Manual FTD Urethritis plus
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1 Manual 32 reactions (catalog no. FTD ) 64 reactions (catalog no. FTD ) Qualitative assay for in vitro diagnostics For use with the ABI 7500, ABI 7500 Fast, ViiA 7, Bio-Rad CFX96, LightCycler 480, RotorGene 3000/6000/Q and SmartCycler 0123 FTD , FTD Fast Track Diagnostics Luxembourg S.à.r.l.; 29, rue Henri Koch; L-4354 Esch-sur-Alzette; Luxembourg FTD _64 MANUAL- v3 2016_12 EN
2 Table of Contents 1. IDENTIFICATION OF THE MANUFACTURER IDENTIFICATION OF THE PRODUCT INTENDED USE PATHOGEN INFORMATION CONTENTS PRECAUTIONS AND WARNINGS SAFETY INFORMATION HANDLING REQUIREMENTS SAFE WASTE DISPOSAL STORAGE AND STABILITY CONDITIONS PRINCIPLE OF THE METHOD ADDITIONALLY REQUIRED EQUIPMENT SAMPLES PROCEDURE PRELIMINARY EXTRACTION PROCEDURE USING THE EASYMAG MAIN PCR SETUP PROCEDURE PROGRAMMING OF THE THERMOCYCLER ASSAY VALIDATION SETUP ON THE ABI INTERPRETATION OF RESULTS TROUBLESHOOTING VALIDATION LEGEND OF SYMBOLS FTD _64 MANUAL- v3 2016_12 EN - 2 -
3 1. Identification of the manufacturer Fast Track Diagnostics Luxembourg S.à.r.l. 29, rue Henri Koch L-4354 Esch-sur-Alzette Tel.: Fax: Identification of the product Category: Multiplex Real-Time PCR for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum and internal control. Reference: FTD Test for 32 reactions. FTD Test for 64 reactions. Reagents in the kits are sufficient for 32 or 64 reactions. These kit sizes allow maximal flexibility from 1 to 30 patients in FTD and from 1 to 62 patients in FTD According PCR run amounts are shown in Table 1. Table 1: Minimum and maximum patient amounts and according run amounts possible for FTD and FTD FTD minimum maximum amount patients 1 30 amount runs 10 1 FTD minimum maximum amount patients 1 62 amount runs 20 1 Indication: For in vitro diagnostics. FTD _64 MANUAL- v3 2016_12 EN - 3 -
4 3. Intended use is an in vitro test for the detection of bacterial nucleic acid in swab samples, culture and urine as an aid to the evaluation of infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum and Ureaplasma parvum. 4. Pathogen information Chlamydia trachomatis (CT), an obligate intracellular Gram-negative bacterium, is one of the most commonly reported sexually transmitted diseases causing serious reproductive morbidity. The majority of persons with CT infection are not aware of their infection because they do show clinical symptoms. Untreated, it can lead to serious complications. In men, CT is associated with nongonococcal urethritis and epididymitis. In women, a chlamydia infection can lead to serious complications, including pelvic inflammatory disease and its subsequent sequelae, including ectopic pregnancy, infertility, and chronic pelvic pain transmitted disease. Chlamydial infection in newborns can cause ophthalmia neonatorum. Neisseria gonorrhoeae (gonococcus, GC) is a Gram-negative diplococcus of the Neisseria genus with a circular DNA genome. An important attribute of the virulence of Neisseria gonorrhoeae is its phenotypic variability. It is the secondmost-prevalent bacterial sexually transmitted infection worldwide. The disease is associated with high morbidity and socioeconomic consequences and remains a public health problem. Gonorrhea can cause severe complications, such as epididymitis in men and pelvic inflammatory disease in women leading to involuntary infertility and ectopic pregnancy. Extragenital sites of infection include the anorectum, oropharnyx and eyes. Mycoplasma genitalium (MG), a genital tract microorganism, belongs to the Mycoplasma species. It has been associated with significant reproductive tract inflammatory syndromes. In men, M. genitalium is a recognized as an important cause of non-gonococcal urethritis. In women, it can cause cervicitis, endometritis, urethritis and pelvic inflammatory disease. Additional evidence suggests that M. genitalium infects extragenital sites, such as mucous membranes of the respiratory and gastrointestinal tracts. FTD _64 MANUAL- v3 2016_12 EN - 4 -
5 Trichomonas vaginalis (Tvag) is an anaerobic, flagellated protozoan causing trichomoniasis, a sexually transmitted infection of the urogenital tract. Trichomoniasis is a common cause of vaginitis in women, while men with this infection can display symptoms of urethritis. An unusual genital discharge is usually noticed. Without treatment, trichomoniasis can increase a person s risk of acquiring HIV. Pregnant women with trichomoniasis can deliver premature, low birth weight babies. The WHO has estimated that 160 million cases of infection are acquired annually worldwide. Mycoplasma hominis (Mhom) is a small opportunistic bacterium without a cell wall and is known to frequently colonise the genital tract of sexually active men and women. It can be commensal or pathogenic. It can be a causal agent in pelvic inflammatory disease and infections during pregnancy, and it has been associated with bacterial vaginosis. It is associated with post-abortal and postpartum fever. In newborns, it can cause pneumonia, meningitis or abscesses. It has also been involved in extragenital infections, especially in immunocompromised patients. Ureaplasma urealyticum/parvum (Uurea/Uparv) are Gram-negative bacteria belonging to the family of Mycoplasmataceae lacking a cell wall. Ureaplasma are part of the normal genital flora of both men and women. In 2002, it was proposed subdividing this species into Uparv, comprising serotypes 1, 3, 6, and 14; and Uurea, comprising serotypes 2, 4, 5, and 7 through 13. These subtypes cannot be distinguished from each other with routine microbiological methods and are, therefore, usually referred to as Ureaplasma species. They are found in the lower genital tract of nearly 50% of pregnant women as part of the normal vaginal flora. Ureaplasma have been described to be associated with a number of diseases, including non-specific urethritis, infertility, chorioamnionitis, stillbirth, premature birth, and, in the perinatal period, pneumonia, bronchopulmonary dysplasia and meningitis. FTD _64 MANUAL- v3 2016_12 EN - 5 -
6 5. Contents Table 2: URscreen PP UTriMyc PP URETH PC UTriMyc PC Table of contents: PP= primer and probe, IC= internal control, PC= positive control, NC= negative control. Contents FTD FTD Primer/Probe mix CT, GC, MG, mcmv (IC) 1 x 48 µl 2 x 48 µl Primer/probe mix for Tvag, Mhom, Uurea, Uparv 1 x 48 µl 2 x 48 µl Positive control 1 containing plasmids for: CT, GC, MG Positive control 1 containing plasmids for: Tvag, Mhom, Uurea, Uparv 1 x 150 µl 2 x 150 µl 1 x 150 µl 2 x 150 µl NC Negative control 1 x 2000 µl 1 x 4000 µl IC Internal control 1 x 128 µl 2 x 128 µl Enzyme 25x RT-PCR Enzyme mix (Fast-track mastermix) 1 x 64 µl 2 x 64 µl Buffer 2x RT-PCR Buffer (Fast-track mastermix) 1 x 800 µl 2 x 800 µl Each vial contains additional volume for pipetting inaccuracy. The box itself, the cover of the box and each vial are labeled with a lot number. FTD _64 MANUAL- v3 2016_12 EN - 6 -
7 6. Precautions and warnings 6.1 Safety information Warning notice: the negative control contains lysis buffer. Hazardous pictogram: Signal word: Warning Hazardous statements: H315: Causes skin irritation. H317: May cause an allergic skin reaction. H319: Causes serious eye irritation. Precautionary statements: Prevention: P280: Wear protective gloves/protective clothing/eye protection/face protection. 6.2 Handling requirements Use of this product should be limited to personnel trained in the techniques of PCR. This product should be used in accordance with Good Laboratory Practice. Take the normal precautions required for handling all laboratory reagents. Do not mix reagents from different lots. Do not use the product after its expiration date. 6.3 Safe waste disposal Dispose of unused reagents and waste in accordance with country, state or local regulations. FTD _64 MANUAL- v3 2016_12 EN - 7 -
8 7. Storage and stability conditions The components of the FTD product should be stored in the original packaging at 20 C and are stable until the expiration date stated on the label. The product is shipped in frozen packages which should ensure a transport temperature under +10 C (satisfactory, according to stability studies). The reagents within a kit are suitable for 32 or 64 reactions. Freeze the product immediately after usage. More than 9x thawing and freezing of the reagents per tube should be avoided, as this may reduce assay sensitivity. We recommend aliquoting the reagents according to your needs after the first thawing. For stability performance data, please refer to 8. Principle of the method The bacterial/protozoan DNA of different pathogens is amplified in the same tube by polymerase chain reaction. The presence of specific bacterial sequences in the reaction is detected by an increase in fluorescence observed from the relevant dual-labeled probe, and is reported as a cycle threshold value (Ct) by the Real- Time thermocycler. The assay uses murine CMV (mcmv) as an internal control (IC), which is introduced into each sample and the negative control at the lysis buffer stage of the extraction process. FTD _64 MANUAL- v3 2016_12 EN - 8 -
9 9. Additionally required equipment FTD kits are suited for use with the Applied Biosystems 7500/7500Fast (Thermo Fisher Scientific), CFX96 (BIO-RAD), LightCycler 480 (Roche) and Rotor-Gene 3000, 6000, Q (Qiagen) and SmartCycler (Cepheid; in combination with Life Science software 2.0d). The assay has been fully validated on an Applied Biosystems 7500 with Fast-track mastermix and with the NucliSENS easymag (biomérieux). If you want to use different extraction methods, please firstly check their compatibility with FTD. For using the SmartCycler we recommend the FTD smartmix. Disposable powder-free gloves Pipettes (adjustable) Sterile pipette tips with filters Vortex mixer Desktop centrifuge For the ABI 7500, CFX96 and LightCycler 480, 96 well PCR plates and plate sealers are recommended. For the usage of the Rotor-Gene 3000/6000/ Q and SmartCycler use appropriate tubes and caps. Sample rack The validation file of Fast-track mastermix and a detailed compatibility list are available under Samples This test is for use with extracted nucleic acid from ( first catch ) urine samples, genital and rectal swabs of human origin. For long term storage FTD recommends storage of all samples at -20 C until extraction. FTD _64 MANUAL- v3 2016_12 EN - 9 -
10 11. Procedure 11.1 Preliminary extraction procedure using the easymag If you want to use different extraction methods, please firstly check their compatibility at Extraction of specimens and negative control with the easymag : 1. Thaw the negative control (NC, white cap) and the internal control (IC, dark blue cap). Before use, the reagents have to be thawed completely, mixed (by short vortexing) and spun down briefly. 2. Extract your samples and the NC. We recommend a starting volume for the extraction of 200 µl and an elution volume of 55 µl. It is well recognised that sensitivity is increased if a larger volume of clinical material is extracted into a small volume of eluate. This is particularly important with samples where pathogen load is expected to be low, such as CSF (and others). In this circumstance, we recommend an input volume of at least 500 µl. Where extreme sensitivity is required, such as certain clinical situations involving HIV or hepatitis viruses in blood, up to 1ml should be extracted. Please follow manufacturer`s extraction kit recommendations. 3. Add 2 µl internal control (IC, blue cap) directly to the lysis buffer of each extraction. Never add the internal control directly to the sample unless they are in lysis buffer. Adding the internal control to each of the samples and to the negative control is a very important step to see if the nucleic acid isolation has been successful and to check for possible PCR inhibition. 4. Do not extract positive controls as they are plasmids and will be inhibited. 5. Make sure to refreeze the left over volumes of NC and IC right after usage. FTD _64 MANUAL- v3 2016_12 EN
11 11.2 Main PCR setup procedure Preparation of PCR with Fast-track mastermix: 1. Thaw reagents for the reaction: URscreen PP and UTriMyc PP, the positive controls (PC) and 2x RT-PCR buffer (Fast-track mastermix, light blue cap) of Fast-track mastermix. The PC and the extracted NC have to be included in each run. Before use, the reagents have to be thawed completely, mixed (by short vortexing) and spun down briefly. The positive controls need to be thawed at room temperature for minutes and vortexed thoroughly right before use. Make sure to keep 25x RT-PCR enzyme (Fast-track mastermix, orange cap) of Fast-track mastermix in a freezer or on a cooling block at all times. 2. Pipette the required amount of 2x RT-PCR buffer in a 1.5ml tube. Do not immerge the whole tip into the liquid when pipetting 2x RT-PCR buffer to avoid waste of material and to obtain accurate volumes. Pipetting must be done very slowly to prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing. 3. Add according amount (see Table 3) of URscreen PP and UTriMyc PP to 2x RT-PCR buffer. Take care to change the tips after each pipetting step. 4. Pipette the required amount (see Table 3) of 25x RT-PCR enzyme to URscreen PP and UTriMyc PP with 2x RT-PCR buffer (reaction mix). Do not immerge the whole tip into the liquid when pipetting 25x RT-PCR enzyme to avoid waste of material and to obtain accurate volumes. Pipetting must be done very slowly to prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing. Take care to change the tips after each pipetting step. Vortex the complete master mix briefly and spin it down. If you use the SmartCycler please add per reaction 4µl of FTD smartmix to reaction mix. 5. Make sure to refreeze the remaining volumes of PP, PC and 2x RT-PCR buffer (Fast-track mastermix) after usage. FTD _64 MANUAL- v3 2016_12 EN
12 Table 3: Shown are the amounts of reagents that are needed for 1, 15, 32 and 64 wells. FTD : Each PPmix is sufficient for 32 reactions (+ pipetting inaccuracy). A minimum of 1 patient up to maximum 30 patients plus PC and NC is possible. FTD : Each PPmix is sufficient for 64 reactions (+ pipetting inaccuracy). A minimum of 1 patient up to maximum 62 patients plus PC and NC is possible. Number of reactions FTD /64 Buffer 12.5 µl µl 400 µl 800 µl PPmix 1.5 µl 22.5 µl 48 µl 96 µl Enzyme 1 µl 15 µl 32 µl 64 µl Total 15 µl 225 µl 480 µl 960 µl Preparation of a 96 well plate for the ABI 7500: All our tests are validated on ABI 7500, Bio-Rad CFX96, LightCycler 480 and RotorGene. If you intend to use the Bio-Rad CFX96 or the LightCycler 480 you must use appropriate plates and adhesive films. For RotorGene and SmartCycler use adequate tubes and caps. If you intend to run our tests on a different cycler, please firstly refer to: Preparation of a 96 well plate for ABI Take a 96 well plate which is compatible with the ABI Pipette 15 µl of the reaction mix (URscreen PP) in the wells. 3. Pipette 15 µl of the reaction mix (UTriMyc PP) in the wells. 4. Add 10 µl of the extracted samples, the extracted negative control and the positive control (which is not extracted; thaw at room temperature for minutes and vortex thoroughly right before use). Each run must include a negative and a positive control. 5. Mix briefly by pipetting up and down. 6. Close the plate with the ABI optical adhesive film. 7. Slightly vortex the plate and centrifuge briefly afterward. 8. Put the plate in the ABI Figure 1 (12 patients + PC + NC) shows an example for location of samples and controls on an ABI 7500 plate. FTD _64 MANUAL- v3 2016_12 EN
13 Figure 1: Schematic presentation of an example for location of samples and controls on a 96 well plate for the ABI Rows A-H; columns 1-12= layout of the 96 well plate S1; S2; S3; S4,, S12= reaction mix and samples 1-12 PC= reaction mix and positive control (C-D1) NC= reaction mix and negative control (C-D2) yellown background= reaction mix with URscreen PP (A1-12, C1-2) green background= reaction mix with UTriMyc PP (B1-12, D1-2) FTD _64 MANUAL- v3 2016_12 EN
14 12. Programming of the thermocycler Pay particular attention to the settings for the detectors: Table 4: Settings of the detectors. PP mix Pathogen Dye Detection wavelength (nm) * Neisseria gonorrhoeae green 520 URscreen Mycoplasma genitalium yellow 550 Chlamydia trachomatis orange 610 mcmv (IC) red 670 Trichomonas vaginalis green 520 UTriMyc Ureaplasma urealyticum yellow 550 Ureaplasma parvum orange 610 Mycoplasma hominis red 670 *The mentioned detection wavelengths are from the ABI They can be slightly different on other machines. NEW! Fast-track mastermix PCR programme 2: 50 C for 15 minutes hold 94 C for 1 minute hold 40 cycles of: 94 C for 8 second 60 C for 1 minute FTDliquid kits and FTlyo kits usage: To use FTlyo and FTDliquid kits on one plate or FTlyo kits alone, use the PCR programme 2. To use only FTDliquid kits, FTD is recommending the optimized PCR programme 2, as all validations are done with this program. But be aware, that you still can use the former PCR programme 1 as well. Fast-track mastermix PCR programme 1: 42 C for 15 minutes hold 94 C for 3 minutes hold 40 cycles of: 94 C for 8 seconds 60 C for 34 seconds FTD _64 MANUAL- v3 2016_12 EN
15 Detailed information on programming of the thermocyclers is provided in the instruction manuals of the cyclers which can be downloaded from our homepage IMPORTANT NOTES: If you use the ABI 7500, it is necessary to change the setting for the passive reference dye. (By default, the ROX dye is selected). After the step specifying the detectors and task for each well, click finish and the software will create the plate document. Click on a well, or click-drag, to select replicate wells. Enter the sample name and change the passive reference to none. If you use the ABI 7500 Fast, do NOT use the fast programme. If you use the RotorGene turn off auto-gain optimization and set gains for yellow, orange, red and green channels on 5. If you use the LightCycler 480, it is necessary for you to perform one FTD color compensation run before you start using FTD tests. FTD advises to run a new FTD color compensation annually and after each maintenance of your device. The reagents for FTD color compensation are supplied for free by FTD. Use Abs Quant/Fit Points for analysis of the run. FTD recommends the usage of transparent 96well plates. If you use the SmartCycler, please be aware that it is currently validated in combination with the Cepheid, Life Science software 2.0d only and with the FTD smartmix (FTD). If you want to use different enzymes please refer to FTD. FTD _64 MANUAL- v3 2016_12 EN
16 13. Assay validation Set a threshold as follows: 1. All negative controls should be below the threshold. If there is a potential contamination (appearance of a curve in the negative control or a cluster of curves in specimens at high Ct for example above 36), results obtained are not interpretable and the whole run (including extraction) has to be repeated. 2. All the positive controls must show a positive (i.e. exponential) amplification trace. The positive controls must fall below a Ct of 33 (detailed information see Interpretation of results). 3. Check the component trace before accepting the exponential trace as real. Contact the equipment manufacturer or FTD for advice (support@fasttrackdiagnostics.com). 4. All internal controls must show a positive (i.e. exponential) amplification trace. The internal control must fall below a Ct of 33. If the internal control is above CT 33, this points to a purification problem or a strong positive sample that can inhibit the IC. FTD _64 MANUAL- v3 2016_12 EN
17 14. Setup on the ABI Open your experiment 2. On the drop down menu on the left choose Analysis [A] and Amplification Plot [B]. 3. Modify the Graph Type [C] as you prefer to Linear or Log and the Color [D] to Target. FTD _64 MANUAL- v3 2016_12 EN
18 4. In the top right corner of your screen choose Analysis settings [E]. 5. A new window opens: Analysis settings; highlight all targets of all tests [F]. 6. Unclick Use Default Settings, Automatic Threshold and Automatic Baseline [G] and Apply Analysis Settings [H]. FTD _64 MANUAL- v3 2016_12 EN
19 7. For advanced analysis, you can also change your settings for each target in the options window. Here you can modify the threshold and baseline for every single parameter [I]. 8. Check the positive controls, negative controls and internal controls first. They have to follow the specifications mentioned in point 13 (Assay validation). 9. If all controls meet the specified ranges, check your samples for positive traces. 10. Your Ct results for all color channels will be displayed on the View Well Table window. 15. Interpretation of results The positive controls and any positive samples will show an exponential fluorescence trace. Any specimen displaying an exponential trace is considered as positive. For example, if a sample (e.g. with the URscreen PP) shows an exponential fluorescence trace at a wavelength ~520 (green channel) it means that it contains Neisseria gonorrhoeae DNA. FTD _64 MANUAL- v3 2016_12 EN
20 16. Troubleshooting No signal with positive controls Incorrect programming of the temperature profile of the thermocycler Compare the temperature profile to the manual. Incorrect configuration of the PCR reaction Check your work steps by means of the pipetting scheme and repeat the PCR if necessary. Check calibration of pipettes. Incorrect handling of the positive controls Inadequate or no vortexing and thawing at room temperature The storage conditions for one or more product components did not comply with the instructions or the FTD kit has expired. Please, check the storage conditions and the expiration date (see the product label) of the reagents and use a new test, if necessary. Weak or no signal of the internal control The PCR conditions do not comply with the protocol. Check the PCR conditions and repeat the PCR with correct settings if necessary. The PCR was inhibited or no / too little internal control was added during the extraction. Make sure that your extraction method is compatible with FTD kits. A strong positive signal of a pathogen can occasionally inhibit the fluorescence of an internal control. Signals within the negative control A contamination occurred during preparation of the PCR or during extraction Repeat the PCR with new reagents in replicates. We recommend to pipette the positive controls last. Make sure that work space and instruments are decontaminated at regular intervals. If you have any further questions or if you encounter problems, please contact support@fast-trackdiagnostics.com. FTD _64 MANUAL- v3 2016_12 EN
21 17. Validation For detailed validation data such as sensitivity, specificity, clinical studies and external quality panel results, please refer to the related validation file at Legend of symbols FTD _64 MANUAL- v3 2016_12 EN
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