HSV I/II Typing Real-TM ,sacace-100t

Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 IVD For in Vitro Diagnostic Use HSV I/II Typing Real-TM Handbook Real Time PCR kit for qualitative detection and differentiation of Herpes Viruses I and II REF V38-100FRT 100 Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 NAME HSV I/II Typing Real-TM INTRODUCTION Herpes simplex virus-1 and -2 belong to the Alpha-herpesvirinae of the family Herpesviridae. Their genomes consist of double-stranded DNAs. HSV is one of the most widespread viruses with a prevalence of 95 % and a presence of HSV-2 in 30 – 50 % of human population. HSV-1, the so called oral strain, causes herpes labialis, herpetic stomatitis and keratitis. HSV-2 is called genital strain and causes predominantly genital herpes. In terms of localization of herpes lesions, two forms of herpes are distinguished, the labial herpes with herpes lesions on the upper part of the body (lips, face, mucous membranes of the mouth); and the genital herpes with herpes lesions on the lower part of the body (mucous membranes of the genitals, buttocks, legs). It was believed in the past that only HSV-I could induce labial herpes and only HSV-II could induce genital herpes. The present data disprove this point of view demonstrating tropism common for both types of the virus. The method of polymerase chain reaction (PCR) in the HSV diagnosis is highly sensitive, specific and less laborious and it has become very important in the laboratory diagnostics of HSV. INTENDED USE kit HSV I/II Typing Real-TM is a test for the qualitative detection and differentiation of Herpes Simplex Virus type I and Herpes Simplex Virus type II DNA in the whole blood, plasma, liquor, urogenital swabs, urine, prostatic liquid and other biological samples. PRINCIPLE OF ASSAY kit HSV I/II Typing Real-TM is based on two major processes: isolation of DNA from specimens and Real Time amplification. Herpes Simplex Virus DNA is extracted from the specimens, amplified using Real-Time amplification and detected fluorescent reporter dye probes specific for HSV DNA I, II and Internal Control. Internal Control (IC) serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition. IC is detected in a channel other than the HSV. Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 MATERIALS PROVIDED  PCR-mix-1-FRT, 1,2 ml;  PCR-mix-2, 2 x 0,3 ml;  TaqF Polymerase, 2 x 0,03 ml;  Pos C+, 0,2 ml;  Negative Control C-, 1,2ml;*  Internal Control IC, 1,0 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 during the DNA isolation directly to the sample/lysis mixture (see DNA-Sorb-B REF K-1-1/B protocol). 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 HSV I/II Typing Real-TM must be stored at 2-8°C except for PCR-mix-2 and TaqF Polymerase that must be stored at -16°C. The kits 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 HSV I/II Typing 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. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. 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™ HSV I/II Typing Real-TM VER. 20.10.2022 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. 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. Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 SAMPLE COLLECTION, STORAGE AND TRANSPORT HSV I/II Typing Real-TM can analyze DNA extracted with DNA-Sorb-B (REF K-1-1/B) from:  whole blood collected in either ACD or EDTA tubes;  plasma collected in either ACD or EDTA tubes;  liquor stored in “Eppendorf” tube;  tissue: 1,0 gr 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;  prostatic liquid stored in “Eppendorf” tube;  seminal liquid: transfer about 30 µl of seminal liquid to a polypropylene tube (1,5 ml) and add 70 µl of sterile saline solution;  cervical, urethral, conjunctival 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. Specimens can be stored at +2-8°C for no longer than 48 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. * recommended kit for DNA extraction: DNA-Sorb-A (REF K-1-1/A) 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) for whole blood, liquor, tissue, etc;  DNA-Sorb-A (Sacace, REF K-1-1/A) for swabs;  SaMag Viral Nucleic Acids Extraction Kit (Sacace, REF SM003) for plasma, liquor;  SaMag STD DNA Extraction Kit (Sacace, REF SM007) for seminal liquid, swabs. Please carry out DNA extraction according to the manufacture’s instruction. Add 10 µl of Internal Control during DNA isolation procedure directly to the sample/lysis mixture. (Note: the Sacace Internal Control is the same for all urogenital infection Real Time kits) Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 PROTOCOL: 1. Prepare required quantity of reaction tubes for samples (N) and controls (N+2). 2. Prepare in the new sterile tube for each sample 10*(N+1) µl of PCR-mix-1-FRT, 5,0*(N+1) of PCR-mix-2 and 0,5*(N+1) µl of TaqF Polymerase. Vortex and centrifuge for 2-3 sec. 3. Add to each tube 15 µl of Reaction Mix and 10 µl of extracted DNA sample to appropriate tube. Mix by pipetting. 4. Prepare for each panel 2 controls: ● add 10 µl of DNA-buffer to the tube labeled Amplification Negative Control; ● add 10 µl of Positive Control C+ to the tube labeled Amplification Positive Control; 5. Insert the tubes in the thermalcycler. Amplification 1. Create a temperature profile on your instrument 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, JOE/Yellow/Hex/Cy3 and Rox(Orange)/TexasRed fluorescence channels. HSV II is detected on the FAM (Green) channel, HSV I is detected on the JOE(Yellow)/HEX/Cy3 channel, IC DNA on the Rox(Orange)/TexasRed 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 4 Fl to 8 Fl 0.1 5 % Off Rox/Orange from 4 Fl to 8 Fl 0.1 5 % Off Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 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. Boundary value of the cycle threshold, Ct Sample Channel Ct for rotor-type instrument Ct for plate-type instrument C+ FAM 33 36 JOE 30 33 ROX 33 36 Samples, C– ROX 33 36 DATA ANALYSIS The fluorescent signal intensity is detected in two channels:  The signal from the HSV II DNA amplification product is detected in the FAM/Green channel;  The signal from the HSV I DNA amplification product is detected in the JOE/Yellow/HEX/Cy3 channel;  The signal from the Internal Control amplification product is detected in the Rox/Orange/TexasRed channel. Interpretation of results The results are interpreted by the software of the instrument by the crossing (or not crossing) of the fluorescence curve with the threshold line. Principle of interpretation:  HSV II DNA is detected in a sample if its Ct value is present in the FAM channel. The fluorescence curve should cross the threshold line in the area of exponential fluorescence growth.  HSV I DNA is detected in a sample if its Ct value is present in the Joe channel. The fluorescence curve should cross the threshold line in the area of exponential fluorescence growth  HSV I/II DNA is not detected in a sample if its Ct value is absent in the FAM/Joe channels (fluorescence curve does not cross the threshold line) while the Ct value in the Rox channel is less than 33.  The result is invalid if the Ct value of a sample in the FAM/Joe channels is absent while the Ct value in the Rox channel is either absent or greater than the specified boundary value (Ct > 36). It is necessary to repeat the PCR analysis of such samples. Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 The result of 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. Control Stage for control Ct channel Fam Ct channel Joe Ct channel Rox Interpretation NCE DNA isolation NEG NEG POS Valid result NCA Amplification NEG NEG NEG Valid result C+ Amplification POS POS POS Valid result QUALITY CONTROL PROCEDURE A defined quantity of Internal Control (IC) is introduced into each sample and control at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition. A negative control of extraction (NCE), negative amplification control (NCA), positive amplification control (C+) are required for every run to verify that the specimen preparation, the amplification and the detection steps are performed correctly. If the controls are out of their expected range (see table Results for Controls), all of the specimens and controls from that run must be processed beginning from the sample preparation step. SPECIFICATIONS Sensitivity The analytical sensitivity of HSV I/II Real-TM Typing PCR kit is specified in the table below. Clinical material DNA extraction kit Analytical sensitivity, GE/ml* Urogenital swabs DNA-sorb-A 5 x 102 * Genome equivalents (GE) of the microorganism per 1 ml of a clinical sample placed in the transport medium specified. Specificity The analytical specificity of HSV I/II Typing Real-TM PCR kit is ensured by selection of specific primers and probes as well as by selection of stringent reaction conditions. The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis. There were no nonspecific responses during examination of human DNA as well as DNA panel of the following microorganisms: Mycoplasma hominis, Lactobacillus spp., Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Candida albicans, Neisseria gonorrhoeae, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma genitalium, Neisseria flava, Neisseria subflava, Neisseria sicca, Neisseria mucosa, Chlamydia trachomatis, Trichomonas vaginalis, Gardnerella vaginalis, Toxoplasma gondii, CMV, and HPV. Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 TROUBLESHOOTING 1. Weak or no signal of the IC (Rox/Orange/TexasRed channel) for the Negative Control of extraction.  The PCR was inhibited.  Make sure that you use a recommended DNA extraction method and follow to the manufacturer’s instructions.  Re-centrifuge all the tubes before pipetting of 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  Improper DNA extraction.  Repeat analysis starting from the DNA extraction stage  The PCR conditions didn’t comply with the instructions.  Check the PCR conditions and select for the IC detection 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 or no signal of the Positive Control.  The PCR conditions didn’t comply with the instructions.  Check the amplification protocol and select the fluorescence channel reported in the manual. 3. Fam (Green) or Joe (Yellow) signal with Negative Control of extraction.  Contamination during DNA 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 between tubes.  Repeat the DNA extraction with the new set of reagents. 4. Any signal with Negative Control of PCR (DNA-buffer).  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 control at last.  Repeat the PCR preparation with the new set of reagents. Sacace™ HSV I/II Typing Real-TM VER. 20.10.2022 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