification.  The laboratory process must be one-directional, it should begin in the Extraction Area and then move to the Amplification and Detection Areas. Do not return samples, equipment and reagents to the area in which the previous step was performed. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 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 HHV6 Real-TM Quant can analyze DNA extracted from:  whole blood collected in either ACD or EDTA tubes (necessary pre-treatment with hemolytic reagent to lyse the erythrocytes);  buffy coat;  sputum: add 1 volume of sputum to 1 volumes of saline water and vortex vigorously. Centrifuge at 10000g/min for 10 min. Discard the supernatant and leave about 100 µl of solution for DNA extraction.  swabs: insert the swab into the nuclease-free 1,5 ml tube and add 0,2 ml of Transport medium. Vigorously agitate swabs in medium for 15-20 sec.  Saliva  Cerebrospinal fluid (liquor)  Viscera biopsy material It is recommended to process samples immediately after collection. Store samples at 2–8 °C for no longer than 24 hours, or freeze at –20/80°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)  Ribo Virus 50– spin column extraction kit (Sacace, REF K-2-C)  SaMag Viral Nucleic Acids Extraction Kit (Sacace, REF SM003) for cerebrospinal fluid and cell free body fluids;  SaMag STD DNA Extraction Kit (Sacace, REF SM007) for swabs. Please carry out the DNA extraction according to the manufacturer’s instructions. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 PROTOCOL: Reaction volume = 25 µl 1. Prepare required quantity of tubes (N + 6 controls (4 standards, 1 Neg. control, 1 Pos. Control). 2. Prepare in the new sterile tube for each sample 10*N µl of PCR-mix-1 “HHV6/Glob”, 5,0*N of PCR-Buffer-FRT and 0,5*N µl of TaqF Polymerase. Vortex and centrifuge for 2-3 sec. 3. Add 15 µl of Reaction Mix and 10 µl of extracted DNA sample to appropriate tube. Mix by pipetting. 4. Prepare for qualitative run 1 positive control and 1 negative control:  add 10 µl of QS2 to the tube labeled Cpos;  add 10 µl of DNA-buffer to the tube labeled Cneg; 5. For quantitative analysis prepare 1 negative control (add 10 µl of DNA-buffer to the tube labeled Cneg) and 4 tubes for standards. Perform QS1 and QS2 standards twice by adding 10 µl of Quantitation Standards HHV6 & Glob (QS1, QS2) into 4 labelled tubes; Amplification 1. Close tubes and transfer them into the Real Time ThermalCycler. 2. Program position of the samples, controls and standards. 3. Program the instruments as follows: Rotor-type Instruments1 Plate- or modular type Instruments2 Step Тemperature, °С Time Repeats Тemperature, °С Time Repeats 1 95 15 min 1 95 15 min 1 2 95 5 s 5 95 5 s 60 20 s 60 20 s 5 72 15 s 72 15 s 3 95 5 s 40 95 5 s 60 40 20 s fluorescent signal detection 60 30 s fluorescent signal detection 72 15 s 72 15 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) Fluorescence is detected at the 2nd step of Cycling 2 stage (60 °C) in FAM/Green and JOE/Yellow/Hex/Cy3 fluorescence channels. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 INSTRUMENT SETTINGS Rotor-type instruments Channel Calibrate/Gain Optimisation… Threshold More Settings/ Outlier Removal Slope Correct FAM/Green from 5 Fl to 10 Fl 0.03 10 % On JOE/Yellow from 4 Fl to 8 Fl 0.03 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. RESULT ANALYSIS: The results are interpreted with the software of instrument through the presence of crossing of fluorescence curve with the threshold line. Internal Control (Human DNA) is detected on the FAM/Green channel and HHV6 on the Joe/HEX/Yellow channel . Calculate the concentration of copies HHV6 DNA in 106 cells using the following formula: In quantitative analysis, if total DNA is isolated from whole human blood, white blood cells, or viscera biopsy material, the logarithmic concentration of HHV6 DNA copies per the standard cell quantity (105) in samples is calculated using the following formula: If total DNA is isolated from saliva, oropharyngeal swabs, and cerebrospinal fluid (liquor), the concentration of HHV6 DNA per ml of sample (Copies HHV6 DNA) is calculated using the following formula: Copies HHV6 DNA = Calc HHV6 DNA х 100 (copies/ml), where Calc HHV6 DNA is the number of HHV6 DNA copies/reaction calculated in Joe/HEX/Yellow channel. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 The calculation can be made manually or using Microsoft® Excel program: Name Type Copies HHV6 (Joe/Yellow)/ reaction Copies Glob (Fam/Green)/ reaction lg HHV-6/10*5 cells copies HHV6/10*6 cells A B C D E=LOG(C/D*200000) F=C/D*2000000 1 Unknown 2907 56303 4.0 1.03E+05 1 Unknown 2958 52432 4.1 1.13E+05 2 Unknown 306 57845 3.0 1.06E+04 2 Unknown 309 52206 3.1 1.18E+04 3 Unknown 37 66964 2.0 1.11E+03 3 Unknown 39 69013 2.1 1.13E+03 C- Unknown C- Unknown C+ Unknown 296 9519 3.8 6.22E+04 C+ Unknown 269 9455 3.8 5.69E+04 QS1 Standard 9968 9894 QS1 Standard 10024 10106 QS2 Standard 97 105 QS2 Standard 102 98 The difference between 2 values of “Pos HHV6 & Human DNA C+” in one run doesn’t have to be higher than 25%. PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples. They did not generate any signal with the specific Human Herpes Virus 6 primers and probes. The specificity of the kit HHV6 Real-TM Quant was 100%. The potential cross-reactivity of the kit HHV6 Real-TM Quant was tested against the group control. It was not observed any cross-reactivity with other pathogens. Analytical sensitivity The kit HHV6 Real-TM Quant allows to detect Human Herpes Virus 6 DNA in 100% of the tests with a sensitivity of not less than 200 copies/ml or 5 copies of HHV6 DNA per 105 cells. The linear range of HHV6 Real – TM Quant PCR kit is 500-10.000.000 HHV6 DNA copies/ml. If the result is less than 500 copies/ml, the results should be indicated as “less than 500 HHV6 copies/ml”. Target region: polymerase gene HHV6 Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 TROUBLESHOOTING Results of analysis are not taken into account in the following cases: 1. The appearance in the results grid of a Ct value in JOE/Yellow/HEX and FAM/Green channels for the negative control of amplification (NCA) and a Ct value in the JOE/Yellow/HEX channel for the negative control of extraction (C–) indicates contamination of reagents or samples. Repeat PCR analysis of all samples in which HHV6 DNA was detected starting from the DNA extraction stage. 2. In qualitative analysis, if the Ct value for the positive control of amplification (C+, QS2) in the JOE/Yellow/HEX (HHV6) or FAM/Green channel is absent in the results grid, repeat amplification of all samples in which HHV6 DNA was not detected. 3. If the Ct value for the positive control of extraction (PCE, Positive Control DNA HHV-6 and human DNA) in JOE (HHV6) or FAM channel is absent, the results of analysis of all samples are considered invalid. Repeat analysis of all samples starting from the DNA amplification stage. 4. If the Ct value for the sample in the JOE/Yellow/HEX channel is absent repeat analysis of the sample starting from the DNA extraction stage. This may be caused by errors in preparation of clinical material, which entailed the loss of DNA, or by the presence of inhibitors. 5. If the Ct value for a clinical sample in the JOE/Yellow/HEX channel (HHV6 DNA) exceeds the boundary Ct value (>36), the result of analysis of such samples is considered equivocal. Repeat analysis of such samples in duplicate. If a positive result is obtained in both replicates, the result of analysis is considered as positive. If the results in two replicates are discrepant, the result of analysis of such samples is considered equivocal. 6. If the number of copies per reaction in DNA calibrators in quantitative tests exceeds the specified value by more than 30%, check the order of placing the tubes in the rotor (DNA calibrators should be inserted into the cells named “Standard” in the table of samples and cell no. 1 in rotor-type instruments should not be empty (fill it with any test tube). 7. If the correlation coefficient R in the Standard Curve window in quantitative tests is less than 0.9, this indicates error in calibration. Check whether the settings for DNA calibrators are correct and change them, if necessary. If this does not help, run PCR for all samples and calibrators. Sacace™ HHV6 Real-TM Quant ver. 05.05.2022 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