GUIDELINES. End-Point PCR Detection of STIs and Other Reproductive Tract Infections

Transcription

1 For Professional Use Only GUIDELINES End-Point PCR Detection of STIs and Other Reproductive Tract Infections AmpliSens Ecoli s.r.o., Studenohorska Bratislava 47 Slovak Republic Tel.: Fax: Federal Budget Institute of Science Central Research Institute for Epidemiology 3A Novogireevskaya Street Moscow Russia

2 TABLE OF CONTENTS 1. INTENDED USE PRINCIPLE OF PCR DETECTION CONTENT ADDITIONAL REQUIREMENTS GENERAL PRECAUTIONS SAMPLING AND HANDLING WORKING CONDITIONS PROTOCOL DNA Extraction DNA extraction with DNA-sorb-AM reagent kit DNA extraction with DNA-sorb-B reagent kit DNA extraction by express method with the EDEM regent kit Amplification A. Preparing tubes for PCR B. Amplification DATA ANALYSIS TROUBLESHOOTING PROGRAMMING ALA-1/4 FLUORESCENCE DETECTOR AND INTERPRETATION OF RESULTS KEY TO SYMBOLS USED VER / Page 2 of 29

3 The list of reagent kits «AmpliSens» manufactured by FBIS CRIE for detection of STIs and other reproductive tract infections by the polymerase chain reaction (PCR) with end-point hybridization-fluorescence detection Detectable microorganisms Reagents kits (infectious agents) Bacterial infections AmpliSens Chlamydia trachomatis-fep Chlamydia trachomatis MULTIPRIME series kits AmpliSens Neisseria gonorrhoeae-screen-fep Neisseria gonorrhoeae AmpliSens Neisseria gonorrhoeae-test-fep MULTIPRIME series kits AmpliSens Treponema pallidum-fep Treponema pallidum MULTIPRIME series kits AmpliSens Mycoplasma genitalium-fep Mycoplasma genitalium Ureaplasma parvum, Ureaplasma urealyticum Mycoplasma hominis Gardnerella vaginalis HSV I, II CMV Trichomonas vaginalis Candida albicans MULTIPRIME series kits AmpliSens Ureaplasma spp.-fep MULTIPRIME series kits AmpliSens Mycoplasma hominis-fep MULTIPRIME series kits AmpliSens Gardnerella vaginalis-fep MULTIPRIME series kits Virus infections herpesviruses AmpliSens HSV I, II-FEP AmpliSens HSV-typing-FEP MULTIPRIME series kits AmpliSens CMV-FEP MULTIPRIME series kits Protozoal infections AmpliSens Trichomonas vaginalis-fep MULTIPRIME series kits Mycotic infections AmpliSens Candida albicans-fep VER / Page 3 of 29

4 Simultaneously detectable microorganisms Chlamydia trachomatis / Ureaplasma spp. / Mycoplasma genitalium Chlamydia trachomatis / Ureaplasma spp. / Mycoplasma hominis Trichomonas vaginalis / Neisseria gonorrhoeae / Chlamydia trachomatis Neisseria gonorrhoeae / Chlamydia trachomatis / Mycoplasma genitalium HSV I / HSV II Ureaplasma parvum / Ureaplasma urealyticum Trichomonas vaginalis / Neisseria gonorrhoeae Chlamydia trachomatis / Ureaplasma spp. Chlamydia trachomatis / Mycoplasma genitalium HSV / CMV Mycoplasma hominis Gardnerella vaginalis Reagents kits of MULTIPRIME series AmpliSens C. trachomatis / Ureaplasma / M. genitalium-multiprime-fep AmpliSens C. trachomatis / Ureaplasma / M. hominis-multiprime-fep AmpliSens T.vaginalis / N.gonorrhoeae / C.trachomatis-MULTIPRIME-FEP AmpliSens N.gonorrhoeae / C.trachomatis / M.genitalium-MULTIPRIME-FEP AmpliSens HSV-typing-FEP AmpliSens U.parvum / U.urealyticum-FEP (detection and differentiation) AmpliSens T.vaginalis / N.gonorrhoeae- MULTIPRIME-FEP AmpliSens C.trachomatis / Ureaplasma- MULTIPRIME-FEP AmpliSens C.trachomatis / M.genitalium- MULTIPRIME-FEP AmpliSens HSV / CMV-MULTIPRIME-FEP AmpliSens M.hominis / G.vaginalis-MULTIPRIME-FEP VER / Page 4 of 29

5 Recommendations for the use of AmpliSens reagent kits for qualitative detection of Neisseria gonorrhoeae DNA For the laboratory diagnosis of gonorrhea, it is recommended to carry out a complex study by two independent laboratory methods. In accordance with current recommendations, if molecular biological techniques (particularly PCR) reveal the presence of Neisseria gonorrhoeae, the diagnosis should be confirmed bacteriologically. If a bacteriological study cannot be performed or if it gives a negative result, a second independent molecular biological test (using a different genetic target or another principle of amplification) should be performed to confirm the presence of Neisseria gonorrhoeae. In view of this, FBIS CRIE has developed and produces two kits for qualitative detection of Neisseria gonorrhoeae DNA AmpliSens Neisseria gonorrhoeae-screen-fep and AmpliSens Neisseria gonorrhoeae-test-fep. Each of these kits has a high specificity and contains different specific regions of the Neisseria gonorrhoeae genome that are used as a target. Thus, two independent PCR tests for detecting this pathogen can be performed with the use of these two kits. It is recommended to use the AmpliSens Neisseria gonorrhoeae-screen-fep PCR kit or a kit of the MULTIPRIME AmpliSens series (in which the same region of Neisseria gonorrhoeae DNA is used as a target) for the first test. If the Neisseria gonorrhoeae DNA is detected in the first test, the presence of the pathogen should be confirmed in the second test by using the AmpliSens Neisseria gonorrhoeae-test-fep PCR kit. VER / Page 5 of 29

6 1. INTENDED USE The Guidelines describe the procedure of detection of STIs and other reproductive tract infections in clinical material by the polymerase chain reaction (PCR) with hybridizationfluorescence detection using end-point fluorescence detector ALA 1/4 (Biosan, Latvia). 2. PRINCIPLE OF PCR DETECTION Detection of microorganisms by the polymerase chain reaction (PCR) is based on the amplification of pathogen genome specific region using specific primers. In Fluorescent End-Point PCR, the amplified product is detected by using fluorescent dyes. These dyes are linked to oligonucleotide probes that bind specifically to the amplified product during thermocycling. A multichannel rotor-type fluorometer is specially designed to detect fluorescence emission from the fluorophores in a reaction mixture after PСR. It allows detection of the accumulating product without re-opening the reaction tubes after the PCR run. The Internal Control (IC) is used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition. It is possible to automate data analysis and reduce the subjectivity in the interpretation of results. Simultaneous amplification and detection of several DNA targets in a single reaction is possible. Optimization ensures a high sensitivity to each DNA target. The use of multiplex PCR allows a 3 4-fold increase in the efficiency of analysis without extending the instrument base. Reagent kits of the MULTIPRIME series are intended for multiplex PCR analysis. VER / Page 6 of 29

7 3. CONTENT PCR kit variant FEP includes: PCR-mix-1-FL Reagent Description Volume, ml Quantity PCR-mix-2-FL-red Solution containing primers, dntp and oligonucleotide probes tubes of 0.5 or 0.2 ml Buffer solution containing Taqpolymerase, Mg tube PCR-mix-Backgroundred 1 Buffer solution tube Mineral oil for PCR 2 Positive Control complex (C+) Oil preventing the PCR-mix evaporation Solution containing specific fragments of DNA of analyzed microorganisms (in the content of genetically engineered structures) dropper bottle tube DNA-buffer Buffer solution tube Negative Control (C )* Buffer solution tube Internal Control-FL (IC)** Phage (λgt67) particle solution containing a cloned genetically engineered construct with an artificial nucleotide sequence nonhomologous to known microorganisms and viruses and complementary to the fluorescent probe which is included in the PCR kit tube * must be used in the extraction procedure as Negative Control of Extraction. ** add 10 µl of Internal Control-FL during the DNA extraction directly to the sample/lysis mixture (see DNA-sorb-AM REF K CE, REF K CE, DNA-sorb-В REF K CE, REF K CE protocols). PCR kit is intended for 110 reactions (including controls). 4. ADDITIONAL REQUIREMENTS DNA extraction kit. Disposable powder-free gloves and laboratory coat. Pipettes (adjustable). Sterile pipette tips with aerosol filters (up to 200 µl). Tube racks. Vortex mixer. 1 Reagent is used for samples extracted by DNA-sorb-AM or DNA-sorb-B reagents kits 2 Must be used for thermocyclers without a constant-temperature lid. VER / Page 7 of 29

8 Desktop centrifuge with rotor for 2-ml reaction tubes. PCR box. Personal thermocyclers (for example, Gradient Palm Cycler (Corbett Research, Australia), MaxyGene (Axygen, USA), GeneAmp PCR System 2700 (Applied Biosystems, USA)). Fluorometer ALA-1/4 (BioSan, Latvia). Disposable polypropylene tubes for PCR (0.1- or 0.2-ml). Refrigerator for 2 to 8 C Deep-freezer for the temperature from minus 24 to minus 16 C. Reservoir for used tips. 5. GENERAL PRECAUTIONS The user should always pay attention to the following: Use sterile pipette tips with aerosol filters and use a new tip for every procedure. Store all extracted positive material (samples, controls and amplicons) away from all other reagents and add it to the reaction mix in a distantly separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Use disposable protective gloves and laboratory coats, and protect eyes while samples and reagents handling. Thoroughly wash hands afterward. 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 samples and unused reagents in accordance with local regulations. Samples 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 another suitable disinfectant. Avoid samples and reagents contact with the skin, eyes and mucous membranes. If these solutions come into contact, rinse the injured area immediately with water and seek medical advice immediately Safety Data Sheets (SDS) are available on request. Use of this product should be limited to personnel trained in the DNA amplification techniques. VER / Page 8 of 29

9 Workflow in the laboratory must be one-directional, beginning in the Extraction Area and moving 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. 6. SAMPLING AND HANDLING Obtaining samples of biological materials for PCR-analysis, transportation and storage are described in the manufacturer s handbook [1]. It is recommended that this handbook is read before starting work. The following material is used for analysis: urogenital, rectal, and oropharyngeal swabs; conjunctival secretion; erosive-ulcerative lesions of skin and mucous membranes; urine sediment (use the first portion of the morning specimen); and prostate secretion. The following clinical material is used: 1. Women: cervical, vaginal, and urethral swabs and urine. 2. Men: urethral swabs, urine, and prostate secretion. 3. Children: urine, conjunctival secretion. Clinical material should be placed into tubes with a transport medium recommended by FBIS CRIE. The obtained samples (except for urine) can be transported and stored under the following conditions: At the room temperature for 48 hours; At 2 8 C for two weeks; At 20 C for the month; At 68 C for a long time; Samples placed to the transport medium with mucolytic agent can be transported and stored under the following conditions: At the room temperature (18-25 C) for 28 days; At 2 8 C for 3 months; At 20 C for a long time. Only one freeze thaw cycle of clinical material is allowed. The obtained urine samples can be transported and stored under the following conditions: At the room temperature for 6 hours; VER / Page 9 of 29

10 At 2 8 C for 24 hours; Clinical samples should be transported in a special container with cooling elements. The following types of clinical material are used for microorganism DNA detection: urogenital swabs, urine sediment, and prostate secretion are used for detection of Chlamydia trachomatis; Neisseria gonorrhoeae; Mycoplasma genitalium, M.hominis; Trichomonas vaginalis; Ureaplasma spp., U. parvum, U.urealyticum DNA; conjunctival secretion as well as rectal and oropharyngeal swabs are used for detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA; urogenital, rectal, and oropharyngeal swabs as well as exudate of blisters and erosiveulcerative lesions of skin and mucous membranes are used for detection of HSV I, II and Treponema pallidum DNA. Whole blood, cerebrospinal fluid and conjunctival secretion are used for detection of HSV I, II DNA as well; urogenital swabs, urine, saliva, and whole blood are used for detection of CMV DNA; urogenital and oropharyngeal swabs and urine are used for detection of Candida albicans, C.glabrata, and C.kruzei DNA; vaginal swabs are used for detection of Gardnerella vaginalis DNA. The following transport media (manufactured by FBIS CRIE) are recommended for transportation and storage of clinical material: Transport Medium with Mucolytic Agent, REF 952-CE. Transport Medium for Swabs, REF 956-CE, 987-CE. If DNA is extracted with EDEM Reagent Kit (manufactured by FBIS CRIE), Transport Medium TM-EDEM, REF 1533-CE is recommended for pretreatment of urine samples. Pretreatment (only for urine samples) A. Pretreatment of urine samples with the DNA-sorb-AM reagent kit for subsequent DNA extraction 1. Shake the vial with urine. 2. Transfer 1 ml of urine to a1.5-ml sterile disposable tube using a new tip with aerosol filter for each sample. 3. Centrifuge the tube at 10,000 g (12,000 rpm at MiniSpin centrifuge, Eppendorf) for 5 min. If urine contains excess salts, resuspend only the upper layer of salt pellet in 1 ml and centrifuge again. 4. Discard the supernatant using a vacuum aspirator with a trap flask without disturbing VER / Page 10 of 29

11 the pellet; use a new tip without aerosol filter for each sample. 5. Add the transport medium to the pellet (final volume, 0.2 ml). Mix thoroughly the content of the tubes using a vortex mixer. Thus pretreated urine samples (urine pellet in the transport medium) can be stored: at 2 8 C for 24 h; at 20 C for one week; at 68 C for a long time. B. Pretreatment of urine samples with the EDEM reagent kit for subsequent DNA extraction 1. Shake the vial with urine. 2. Transfer 1 ml of urine to a tube with 0.5 ml of TM-EDEM transport medium using a new tip with the aerosol filter for each sample. 3. Centrifuge the tubes containing TM-EDEM and urine at 12,000 rpm for 5 min in a MiniSpin centrifuge (Eppendorf). 4. Discard the supernatant using a vacuum aspirator with a trap flask without disturbing the pellet; use a new tip without aerosol filter for each sample. 5. Add 0.5 ml of TM-EDEM to each tube with urine pellet using a new tip for each tube. Close tubes tightly. Mix thoroughly the content of the tubes on a vortex mixer to resuspend the pellet. Centrifuge at rpm for 2-3 s to spin down the drops from the walls of the tube and the cap. 6. Obtained samples of urine pellet in the TM-EDEM should be used for DNA extraction procedure. Thus obtained urine pellet in the TM-EDEM can be stored: at the room temperature (18 25 C) for 48 h; at 2 8 C for 14 days; at 20 C for a long time; 7. WORKING CONDITIONS Reagent kits should be used at C. 8. PROTOCOL 8.1. DNA Extraction It is recommended to use the following nucleic acid extraction kits: DNA-sorb-AM, REF K CE, REF K CE; VER / Page 11 of 29

12 DNA-sorb-В, REF K CE, REF K CE (for prostate secretion, whole blood, or spinal fluid); EDEM, REF K CE. Extract DNA according to the manufacturer s instructions DNA extraction with DNA-sorb-AM reagent kit DNA-sorb-AM nucleic acid extraction kit is a reagent kit for rapid and efficient manual extraction and purification of DNA from various clinical materials. Lysis solution contains a chaotropic agent (guanidine chloride) that lyses cells and denatures cell proteins. The nucleic acids are then adsorbed on silica particles. DNA extracted from clinical samples can be used for PCR diagnostic tests. DNA-sorb-AM nucleic acid extraction kit variant 50 or 100 includes: Reagent Lysis Solution Washing Buffer Universal Sorbent TE-buffer for DNA elution Description solution containing chaotropic agent solution containing isopropanol suspension of silica particles buffer solution for DNA elution Variant 50 Variant 100 Volume, ml Quantity Volume, ml Quantity 15 1 vial 30 1 vial 50 1 vial vial tube tubes tube tubes Preparation to DNA extraction 1. Turn on the thermostat and set the temperature at 65 C. 2. Lysis Solution (if stored at 2 8 С) should be heated to 65 С until the ice crystals disappear (ice crystals can appear at the bottom of the vial). 3. Take the required number of 1.5-ml disposable sterile tubes, label them, and place in a tube rack. 4. Centrifuge the tubes with clinical samples at rpm for 5 s, then carefully mix using a vortex mixer, and place in a tube rack. 5. When using the first portion of the morning specimen for analysis, pretreat it to obtain the urine pellet in the transport medium as described above. DNA extraction procedure 1. Add 10 µl of Internal Control-FL to each sterile disposable tube. VER / Page 12 of 29

13 Internal Control-FL and Negative Control (C ) are included in the PCR kit. 2. Thoroughly resuspend Universal Sorbent on a vortex mixer. Add 20 µl of Universal Sorbent and 300 µl of Lysis Solution to each test tube using tips with aerosol filter. If the number of clinical samples exceeds 50, it is recommended that the whole volume of sorbent and IC are transferred to the tube with Lysis Solution (2 ml of Universal Sorbent and 1 ml of IC per 30 ml of Lysis Solution). Thoroughly stir this suspension and transfer 330 µl of it to the tubes. The prepared mixture can be stored at room temperature for 2 days. Stir well before use. 3. Add 100 µl of a sample to the tube using a tip with aerosol filter. Add 100 µl of Negative Control (C ) to the tube with Negative Control of Extraction (C ). 4. Tightly close the caps, thoroughly mix the tubes on a vortex mixer, and incubate them at 65 C for 5 min in a thermostat. After incubation, mix the contents of the tubes on a vortex once again and incubate at room temperature for another 2 min. 5. Centrifuge all tubes at 10,000 rpm for 30 s and carefully remove the supernatant from each tube with a vacuum aspirator without disturbing the pellet. Use a new tip (without aerosol filter) for every tube. 6. Add 1 ml of Washing Buffer into each tube. Vortex until the sorbent is completely resuspended. 7. Repeat step Incubate all tubes with open caps at 65 C for 5 10 min (for sorbent predrying). 9. Add 100 µl of TE-buffer for DNA elution using tip with aerosol filter. Vortex until the sorbent is completely resuspended. Incubate tubes at 65 C for 5 min. The elution volume can be adjusted to 150 µl. 10. Centrifuge tubes at 12,000 rpm for 1 min. The supernatant contains purified DNA and is ready for PCR amplification. The purified DNA can be stored: at 2 8 C for 1 week; at 16 C for 1 year. If samples are analyzed once again, mix the content of the tubes on a vortex mixer and repeat centrifugation in accordance with item DNA extraction with DNA-sorb-B reagent kit The principle of extraction with the DNA-sorb-B reagent kit corresponds to the principle specified above for the DNA-sorb-AM reagent kit. VER / Page 13 of 29

14 DNA-sorb-B nucleic acid extraction kit variant 50 or 100 includes: Reagent Lysis Solution Washing Solution 1 Washing Solution 2 Universal Sorbent TE-buffer for DNA elution Preparation to DNA extraction Description solution containing chaotropic agent solution containing chaotropic agent and isopropanol solution containing isopropanol suspension of silica particles buffer solution for DNA elution 1. Turn on the thermostat and set temperature at 65 C. Variant 50 Variant 100 Volume, ml Quantity Volume, ml Quantity 15 1 vial 30 1 vial 15 1 vial 30 1 vial 50 1 vial vial tube tubes tube tubes 2. Lysis Solution and Washing Solution 1 (if stored at 2 8 С) should be heated to 65 С until ice crystals disappear. 3. Take the required number of 1.5-ml sterile disposable tubes, label them, and place in a tube rack. 4. Centrifuge the tubes with clinical samples at rpm for 5 s, then carefully mix using a vortex mixer, and place in a tube rack. DNA extraction procedure 1. Add 10 µl of Internal Control-FL to each sterile disposable tube. Internal Control-FL and Negative Control (C ) are included in the PCR kit. 2. Add 300 µl of Lysis Solution to each prepared tube. 3. Add 100 µl of a sample to the tubes with Internal Control and Lysis Solution. Add 100 µl of Negative Control to the tube labeled C. 4. Vortex the tubes and then incubate at 65 C for 5 min. Centrifuge all tubes at 5000 rpm for 5 s. If a sample hasn t dissolved completely, centrifuge the tube at 12,000 rpm for 5 min, transfer the supernatant to a new tube, and use for DNA extraction. 5. Thoroughly resuspend Universal Sorbent on a vortex mixer. Add 25 µl of Universal Sorbent into each test tube using a new tip. Vortex the tubes, then place them in a tube rack for 2 min. Vortex once again and place the tubes for 5 min in a tube rack. 6. Centrifuge all tubes at 5000 rpm for 30 s. Discard the supernatant using a vacuum aspirator. Use a new tip for every tube. VER / Page 14 of 29

15 7. Add 300 µl of Washing Solution 1 to each tube. Vortex until the sorbent is completely resuspended. 8. Repeat step Add 500 µl of Washing Solution 2 to each tube. Vortex until the sorbent is completely resuspended. 10. Centrifuge at 10,000 rpm for 30 s. Discard the supernatant using a vacuum aspirator. Use a new tip for every tube. 11. Repeat steps Remove the supernatant entirely. 12. Incubate all tubes with open caps at 65 C for 5-10 min. 13. Add 50 µl of TE-buffer for DNA elution. Mix the contents of the tubes on a vortex mixer. Incubate the tubes at 65 C for 5 min, vortex occasionally while incubating. 14. Centrifuge tubes at 12,000 rpm for 1 min. The supernatant contains purified DNA and is ready for PCR amplification. The purified DNA can be stored: at 2 8 C for 1 week; at 16 C for 1 year DNA extraction by express method with the EDEM regent kit Reagent kit for Extraction of DNA by Express Method (EDEM) is intended for the treatment of different types of clinical materials (urogenital, oropharyngeal, and conjunctival swabs; erosive-ulcerative lesions of skin and mucous membranes; and first portions of human urine samples 3 ) with subsequent tests for the presence of STIs and other reproductive tract infections by using hybridization-fluorescence detection and PCR kits manufactured by FBIS CRIE (including the MULTIPRIME series kits). The reagent kit EDEM is intended for qualitative PCR-analysis and primary screening of patients. This reagent kit is not intended for quantitative PCR analysis or monitoring after treatment (for these purposes, DNA-sorb-AM reagent kit is used for DNA extraction). Samples must be placed into tubes with TM-EDEM transport medium only (the EDEM reagent kit contains TM-EDEM). Clinical material obtained from a patient is transferred into TM-EDEM transport medium, in which it is stored and transported to a laboratory. For DNA extraction, an aliquot of a clinical sample is transferred into a tube with IC-diluent, after which it is thermally processed to destroy cell membranes, viral coats, and other biopolymer complexes, and to 3 Urine samples should be pretreated. VER / Page 15 of 29

16 ensure DNA release. Insoluble components are pelleted on the tube bottom by centrifuging; the supernatant containing DNA is used for PCR. The internal control sample (IC) contained in IC-diluent is isolated simultaneously with DNA from clinical material and, thereby, is a quality marker of laboratory analysis of clinical samples. EDEM reagent kit includes: Reagent Description Volume, ml Quantity Transport mediumtm-edem colorless clear liquid tubes IC-diluent colorless clear liquid tubes PCR-buffer-Background colorless clear liquid tubes Preparation to DNA extraction 1. Switch on the thermostat and set the temperature at 95 С. 2. Prepare and place the required number of tubes with IC-diluent into the tube rack and label them. Spin down the drops of solution from tube walls and caps by short centrifuging at rpm for 2 3 s. 3. Before starting DNA extraction, mix the content of tubes with clinical material in TM- EDEM transport medium by vortexing and spin down the drops of material from tube walls and caps by short centrifugation at rpm for 2 3 s. Place the prepared tubes into a tube rack. 4. Urine samples should be preliminary treated in accordance with chapter Pretreatment of urine samples with the EDEM reagent kit for subsequent DNA extraction to obtain the urine pellet in the TM-EDEM transport medium. To do this, the additional reagent TM-EDEM transport medium (50 ml) is to be used. DNA extraction procedure 1. Transfer 100 µl of clinical material in the TM-EDEM transport medium into the prepared tubes with IC-diluent using a new tip with aerosol filter for each sample. Add 100 µl of TM-EDEM transport medium into the tube for Negative Control of Extraction (C ). 2. Tightly close all tubes, carefully mix the contents by vortexing (prevent spraying), and incubate in a thermostat at 95 С for 5 min. Close tightly the tubes so that they would not open during heating. 3. After the end of incubation, place the tubes into a desktop centrifuge and centrifuge at 14,000 rpm for 1 min. Thus obtained DNA samples are ready for PCR analysis with hybridization-fluorescence detection. DNA samples can be stored for one week at 2 8 С or for one year at 16 C (it is VER / Page 16 of 29

17 necessary to vortex and recentrifuge the tube contents according to item 3 if PCR analysis of DNA samples is performed once again). 8.2 Amplification In case of invalid or equivocal result of PCR analysis obtained with the use of EDEM reagent kit, repeat DNA extraction procedure. To do this, 100 µl of clinical material in TM-EDEM transport medium should be treated with the DNAsorb-AM reagent kit according to its instruction manual. The total reaction volume is 30 µl, the volume of DNA sample is 10 µl. A. Preparing tubes for PCR The type of the tubes depends on the used thermocycler. Use the disposable tips with aerosol filters for reagents, clinical and control samples. 1. Prepare the required number of tubes with PCR-mix-1-FL for amplification of DNA from clinical and control samples. 2. Add 10 µl of PCR-mix-2-FL-red to the surface of the wax layer of each tube ensuring that it does not fall under the wax and mix with PCR-mix-1-FL. 3. Add above 1 drop of mineral oil for PCR (about 25 µl). 4. Prepare 1 tube with PCR-mix-1-FL tube and mark it as Background («Blank» or «B»). Add 20 µl of PCR-mix-Background-red to the surface of the wax layer, ensuring that it does not fall under the wax and mix with PCR-mix-1-FL. Add above 1 drop of mineral oil for PCR (about 25 µl). Use mineral oil for PCR if working with thermocyclers without a constanttemperature lid. PCR-mix-Background-red is to be used if DNA was extracted using DNA-sorb- AM ( REF K CE) or DNA-sorb-В ( REF K CE). If any other nucleic acid extraction kit (recommended by FBIS CRIE) was used, follow the instructions provided by the manufacturer. If DNA was extracted using EDEM ( REF 180-CE) reagent kit, the tube Background should be prepared in this way: add 10 µl of PCR-buffer- Background to the tube with PCR-mix-1-FL to the surface of the wax layer, ensuring that it does not fall under the wax and mix with PCR-mix-1-FL, then add 10 µl of Negative Control of Extraction (C ) treated according to this instruction manual. 5. Using tips with aerosol filter add 10 µl of DNA samples obtained from test or control samples at the DNA extraction stage. 6. 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). C Add 10 µl of the sample extracted from the Negative Control of Extraction VER / Page 17 of 29

18 B. Amplification reagent to the tube labeled C (Negative Control of Extraction). 1. Run the following program in the thermocycler (see Table 1). 2. When the temperature reaches 95 C (pause mode), insert tubes into the thermocycler wells and press the button to continue. It is recommended to sediment drops from walls of tubes by short centrifugation before placing them into the instrument. Insert empty tubes at the edges of reaction module in case of incomplete filling of plate-type instrument Table 1 AmpliSens-1-FEP amplification program Thermocyclers with active temperature adjustment GeneAmp PCR System 2700 Step Tempe Time Cycles rature Gradient Palm Cycler, MaxyGene Thermocyclers with block temperature adjustment Uno II Tempe Tempe Time Cycles rature rature Time Cycles 0 95 С pause 95 С pause 95 С pause 1 95 С 5 min 1 95 С 5 min 1 95 С 5 min 1 95 С 20 s 95 С 2 s 95 С 25 s 2 65 С 25 s С 10 s С 40 s С 30 s 72 С 10 s 72 С 40 s 95 С 20 s 95 С 2 s 95 С 25 s 3 60 С 30 s С 15 s С 40 s С 30 s 72 С 10 s 72 С 40 s 4 95 С 20 s 95 С 2 s 95 С 25 s С 30 s 60 С 15 s 60 С 40 s С storage 10 С storage 10 С storage Once amplification is completed, pass to fluorescence detection. 9. DATA ANALYSIS Detection is performed using a fluorescence detector. The obtained results are interpreted automatically by the software of the PCR instrument used. Interpretation is based on the level of the fluorescence signal detected in the control and clinical samples in respective channels relative to the background samples. Please read the fluorescence detector Operating Manual before using this kit. Detection can be performed within 1 day after completion of amplification only if the tubes with the amplified product were stored at 2 8 C in a light-free area. The fluorescent signal intensity is detected in two channels when using reagent kits for detection of DNA of one microorganism: the signal from the microorganism DNA amplification product is detected in the FAM channel (or analogous, depending on the detector model); VER / Page 18 of 29

19 the signal from the IC DNA amplification product is detected in the HEX channel (or analogous, depending on the detector model). Threshold values for test settings used for detection are specified in Table 2 and in the Important Product Information Bulletin for used reagent kits. Threshold values for reagent kits for detecting DNA of one microorganism Name of the test Threshold of negative result Threshold of positive result Chlamydia trachomatis Neisseria gonorrhoeae-screen Neisseria gonorrhoeae-test Mycoplasma genitalium Treponema pallidum Trichomonas vaginalis HSV I, II CMV Ureaplasma species Mycoplasma hominis Candida albicans Gardnerella vaginalis For all above listed tests threshold for Internal Control (in 3.0 the HEX channel) Table 2 The fluorescent signal intensity is detected in three or four channels (depending on the used reagent kits) when reagent kits of the MULTIPRIME series (for detection of several microorganisms DNA) are used: the signal from the IC amplification product is detected in one of the channels; the signals from the corresponding microorganism DNA amplification products are detected in other channels. The assignment of detection channels and threshold values for test settings for reagent kits of the MULTIPRIME series are specified in Tables 3 and 4 and in Important Product Information Bulletins for used reagent kits. VER / Page 19 of 29

20 Table 3 Threshold values and assignment of channels for reagent kits of the MULTIPRIME series Microorganism Channel for Threshold of Threshold of fluorophore negative result positive result C.trachomatis / Ureaplasma / M.genitalium «Ch.t./Ure /M.gen.» test Chlamydia trachomatis FAM Ureaplasma spp. HEX Mycoplasma genitalium ROX C. trachomatis / Ureaplasma / M.hominis «Ch.t./Ure /M.hom.» test Chlamydia trachomatis FAM Ureaplasma spp. HEX Mycoplasma hominis ROX N. gonorrhoeae / C. trachomatis / M.genitalium «N.gon./Ch.t./M.gen.» test Neisseria gonorrhoeae FAM Chlamydia trachomatis HEX Mycoplasma genitalium ROX T. vaginalis / N. gonorrhoeae/ C. trachomatis «T.v./N.gon./Ch.t.» test Trichomonas vaginalis FAM 3,0 3,5 Neisseria gonorrhoeae HEX 3,0 3,5 Chlamydia trachomatis ROX 3,0 3,5 C. trachomatis / Ureaplasma «Ch.t./Ure» test Chlamydia trachomatis FAM Ureaplasma spp. HEX C.trachomatis / M.genitalium «Ch.t./M.gen.» test Chlamydia trachomatis FAM Mycoplasma genitalium HEX T. vaginalis / N. gonorrhoeae «T.v./N.g.» test Trichomonas vaginalis FAM Neisseria gonorrhoeae HEX HSV-typing «HSV-typing» test HSV II FAM HSV I HEX U.parvum / U.urealyticum «U.p./U.u.» test Ureaplasma parvum FAM Ureaplasma urealyticum HEX M.hominis/ G.vaginalis «M.h./G.v.» test Mycoplasma hominis FAM Gardnerella vaginalis HEX VER / Page 20 of 29

21 Table 4 Test settings: threshold values for IC when using reagent kits of MULTIPRIME series Channel for registration of IC Tests (kits) Threshold for IC DNA amplification first group for detection of three microorganisms second group for detection of two microorganisms Сy5 3.0 ROX 3.0 The fluorescence detection procedure and interpretation of results are specified in detail in item 11 Programming ALA-1/4 fluorescence detector and interpretation of results. Prior to detection, all settings should be entered and saved (see tables 2, 3, 4 and Important Product Information Bulletins). The procedure of entering and checking test settings is specified in item 11 Programming ALA-1/4 fluorescence detector and interpretation of results. Interpretation of results The results are interpreted by the software of the PCR instrument by the crossing (or not crossing) of the fluorescence curve with the threshold line. 1. For reagent kits intended for detection of one microorganism DNA, the principle of results interpretation is the following: The microorganism DNA is detected in a sample if the signal in the FAM channel is greater than the specified threshold value of the positive result. The microorganism DNA is not detected in a sample if the signal in the FAM channel is less than the specified threshold value of the negative result whereas the signal in the HEX channel is greater than the specified threshold value. The result is invalid in a sample if the signal in the FAM channel is less than the specified threshold value of the negative result and the signal in the HEX channel is less than the specified threshold value. The result is equivocal if the signal in the FAM channel is greater than the specified threshold value of the negative result but less than the threshold value of the positive result (the signal is between thresholds). If the result is invalid or equivocal, PCR should be repeated once again. 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 (Table 5). VER / Page 21 of 29

22 Control Stage for control C DNA extraction NCA PCR C+ PCR Results for controls Result of automatic interpretation FAM channel < threshold of negative result < threshold of negative result > threshold of positive result HEX channel > threshold < threshold > threshold Table 5 2. For reagent kits of the MULTIPRIME series, the principle of interpretation of results is the following: The microorganism DNA is detected in a sample if its signal in the channel assigned for detecting the microorganism DNA amplification is greater than the specified threshold value of the positive result. The microorganism DNA is not detected in a sample if the signal in the channel for detecting the microorganism DNA amplification is less than the specified threshold value of the negative result whereas the signal in channel for detecting the IC DNA amplification is greater than the specified threshold value. The result is invalid in a sample if the signal in the channel for detecting the microorganism DNA amplification is less than the specified threshold value of the negative result and the signal in the channel for detecting the IC DNA amplification is less than the specified threshold value. The result is equivocal if the signal of a sample in the channel for detecting the microorganism DNA amplification is greater than the specified threshold value of the negative result but less than the threshold value of the positive result (the signal is between thresholds). If the result is invalid or equivocal, PCR should be repeated once again. The result of analysis is considered reliable only if the results obtained for both Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct (Table 6). VER / Page 22 of 29

23 Control Results for controls when using MULTIPRIME series kits Stage for control C DNA extraction NCA PCR C+ PCR 10. TROUBLESHOOTING Result of automatic interpretation Channel for results of microorganism DNA amplification < threshold of negative result < threshold of negative result > threshold of positive result Channel for results of IC DNA amplification > threshold < threshold > threshold Table 6 For reagent kits for detection of one microorganism DNA, the results of analysis are not taken into account in the following cases: If the signal of the Positive Control of amplification (C+) in the FAM channel is less than the threshold of the positive result, repeat PCR and detection for all samples in which microorganism DNA was not detected. If the signal of the Negative Control of extraction (C ) and/or Negative Control of amplification (NCA) in the FAM channel is greater than the threshold of the positive result, repeat PCR beginning from the extraction stage for all samples in which the microorganism DNA was detected. If the signal of the Negative Control of extraction (C ) and/or Negative Control of amplification (NCA) is greater than the threshold of the positive result once again, it can indicates contamination of laboratory work areas and/or reagents by amplification products or microorganisms DNA. In this case measures for detecting and eliminating the contamination source must be taken. For reagent kits of the MULTIPRIME series, the results of analysis are not taken into account in the following cases: If the signal of the Positive Control of amplification (C+) in one or several channels for detection of the microorganism DNA amplification (FAM and/or HEX and/or ROX) is less than the threshold of the positive result, repeat PCR and detection for all samples in which the signal for this channel (channels) is less than the threshold of the positive result. If the signal of the Negative Control of extraction (C ) and/or Negative Control of amplification (NCA) in one or several channels for detection of the microorganism DNA amplification is greater than the threshold of the positive result, repeat PCR beginning from the extraction stage for all samples in which the signal for those channels is greater than the threshold of the positive result. VER / Page 23 of 29

24 If the signal of the Negative Control of extraction (C ) and/or Negative Control of amplification (NCA) in one or several channels for detection of the microorganism DNA amplification is greater than the threshold of the positive result once again, it can indicates contamination of laboratory work areas and/or reagents by amplification products or microorganisms DNA. In this case measures for detecting and eliminating the contamination source must be taken. 11. PROGRAMMING ALA-1/4 FLUORESCENCE DETECTOR AND INTERPRETATION OF RESULTS Detection with the fluorescence detector (automatic luminescence analyzer) ALA 1/4 is performed in accordance with the instruction manual to this instrument. Before starting work enter and save in the ALA1 program the test settings with threshold values for used reagent kits. The use of ALA1 program ver and higher. To import the test settings select Settings Import tests. A. Fluorescence detection 1. Switch on the instrument and start Analyser ALA_1 software on the computer connected with the PCR instrument. 2. Create a new protocol by pressing the New protocol button. Select the type of the rotor in the Rotor type field (36 wells or 48 wells) Select the From LIS (Laboratory Information Systems) button to load the protocol of detection (variant LIS should be activated in the Settings/Programm settings/lis submenu) Select the necessary test from the list of the available tests and press the Add arrow. In the Samples field, set the number of samples without the control samples (C, NCA, C+) and the blanc-tubes. Press the Add button In the Blank-tubes for reaction mixes field, select the necessary number of Background (Blank) tubes Define the names of the samples Add the required number of controls C, C+, NCA in the Samples, controls, blank-tubes table (using buttons with corresponding designations over this table) Press the OK and Save buttons. 3. Insert the tubes into the wells of the removed rotor of the ALA-1/4 instrument strictly in accordance with the entered order. Place the rotor into the instrument module, fix it, and close the lid. Press the Measure button (at the top of the display) to start VER / Page 24 of 29

25 detection process. 4. If more than 36 (48) samples are detected within the same protocol, after the detection of 36 (48) samples is completed, take the detected tubes out, insert the rest of the samples into the rotor, and press the Continue button in the program window. 5. The results grid will appear on the display after detection is completed. Press the Results button at the active button panel if it is necessary to open the Results grid once again. 6. Results of detection are saved automatically in the file with the name including date and time of detection in the ALA1/Result directory. To save file with the other name or to other directory select the Save button and indicate the file name and required directory. When working with LIS support, press the Send to LIS button. B. Interpretation of results Data are analyzed automatically, the result appears in the Result column. Interpretation of results for reagent kits for detection of one microorganism DNA 1.1 Positive (name of microorganism) is indicated for the samples where the microorganism DNA was detected. 1.2 Negative is indicated for the samples where the microorganism DNA was not detected. 1.3 Controversial is indicated for the samples where the signal cannot be interpreted unambiguously (equivocal result (grey background)). 1.4 Invalid is indicated when an invalid result is obtained (light-yellow background). If the result is invalid or controversial (equivocal), PCR or DNA extraction and PCR should be repeated once again. The result of the analysis is considered reliable only if the results obtained for both Positive and Negative Controls of amplification as well as for the Negative Control of Extraction are correct (Table 7). Example of obtained results Results for controls Control Stage for control Result designation in ALA1 program C DNA extraction OK NCA PCR OK C+ PCR OK Table 7 Results obtained using reagent kits for detection of one microorganism (Chlamydia VER / Page 25 of 29

26 trachomatis) DNA The results for control samples meet the requirements. The results for detected samples are considered reliable. - Microorganism DNA is detected in samples 3 and 5. - In other samples microorganism DNA is not detected. Interpretation of results for reagent kits of the MULTIPRIME series. 1.1 Positive (names of microorganisms) is indicated for the samples where DNA of one or several microorganisms was detected. 1.2 Negative is indicated for the samples where DNA of even one microorganism was not detected. 1.3 Controversial (name of microorganism) is indicated for the samples where the signal can be interpreted unambiguously (equivocal result (grey background)). 1.4 Invalid is indicated when an invalid result was obtained (light-yellow background). If the result is invalid or controversial (equivocal), PCR or DNA extraction and PCR should be repeated once again. The result of the analysis is considered reliable only if the results obtained for both Positive and Negative Controls of amplification as well as for the Negative Control of Extraction are correct (Table 8). Results for controls Control Stage for control Result designation in ALA1 program C DNA extraction OK NCA PCR OK C+ PCR OK Table 8 VER / Page 26 of 29

27 Example of obtained results Results obtained when using reagent kit AmpliSens C. trachomatis / Ureaplasma / M.genitalium-MULTIPRIME-FEP - The results for control samples meet the requirements. The results for detected samples are considered reliable. - Chlamydia trachomatis and Ureaplasma spp. DNA is detected in samples 3 and 9. - Ureaplasma spp. DNA is detected in samples 5 and 7. - In samples 1, 2, 4, 6, 8 analyzed microorganisms DNAs are not detected. VER / Page 27 of 29

28 12. KEY TO SYMBOLS USED Catalogue number Caution Batch code Sufficient for In vitro diagnostic medical device Expiration Date Research use only Version Authorized representative in the European Community Temperature limitation Consult instructions for use Keep away from sunlight Manufacturer Date of manufacture VER / Page 28 of 29

29 VER RT VV VV ME GA РМ List of Changes Made in the Instruction Manual Location of changes Cover page Through the text text Cover page The list of reagent kits Throughout the text DNA extraction Table 1 AmpliSens- 1-FEP amplification program Table 3 Threshold values and assignment of channels for reagent kits of the MULTIPRIME series Cover page DNA extraction with DNA-sorb-AM reagent kit DNA extraction with DNA-sorb-B reagent kit 8.2 Amplification 16. Key to symbols used Essence of changes The name of Institute was changed to Federal Budget Institute of Science Central Research Institute for Epidemiology Abbreviation FBIS was added Subsection Recommendations for the use of AmpliSens reagent kits for qualitative detection of Neisseria gonorrhoeae DNA was added Symbol IVD was deleted PCR kits with FRT format was deleted. AmpliSens Neisseria gonorrhoeae-test-fep and AmpliSens T.vaginalis / N.gonorrhoeae / C.trachomatis- MULTIPRIME-FEP PCR kits were added. AmpliSens N.gonorrhoeae / C.trachomatis / M.genitalium / T.vaginalis-MULTIPRIME-FEP, AmpliSens C.trachomatis / Ureaplasma / M.genitalium / M.hominis- MULTIPRIME-FEP, AmpliSens C.albicans / C.glabrata / C.krusei-MULTIPRIME-FEP and AmpliSens HSV II / HSV I / T.pallidum-MULTIPRIME-FEP PCR kits were deleted The text was corrected in accordance with the instruction manual and guidelines templates. Section Pretreatment (only for urine samples) was transferred from chapter Protocol to chapter Sampling and handling The reagents Internal Control complex (ICc), Internal Control-FL and Negative control was deleted from content of DNA-sorb-AM Amplification program was corrected Information corresponding to the T.vaginalis / N.gonorrhoeae / C.trachomatis test was added The European representative address was added. IVD symbol was added The description of the reagents was corrected. The phrase Insert additional empty tubes at the edges of reaction module in case of incomplete filling of plate-type instrument was added. The chapter was added VER / Page 29 of 29