Rotavirus/Norovirus/Astrovirus Real-TM,sacace-50t

Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 IVD For in Vitro Diagnostic Use Rotavirus/Norovirus/Astrovirus Real-TM Handbook Real Time PCR kit for qualitative detection of Rotavirus A, Norovirus 2 genotype, Astrovirus REF V40-50FRT 50 Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 NAME Rotavirus/Norovirus/Astrovirus Real-TM INTRODUCTION Acute Intestinal Infection (A.I.I.) are one of the primary causes of hospitalization in infectious disease departments. In accordance with the data provided by the contemporary literature, the most often detectable and generally spread etiological agents of A.I.I. are bacterial microorganisms such as Shigella spp. and enteroinvasive E. coli (EIEC), Salmonella spp., thermophilic group of Campylobacter spp., enteropathogenic E.coli (EPEC) and enteroaggregative E. coli (EAEC) and viral agents such as group A rotaviruses, genotype 2 noroviruses, group F adenoviruses (type 40 and 41) and astroviruses. INTENDED USE Kit Rotavirus/Norovirus/Astrovirus Real-TM is a Real-Time test for the qualitative detection and differentiation of Rotavirus A, Norovirus 2 genotype, Astrovirus in the biological materials and in the environment. RNA is extracted from specimens, amplified using RT-amplification and detected using fluorescent reporter dye probes specific for Rotavirus/Norovirus/Astrovirus RNA and IC (Internal Control). PRINCIPLE OF ASSAY Rotavirus/Norovirus/Astrovirus Real-TM Test is based on three major processes: isolation of virus RNA from specimens, one-step reverse transcription of the RNA and Real Time amplification of the cDNA. Rotavirus/Norovirus/Astrovirus 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. Rotavirus/Norovirus/Astrovirus 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™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 MATERIALS PROVIDED “Rotavirus/Norovirus/Astrovirus Real-TM”: Real Time amplification kit  PCR-mix-1 Rotavirus / Astrovirus, 0,6 ml;  PCR-mix-1 Norovirus / IC 0,6 ml;  RT-PCR-mix-2, 2 x 0,3 ml;  Hot Start Taq Polymerase, 2 x 0,03 ml;  M-MLV Revertase, 2 x 0,015 ml;  RT-G-mix-2, 2 x 0,015 ml; Contains reagents for 55 reactions “Controls”  Negative Control C-, 1,6 ml;*  Internal Control (IC RNA), 5 x 0,12 ml.**  Pos cDNA Rotavirus/Astrovirus C+, 0,1 ml;  Pos cDNA Norovirus 2 / Internal Control (IC) C+, 0,1 ml;  DNA-buffer, 0,5 ml; * must be used during the sample preparation procedure: add 100 µl of C– (Negative Control) to labeled Cneg **add 10 µl of Internal Control to all samples during the RNA isolation 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 Rotavirus/Norovirus/Astrovirus Real-TM” must be stored at -20°C; Controls must be stored at 2-8°C. The kits can be shipped at 2-8°C for 3-4 days but should be stored at 2-8°C and -20°C immediately on receipt. STABILITY Rotavirus/Norovirus/Astrovirus 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. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 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. Some components of this kit contain sodium azide as a preservative. Do not use metal tubing for reagent transfer. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 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 Rotavirus/Norovirus/Astrovirus Real-TM can analyze RNA extracted from:  water: centrifuge 10-20 ml for 10 min at maximum speed. Discard the supernatant and leave about 100 µl of solution for DNA extraction;  whole blood collected in EDTA tubes;  feces:  Prepare 20% feces suspension by adding in 5 ml tube of 4ml of Saline Solution and 1,0 gr (approx. 1,0 ml) of feces. Vortex to get the homogeneous suspension and centrifuge for 5 min to 7000-12000g and using a micropipette with a plugged aerosol barrier tip transfer in a new sterile 1,5 ml tube 50 µl of the bacterial fraction (white-yellowish line between the sediment and the supernatant) 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:  Ribo-Sorb- (Sacace, REF K-2-1);  DNA/RNA Prep (Sacace, REF K-2-9). Please carry out the RNA extraction according to the manufacturer’s instructions. Add 10 μl of Internal Control during the RNA isolation procedure directly to the sample/lysis mixture. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 RT AND AMPLIFICATION Total reaction volume is 25 µl, the volume of RNA sample is 10 µl. 1 Prepare required quantity of reaction tubes (2 tubes for each sample + Controls) 2 Prepare the reaction mix for required number of samples. 3 For N reactions mix for each PCR-Mix-1 in a new tube: 10*(N+1) µl of RT-PCR-mix-1 Rotavirus / Astrovirus (or Norovirus / IC) 5.0*(N+1) µl of RT-PCR-mix-2 0.5*(N+1) µl of Polymerase 0.25*(N+1) µl of RT-G-mix-2 0.25*(N+1) µl of MMlv 4 Vortex the tube, then centrifuge shortly. Add 15 µl of prepared reaction mix into each appropriate tube. 5 Using tips with aerosol filter add 10 µl of RNA samples obtained at the stage of RNA isolation and mix carefully by pipetting. N.B. 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 6 Prepare for each panel 3 controls:  add 10 µl of DNA-buffer to the tube labeled Amplification Negative Control;  add 10 µl of Pos cDNA Rotavirus/Astrovirus to the tube with PCR-mix-1 Rotavirus / Astrovirus;  add 10 µl of Pos cDNA Norovirus 2 / IC C+ to the tube with PCR-mix- Norovirus / 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 50 30 min – 1 50 30 min – 1 Hold 95 15 min – 1 95 15 min – 1 Cycling 2 95 10 s – 45 95 10 s – 60 25 s 45 FAM(Green), JOE(Yellow) 60 35 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™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 INSTRUMENT SETTINGS Rotor-type instruments Channel Calibrate/Gain Optimisation… Threshold More Settings/ Outlier Removal Slope Correct FAM/Green from 5 Fl to 10 Fl 0.05 5-10 % On JOE/Yellow from 4 Fl to 8 Fl 0.05 10 % On Plate-type instruments For each channel set the threshold line at the level of 10-20 % of HEX maximum fluorescence obtained for the C+ in the last amplification cycle. Boundary Ct values All types of test material RT-PCR-mix-1-FEP/FRT Ct value FAM/Green JOE/Yellow/HEX RT-PCR-mix-1-FEP/FRT Rotavirus / Astrovirus 40 40 RT-PCR-mix-1-FEP/FRT Norovirus / STI 40 40 RESULTS ANALYSIS 1. The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line.  Internal Control (IC) is detected on the FAM (Green) channel and Norovirus on the JOE (Yellow)/HEX/Cy3 channel with PCR-mix- Norovirus / IC;  Rotavirus А is detected on the FAM (Green) channel and Astrovirus on the JOE (Yellow)/HEX/Cy3 channel with PCR-mix-1 Rotavirus / Astrovirus; 2. The sample is considered to be positive if the value of Ct is different from zero and lower than boundary value. 3. The sample is considered to be negative if the result is positive only on the channel Fam with PCR-mix- Norovirus / IC STI-87-rec and the Ct value is lower than boundary value. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 SPECIFICATIONS Sensitivity The analytical sensitivity of Rotavirus/Norovirus/Astrovirus Real-TM PCR kit is specified in the table below. Pathogen Test material RNA/DNA extraction kit Analytical sensitivity, GE/ml* Rotavirus A Feces DNA/RNA Prep 1 x 103 Norovirus genotype 2 Feces DNA/RNA Prep 5 x 102 Astrovirus Feces DNA/RNA Prep 1 x 103 * Genome equivalents (GE) of the microorganism per 1 ml of a sample. Specificity The analytical specificity of Rotavirus/Norovirus/Astrovirus 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 confirmed on the following microorganism strains: GISK collection: Enterovirus strains (Coxsakie B1, B2, B3, B4, B5, and B6; Polio (Sabin) I, II, and III). Adenovirus serogroups 5 and 7; influenza virus А (H13N2, H9N2, H8N4, H2N3, H4N6, H11N6, H12N5, H3N8, H1N1, H6N2, H10N7, and H5N1) and В; rhinovirus; RS viruses; and human adenovirus types 3, 5, 7, 37, and 40. VGNKI collection: Salmonella enteritidis S-6, S.choleraesuis 370, S.typhimurium 371, S.dublin 373, S.typhi C1, S.abortusovis 372, and S.gallinarum-pullorum;, Shigella flexneri 851b; Campylobacter fetus ssp. fetus 25936 and C.jejuni ssp. jejuni 43435; Clebsiella K 65 SW4; Listeria monocitogenes USKHCH 19 and L.monocitogenes USKHCH 52; Proteus vulgaris 115/98; Pseudomonas aeruginosa DN c1; Staphilococcus aureus 653 and S. aureus 29112; Morganella morganii 619 c 01; and Enterobacter faecalis 356. Center for Disease Control and Prevention (CDC, USA) collection: 44 isolates of norovirus genotype 1 and 2 different genetic clusters; 40 strains of different rotavirus [P]G types, 19 strains of astrovirus serotypes 1, 2, 4, 5, and 8; and 15 strains of different adenovirus types and the following bacterial strains (see table below). Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 Table. The panel of bacterial pathogens Center for Disease Control and Prevention (CDC, USA) Strain ID Organism Strain ID Organism K2033 Salmonella ser. Grumpensis K2015 Salmonella ser. Oranienburg K1806 Salmonella ser. Newport AM01144 Salmonella ser. Newport K2077 Salmonella ser. Enteriditis K1810 Salmonella ser. Anatum 83-99 Salmonella ser. Typhimurium K1991 Salmonella ser. Typhimurium PS505 Shigella boydii K1898 Salmonella ser. Heidelberg PS408 Shigella sonnei PS555 Shigella boydii B4003 Shigella sonnei F6446 Shigella dysenteriae PS801 Shigella dysenteriae S821X1 Shigella dysenteriae type 1 C898 Shigella dysenteriae type1 S177X1 Shigella dysenteriae type 1 F2035 Shigella flexneri S3314 Shigella dysenteriae type 2 E2539-C1 Enterotoxigenic Escherichia coli (ETEC) PS071 Shigella flexneri H10407 Enterotoxigenic Escherichia coli (ETEC) PS050 Shigella flexneri F1008 Enterotoxigenic Escherichia coli (ETEC) F7862 Shigella flexneri EDL 933 Shiga-toxin E. coli (STEC) TX1 Enterotoxigenic Escherichia coli (ETEC) 3543-01 Shiga-toxin E. coli (STEC) 3525-01 Shiga-toxin Escherichia coli (STEC) 4752-71 Proteus vulgaris 25922 Escherichia coli O6:H1 QA/QC Citrobacter freundii 621-64 Citrobacter freundii QA/QC Aeromonas 3910-68 Aeromonas spp. 3043-74 Serratia marcescens E9113 Vibrio cholerae QA/QC Serratia marcescens 501-83 Edwardsiella spp. F7894 Vibrio vulnificus 587-82 Providencia stuartii F8515 Yersinia enterocolitica 27853 Pseudomonas aeruginosa F8510 Yersinia enterocolitica D4989 Helicobacter cineadi K4299 Vibrio parahaemolyticus D6827 Helicobacter pullorum F9835 Vibrio parahaemolyticus D5127 Helicobacter pylori K2023 Salmonella ser. Kentucky D2686 Arcobacter butzleri K1684 Salmonella O-1, 4, 12 gr. B There were no nonspecific test responses during examination of human DNA as well as a DNA panel of the above-mentioned microorganisms. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 TROUBLESHOOTING Results of analysis are not taken into account in the following cases:  If the signal for C– (except for C– in JOE/Yellow/HEX channel for RT-PCR-mix-1-FEP/FRT Norovirus / STI) and/or the signal for NCA in the JOE/Yellow/HEX and/or FAM/Green channel is less than the boundary value, analysis should be repeated starting from the RNA extraction stage.  If no signal is detected for the positive controls of amplification, it may suggest that the programming of the temperature profile of the used Instrument was incorrect, or that the configuration of the PCR reaction was incorrect, or that the storage conditions for kit components did not comply with the manufacturer’s instruction, or that the reagent kit expired. Programming of the used instrument, storage conditions, and the expiration date of the reagents should be checked, and then PCR should be repeated.  If a positive result (the fluorescence curve crosses the threshold line) is detected for a sample that has a fluorescence curve without the typical exponential growth phase (the curve is linear), this may suggest incorrect setting of the threshold line or incorrect calculation of baseline parameters. Such a result should not be considered as positive. Sacace™ Rotavirus/Astrovirus/Norovirus Real-TM VER 02.02.2022 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