STD (C.trach/Ureapl/M.homi/M.genital) Real-TM ,sacace-100t

Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM VER 02.02.2022 For in Vitro Diagnostic Use For Professional Use Only Сhlamydia trachomatis/Ureaplasma/ M.genitalium/M.hominis Real-TM Handbook Multiplex Real Time PCR kit for qualitative detection of Chlamydia trachomatis, Ureaplasma species, Mycoplasma genitalium and hominis REF B60-100FRT 100 Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 NAME Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM INTRODUCTION STDs (sexually transmitted diseases) refer to a variety of bacterial, viral and parasitic infections that are acquired through sexual activity. Some STDs, such as syphilis and gonorrhea, have been known for centuries — while others, such as HIV, have been identified only in the past few decades. STDs are caused by more than 25 infectious organisms. As more organisms are identified, the number of STDs continues to expand. Common STDs include: chlamydia, gonorrhea, herpes, HIV, HPV, syphilis, gardnerella and trichomoniasis. The Chlamydia trachomatis is nonmotile, gram-negative bacterial pathogen and is the most common sexually transmitted bacterial agent. The prevalence of C. trachomatis infection in sexually active adolescent women, the population considered most at risk, generally exceeds 10%, and in some adolescent and STD clinic populations of women, the prevalence can reach 40%. The prevalence of C. trachomatis infection ranges from 4 to 10% in asymptomatic men and from 15 to 20% in men attending STD clinics. Chlamydial infections in newborns occur as a result of perinatal exposure; approximately 65% of babies born from infected mothers become infected during vaginal delivery. The development of tests based on nucleic acid amplification technology has been the most important advance in the field of STD diagnosis. Because nucleic acid amplification is exquisitely sensitive and highly specific, it offers the opportunity to use noninvasive sampling techniques to screen for infections in asymptomatic individuals who would not ordinarily seek clinical care. INTENDED USE Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM PCR kit is an in vitro nucleic acid amplification test for multiplex detection of Chlamydia trachomatis, Ureaplasma (parvum and urealyticum), Mycoplasma genitalium and Mycoplasma hominis DNA in clinical materials (urogenital, rectal and pharyngeal swabs, conjunctival discharge, prostate gland secretion and urine samples) by using real-time hybridization-fluorescence detection. The results of PCR analysis are taken into account in complex diagnostics of disease. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 PRINCIPLE OF PCR DETECTION C.trachomatis / Ureaplasma / M.genitalium/ M.hominis detection by the multiplex polymerase chain reaction (PCR) is based on the amplification of pathogen genome specific region using specific C.trachomatis / Ureaplasma / M.genitalium / M.hominis primers. In real-time PCR, the amplified product is detected using fluorescent dyes. These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling. The real-time monitoring of the fluorescence intensities during the real-time PCR allows the detection of accumulating product without re-opening the reaction tubes after the PCR run. Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM PCR kit is a qualitative test that contains the Internal Control (IC). It must be used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition. Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM PCR kit uses “hot-start”, which greatly reduces frequency of nonspecifically primed reactions. “Hot-start” is guaranteed by chemically modified polymerase (TaqF), which is activated by heating at 95 ºC for 15 min. MATERIALS PROVIDED Reagent Description Volume, ml Quantity PCR-mix-1-FRT C.trachomatis / Ureaplasma / M.genitalium/ M.hominis colorless clear liquid 1.1 1 tube PCR-mix-2-FRT colorless clear liquid 0.6 1 tube Polymerase (TaqF) colorless clear liquid 0.06 1 tube Positive Control complex (C+) colorless clear liquid 0.2 1 tube DNA-buffer colorless clear liquid 0.5 1 tube Negative Control (C–)* colorless clear liquid 1.2 1 tube Internal Control-FL (IC)** colorless clear liquid 1.0 1 tube Contains reagents for 110 tests. * must be used in the extraction procedure as Negative Control of Extraction. ** add 10 µl of Internal Control-FL during the DNA extraction procedure directly to the sample/lysis mixture (see DNA-sorb-A REF K-1-1/A/100 protocol). Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 MATERIALS REQUIRED BUT NOT PROVIDED • DNA extraction kit. • Transport medium. • Disposable powder-free gloves. • Pipettes (adjustable). • Sterile pipette tips with aerosol barriers. • Disposable polypropylene 1,5/2,0 ml tubes. • 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. 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. 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™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 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™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 STORAGE INSTRUCTIONS The components of Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis RealTM PCR kit must be stored at 2–8 ºC excepting Polymerase (TaqF) and PCR-mix-2-FRT that must be stored at -16°C or below. The kit can be shipped at 2-8°C no longer than 5 days but should be stored at 2-8°C and – 16°C or below immediately on receipt. STABILITY Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis 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. The shelf life of reagents before and after the first use is the same, unless otherwise stated. SAMPLE COLLECTION, STORAGE AND TRANSPORT Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM can analyze DNA extracted from: • cervical, urethral, rectal, conjunctival swabs: insert the swab into the nuclease-free 1,5 ml tube and add 0,2-0,5 ml of Transport medium (can be ordered separately, Sacace REF K12-Stab). Vigorously agitate swabs in medium for 15-20 sec. • urine sediment: collect 10-20 ml of first-catch urine in a sterile container. Centrifuge for 30 min at 3000 x g, carefully discard the supernatant and leave about 200 µl of solution. Resuspend the sediment. Use the suspension for the DNA extraction. • prostate gland secretion stored in “Eppendorf” tube; 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. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 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 kit: ⇒ DNA-Sorb-A (Sacace, REF K-1-1/A/100); Please carry out the DNA extraction according to the manufacturer’s instructions. Add 10 μl of Internal Control-FL (IC) during the DNA isolation procedure directly to the sample/lysis mixture. (Note: the Sacace Internal Control is the same for all urogenital infectious kits) REAGENTS PREPARATION (REACTION VOLUME 25 µL): The total reaction volume is 25 µl, the volume of DNA sample is 10 µl. Unfreeze PCR-mix-2-FRT before mixing. 1. Prepare the required number of the tubes for amplification of DNA from clinical and control samples (for RotorGene use 0.2-ml tubes for a 36-well rotor or 0.1-ml strips for a 72-well rotor). 2. Prepare in a new sterile tube the Reaction Mix. For each sample mix 10*(N+1) µl of PCR-mix-1-FRT C.trachomatis / Ureaplasma / M.genitalium / M.hominis, 5.0*(N+1) µl of PCR-mix-2-FRT and 0.5*(N+1) µl of Polymerase (TaqF). 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. Carry out the control amplification reactions: NCA -Add 10 µl of DNA-buffer to the tube labeled NCA (Negative Control of Amplification). C+ -Add 10 µl of Positive Control complex to the tube labeled C+ (Positive Control of Amplification). 5. Insert the tubes in the thermalcycler. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 Amplification Create a temperature profile on your instrument as follows: Step Real Time PCR instruments1 Temperature, °С Time Cycles Hold 95 15 min 1 Cycling 95 5 sec 60 20 sec 5 72 15 sec Cycling 2 95 5 sec 60 40 20 sec fluorescence detection 72 15 sec 1 For example Rotor-Gene™ 3000/6000/Q (Qiagen) or equivalent. Fluorescence is detected at the Step 3 (60 °C) in FAM/Green, JOE/Yellow/HEX/Cy3, ROX/Orange/Texas Red, Cy5/Red and Cy5.5/Crimson channels. INSTRUMENT SETTINGS Rotor-type instruments Channel Calibrate/Gain Optimization Threshold More Settings/ Outlier Removal Slope Correct FAM/Green from 5FI to 10FI 0.1 0 % Off JOE/Yellow from 4FI to 8FI 0.1 5 % Off Rox/Orange from 4FI to 8FI 0.1 5 % Off Cy5/Red from 4FI to 8FI 0.07 5 % On Cy5.5/Crimson from 4FI to 8FI 0.1 20% On DATA ANALYSIS • Chlamydia trachomatis DNA PCR product is detected in the FAM/Green channel, • Ureaplasma DNA is detected in the JOE/HEX/Yellow channel, • Mycoplasma genitalium DNA is detected in the ROX/Orange channel, • Mycoplasma hominis DNA is detected in the Cy5.5/Crimson channel, • Internal Control DNA is detected in the Cy5/Red channel. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 RESULTS ANALYSIS: The results are interpreted with the software through the presence of crossing of fluorescence curve with the threshold line. The results of the analysis are considered reliable only if the results obtained for Positive and Negative Controls are correct. Results for controls Control Stage for control Ct FAM/ Green Ct JOE/HEX/ Yellow Ct ROX/ Orange Ct Cy5.5/ Crimson Ct Cy5/ Red Interpretation NCE DNA isolation Neg Neg Neg Neg Pos Valid result NCA Amplification Neg Neg Neg Neg Neg Valid result Pos C+ Amplification Pos Pos Pos Pos Pos Valid result 1. The sample is considered to be positive for Chlamydia trachomatis if its Ct value is defined in the results grid (the fluorescence curve crosses the threshold line) in the Green channel. 2. The sample is considered to be positive for Ureaplasma if its Ct value is defined in the results grid (the fluorescence curve crosses the threshold line) in the Yellow channel. 3. The sample is considered to be positive for Mycoplasma genitalium if its Ct value is defined in the results grid (the fluorescence curve crosses the threshold line) in the Orange channel. 4. The sample is considered to be positive for Mycoplasma hominis if its Ct value is defined in the results grid (the fluorescence curve crosses the threshold line) in the Crimson channel. 5. The sample is considered to be negative for Chlamydia trachomatis, Ureaplasma, Mycoplasma genitalium and Mycoplasma hominis if its Ct value is not defined in the results grid (the fluorescence curve does not cross the threshold line) in Green, Yellow, Orange and Crimson channels and the Ct value does not exceed the boundary value in the results grid in the Red channel. Boundary value of the cycle threshold, Ct Sample Channel for fluorophore Ct value C+ FAM <30 JOE/HEX <33 ROX <33 Cy5 <33 Cy5.5 <33 Clinical samples, C- Cy5 <33 PERFORMANCE CHARACTERISTICS Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 Sensitivity The analytical sensitivity for Chlamydia trachomatis, Ureaplasma, Mycoplasma genitalium, and Mycoplasma hominis is not less than 5х102 genome equivalents per 1 ml of sample (GE/ml). The analytical sensitivity of each microorganism does not change even at high concentrations of the three other microorganisms. Specificity The analytical specificity of Сhlamydia trachomatis/Ureaplasma/M.genitalium/ M.hominis Real-TM PCR kit is ensured by selection of specific primers and probes as well as stringent reaction conditions. The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis. The clinical specificity of Сhlamydia trachomatis/Ureaplasma/M.genitalium/M.hominis Real-TM PCR kit was confirmed in laboratory clinical trials. Target regions Channel for fluorophore FAM JOE ROX Cy5 Cy5.5 DNA-target Chlamydia trachomatis Ureaplasma spp. Mycoplasma genitalium Internal Control-FL Mycoplasma hominis Target gene cryptic plasmid UreC gyrB gene genetically engineered construction 16s rRNA gene Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 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. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 TROUBLESHOOTING 1. Weak or no signal of the IC (Red 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 • 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. Any signal with Negative Control of extraction (except for Red channel). • 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 between tubes. ⇒ Repeat the DNA extraction with the new set of reagents. 4. Any signal with Negative Control of PCR. • 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 control at last. ⇒ Repeat the PCR preparation with the new set of reagents. Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 REFERENCES • Role of Chlamydia trachomatis in Miscarriage. Baud D, Goy G, Jaton K, Osterheld MC, Blumer S, Borel N, Vial Y, Hohlfeld P, Pospischil A, Greub G. Emerg Infect Dis. 2011 Sep;17(9):1630-5. • Molecular Diagnosis of Genital Chlamydia trachomatis Infection by Polymerase Chain Reaction. Khan ER, Hossain MA, Paul SK, Mahmud MC, Rahman MM, Alam MA, Hasan MM, Mahmud NU, Nahar K. Mymensingh Med J. 2011 Jul;20(3):362-5. • Chlamydia trachomatis prevalence in unselected infertile couples.Salmeri M, Santanocita A, Toscano MA, Morello A, Valenti D, La Vignera S, Bellanca S, Vicari E, Calogero AE. Syst Biol Reprod Med. 2010 Dec;56(6):450-6. Epub 2010 Sep 17. • Urine-based testing for Chlamydia trachomatis among young adults in a population-based survey in Croatia: feasibility and prevalence. Božičević I, Grgić I, Židovec-Lepej S, Čakalo JI, Belak-Kovačević S, Štulhofer A, Begovac J. BMC Public Health. 2011 Apr 14;11:230. • Frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species in cervical samples. Rodrigues MM, Fernandes PÁ, Haddad JP, Paiva MC, Souza Mdo C, Andrade TC, Fernandes AP. J Obstet Gynaecol. 2011;31(3):237-41. • Prevalence of Chlamydia trachomatis: results from the first national population-based survey in France. Goulet V, de Barbeyrac B, Raherison S, Prudhomme M, Semaille C, Warszawski J; CSF group. Sex Transm Infect. 2010 Aug;86(4):263 Sacace™ Сhlamydia trachomatis/Ureaplasma/M.genitalium Real-TM VER 02.02.2022 KEY TO SYMBOLS USED * Rotor-Gene™ Technology is a registered trademark of Qiagen Sacace Biotechnologies Srl via Scalabrini, 44 – 22100 – Como – Italy Tel +390314892927 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 C– Negative control of Extraction Consult instructions for use C+ Positive Control of Amplification Expiration Date IC Internal Control