Influenza A/B Real-TM,sacace -100t

Sacace™ Influenza A B Real-TM VER 25/11/2021 IVD For in Vitro Diagnostic Use Influenza A B Real-TM Handbook Real Time Amplification test for the detection of Influenza A and B Viruses REF V36-100FRT 100 Sacace™ Influenza A B Real-TM VER 25/11/2021 NAME Influenza Virus A B Real-TM INTRODUCTION Influenza virus infection, one of the most common infectious disease, is a highly contagious airborne disease that causes an acute febrile illness and results in variable degrees of systemic symptoms, ranging from mild fatigue to respiratory failure and death. These symptoms contribute to significant loss of workdays, human suffering, mortality, and significant morbidity. Influenza results from infection with 1 of 3 basic types of influenza virus – A, B, or C – which are classified within the family Orthomyxoviridae. These single stranded RNA viruses are structurally and biologically similar but vary antigenically. The most common prevailing influenza A subtypes that infect humans are H1N1 and H3N2. INTENDED USE Influenza Virus A B Real-TM is Real-Time amplification test for the qualitative detection of Influenza A and B RNA in clinical specimens. PRINCIPLE OF ASSAY Influenza Virus A B Real-TM Test is based on three major processes: isolation of virus RNA from specimens, reverse transcription of the RNA, Real Time amplification of the cDNA. Influenza virus A&B detection by the polymerase chain reaction (PCR) is 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. Influenza Virus A B 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™ Influenza A B Real-TM VER 25/11/2021 MATERIALS PROVIDED “Reverta-L ”: Reverse transcription of the RNA  RT-G-mix-1, 10 x 0,01 ml;  RT-mix, 10 x 0,125 ml;  Reverse transcriptase (M-MLV), 2 x 0,03 ml;  TE-buffer, 2 x 1,2 ml. Contains reagents for 120 tests. “Influenza A&B ”: Real Time amplification kit  PCR-mix-1, 5 x 0,2 ml;  PCR-mix-2-FRT, 0,6 ml;  TaqF Polymerase, 0,06 ml;  Pos cDNA Infl. A / Infl. B / IC, 0,2 ml;  Negative Control, 2 x 1,2 ml*;  Internal Control RNA, 10 x 0,12 ml**;  DNA buffer, 0,5 ml; Contains reagents for 110 tests. * must be used in the isolation procedure as Negative Control of Extraction. ** add 10 µl of Internal Control RNA during the RNA purification procedure directly to the sample/lysis mixture MATERIALS REQUIRED BUT NOT PROVIDED  Real Time Thermalcycler  Workstation  Pipettors (capacity 0,5-10 µl; 5-40 µl) with aerosol barrier  Tube racks STORAGE INSTRUCTIONS Influenza Virus A B Real-TM must be stored at 2-8°C excepting Reverta, PCR-mix-1, PCRmix-2-FRT and TaqF Polymerase that must be stored at temperature ≤ -16°C.The kit can be shipped at 2-8°C for 3-4 days but should be stored at 2-8°C and ≤ -16°C immediately on receipt. STABILITY Influenza Virus A B Real-TM 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. 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™ Influenza A B Real-TM VER 25/11/2021 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following:  Clinical specimens from suspect influenza A (H1N1) cases should be performed in a BSL2 laboratory with BSL3 practices (enhanced BSL2 conditions). 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. Some components of this kit contain sodium azide as a preservative. Do not use metal tubing for reagent transfer. Sacace™ Influenza A B Real-TM VER 25/11/2021 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 Influenza Virus A B Real-TM can analyze RNA from:  nasopharyngeal swabs: swab area and place in “Eppendorf” tube with 0,5 ml of saline water or PBS sterile (Sacace Transport medium is recommended). Agitate vigorously. Repeat the swab and agitate in the same tube. Centrifuge at 1000g/min for 5 min. Discard the supernatant and leave about 100 µl of solution for RNA extraction.  aspirate, bronchial lavage, nasal wash: centrifuge at 2000 g/min for 10-15 min. If the pellet is not visible add 10 ml of liquid and repeat centrifugation. Remove and discard the supernatant. Resuspend the pellet in 100 µl of Saline water.  tissue: 1,0 gr (parenchimatous organs, trachea, lung, brain) homogenized with mechanical homogenizer or scalpel, glass sticks, teflon pestles and dissolved in 1,0 ml of saline water or PBS sterile. Vortex vigorously and incubate 30 min at room temperature. Transfer the supernatant into a new 1,5 ml tube; 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. RNA 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:  Ribo-Sorb- (Sacace, REF K-2-1);  Ribo-Virus spin column extraction kit (Sacace, REF K-2-C);  SaMag Viral Nucleic Acids Extraction kit (Sacace, REF SM003); Please carry out the RNA 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™ Influenza A B Real-TM VER 25/11/2021 RT AND AMPLIFICATION Reverse Transcription: 1. Prepare Reaction Mix: for 12 reactions, add 5,0 µl RT-G-mix-1 into the tube containing RTmix and vortex for at least 5-10 seconds, centrifuge briefly. This mix is stable for 1 month at -20°C. Add 6 µl M-MLV into the tube with Reagent Mix, mix by pipetting, vortex for 3 sec, centrifuge for 5-7 sec (must be used immediately after the preparation). (If it is necessary to test less than 12 samples add for each sample (N) in the new sterile tube 10*N µl of RT-G-mix-1 with RT-mix and 0,5*N µl of M-MLV). 2. Add 10 µl of Reaction Mix into each sample tube. 3. Pipette 10 µl RNA samples to the appropriate tube. (If the Ribo-Sorb isolation kit is used as a RNA extraction kit, re-centrifuge all the tubes with extracted RNA for 2 min at maximum speed (12000-16000 g) and take carefully supernatant. N.B. don’t disturb the pellet, sorbent inhibit reaction).Carefully mix by pipetting. 4. Place tubes into thermalcycler and incubate at 37oС for 30 minutes. 5. Dilute 1: 2 each obtained cDNA sample with TE-buffer (add 20 µl TE-buffer to each tube). cDNA specimens could be stored at -20oС for a week or at -70oС during a year. Real Time amplification: Reaction Mix 25 µl 1. Prepare required quantity of tubes or PCR plate. 2. Prepare for each sample in the new sterile tube 10*N µl of PCR-mix-1, 5*N µl of PCR-mix2-FRT and 0,5*N of TaqF Polymerase. 3. Add 15 µl of Reaction Mix into each tube. 4. Add 10 µl of cDNA sample to appropriate tube with Reaction Mix. 5. Prepare for each panel 2 controls:  add 10 µl of DNA-buffer to the tube labeled Amplification Negative Control;  add 10 µl of Pos cDNA Infl. A / Infl. B / IC to the tube labeled Cpos; Sacace™ Influenza A B Real-TM VER 25/11/2021 Amplification 1. Create a temperature profile on your instrument as follows: Rotor type instruments1 Plate type instruments2 Step Temperature, °С Time Cycles Temperature, °С Time Cycles 1 95 15 min 1 95 15 min 1 2 95 10 s 10 95 10 s 54 20 s 54 25 s 10 72 10 s 72 25 s 3 95 10 s 35 95 10 s 54 35 20 s Fluorescence detection 54 25 s Fluorescence detection 72 10 s 72 25 s 1 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), LineGeneK® (Bioer); Modular type instruments Step Temperature, °С Time Cycles Hold 95 900 s (variant FRT-100 F) 1 Stage1 95 15 s 54 42 25 s Fluorescence acquiring 72 25 s For example, SmartCycler® (Cepheid) Influenza Virus A is detected on the Rox (Orange) channel, Influenza Virus B is detected on the JOE (Yellow) channel, IC on the FAM (Green) channel. INSTRUMENT SETTINGS Rotor-type instruments Channel Calibrate/Gain Optimisation… Threshold More Settings/ Outlier Removal Slope Correct FAM/Green from 5 Fl to 10 Fl 0.1 0 % off JOE/Yellow from 5 Fl to 10 Fl 0.1 5 % off Rox/Orange from 5 Fl to 10 Fl 0.1 5 % off Plate- or modular 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. Sacace™ Influenza A B Real-TM VER 25/11/2021 RESULTS ANALYSIS Analysis of results is performed by the software of the real-time PCR instrument used by measuring fluorescence signal accumulation in three channels:  The signal of the IC cDNA amplification product is detected in the channel for the FAM fluorophore.  The signal of the Influenza virus B cDNA amplification product is detected in the channel for the JOE fluorophore.  The signal of the Influenza virus A cDNA amplification product is detected in the channel for the ROX fluorophore. 1. The sample is considered to be positive for Influenza Virus A if in the channel Rox (Orange) the value of Ct is different from zero and is less than the boundary value specified in the table below. 2. The sample is considered to be positive for Influenza Virus B if in the channel Joe (Yellow) the value of Ct is different from zero and is less than the boundary value specified in the table below. 3. The sample is considered to be negative for Influenza A/B if Ct value is not determined (absent) in ROX and JOE channels, whereas the Ct value is determined in FAM channel and is less than the value specified in the table below. Boundary values for rotor type intruments: Sample Stage for control Ct channel Fam (Green) Ct channel Joe (Yellow) Ct channel Rox (Orange) Result NCE RNA isolation < 28 Neg Neg Valid result NCA PCR Neg Neg Neg Valid result Pos cDNA Infl. A / Infl. B / IC PCR < 25 < 25 < 25 Valid result Clinical samples RNA isolation < 28 < 33 < 33 Valid result Boundary values for plate type intruments: Sample Stage for control Ct channel Fam (Green) Ct channel Joe (Yellow) Ct channel Rox (Orange) Result NCE RNA isolation < 30 Neg Neg Valid result NCA PCR Neg Neg Neg Valid result Pos cDNA Infl. A / Infl. B / IC PCR < 28 < 28 < 28 Valid result Clinical samples RNA isolation < 30 < 33 < 33 Valid result Sacace™ Influenza A B Real-TM VER 25/11/2021 Boundary values for modular type intruments: Sample Stage for control Ct channel Fam (Green) Ct channel Joe (Yellow) Ct channel Rox (Orange) Result NCE RNA isolation < 36 Neg Neg Valid result NCA PCR Neg Neg Neg Valid result Pos cDNA Infl. A / Infl. B / IC PCR < 37 < 37 < 37 Valid result Clinical samples RNA isolation < 37 < 40 < 40 Valid result The result of the analysis is considered reliable only if the results obtained for Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct (see the table below). Results for controls: Control Stage for control Ct channel Fam (Green) Ct channel Joe (Yellow) Ct channel Rox (Orange) NCE RNA isolation < boundary value Neg Neg NCA PCR Neg Neg Neg Pos cDNA Infl. A / Infl. B / IC PCR < boundary value < boundary value < boundary value Target region Channel for fluorophore FAM JOE ROX DNA-target Internal Control cDNA Influenza virus B cDNA Influenza virus A cDNA Target gene Artificially synthesized sequence NS gene of Influenza virus B M1 gene of Influenza virus A Sacace™ Influenza A B Real-TM VER 25/11/2021 PERFORMANCE CHARACTERISTICS Sensitivity The kit Influenza A B Real-TM allows to detect Influenza A&B viruses in 100% of the tests with a sensitivity of not less than 1 x 103 GE/ml*. * Genome equivalents (GE) of the pathogen agent per 1 ml of a sample. Specificity The analytical specificity of Influenza A B Real-TM PCR kit is ensured by the selection of specific primers and probes as well as stringent reaction conditions. The primers and probes have been checked for possible homologies to all sequences published in gene banks by sequence comparison analysis. The PCR kit allows detection of cDNA fragments of Influenza virus A and Influenza virus B. The specific activity of the PCR kit was proven in detection of reference strains and isolates of epidemic Influenza viruses А/H1N1 and А/H3N2 extracted in the period from 1977 to 2011 from clinical samples, Influenza viruses A extracted from animals (H1N1, H2N2, Н2N3, H2N9, H3N2, H3N8, H4N6, H5N1, H5N3, H5N2, H5N3, H6N2, H7N1, Н8N4, Н9N2, H10N7, Н11N6, H12N5, Н13N2), Influenza viruses B lineages Yamagata and Victoria as well as A/California/07/2009 strain of pandemic Influenza virus А/H1N1pdm2009. The absence of nonspecific reactions of the kit components was shown for cDNA/DNA of other viral (Human Respiratory-Syncytial virus “Long” strain, Human Rhinoviruses (types 13, 15, 16, 17, 21, 26, and 29), Herpes viruses, Cytomegalovirus, Enteroviruses (types Echo9, Echo30) and bacterial (Streptococcus spp., Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydophila pneumonia, Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila) causative agents of acute respiratory disease; normal microflora of human nasal cavity and oropharynx; and human cDNA/DNA as well as analysis of clinical material containing nucleic acids of Respiratory Syncytial viruses (types A and B), Parainfluenza viruses (types 1- 4), Human Coronavirus OC43, E229, NL63, HKUI, Human Adenoviruses groups B, C, and E, Human Metapneumovirus, and Human Bocavirus. The clinical specificity of Influenza A B Real-TM PCR kit was confirmed by laboratory clinical trials. Sacace™ Influenza A B Real-TM VER 25/11/2021 TROUBLESHOOTING 1. Weak or absent signal of the IC (Fam (Green) channel): retesting of the sample is required.  The PCR was inhibited.  Make sure that you use a recommended RNA extraction method and follow the manufacturer’s instructions.  Re-centrifuge all the tubes before pipetting the extracted RNA 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 RNA extraction procedure. 2. Weak signal on the Joe (Yellow)/Cy3/HEX and Rox/TexasRed channels: retesting of the sample is required. 3. Joe (Yellow)/Cy3/HEX or Rox/TexasRed signal with Negative Control of extraction.  Contamination during RNA extraction procedure. All samples 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 samples 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™ Influenza A B Real-TM VER 25/11/2021 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