User manual of MeningoPlex Version_ MeningoPlex

Transcription

1 MeningoPlex Real-time PCR detection of Haemophilus influenzae, Neisseria meningitides, Streptoccocus pneumoniae and Listeria monocytogenes In vitro diagnostics -80 C to -20 C See cover RT-HNSL-050 See cover Controls DESCRIPTION Multiplex Real-Time PCR kit is dedicated for a detection of Haemophilus influenzae (serological group A to F), Neisseria meningitidis (serological group B and C), Streptococcus pneumoniae and Listeria monocytogenes. Diagnostic kit is dedicated for a molecular-biological diagnostic of main agent of serious bacterial meningitidis. Recommended input material is mainly CSF. We recommend process the biological material using any common commercial procedures for DNA purification. Kit was optimized for the purification of nucleic acids using Qiagen QIAamp Mini Kit. STORAGE CONDITIONS The components of the kit should be stored at 20 C (or lower) and repeated thawing and freezing should be avoided, as this may reduce assay performance. PMX II is light-sensitive and it is highly recommended to expose it to the light only for the time needed for assay preparation. GENERAL RECOMMENDATIONS FOR PCR DIAGNOSTICS PCR is a highly sensitive method capable of detecting even very small amounts of nucleic acid which can serve as a template to amplify and cause contamination analysis. The source of contamination PCR may be in particular: Laboratory table accessories and pipettes contaminated from previous isolation of nucleic acids Contacts between samples PCR products from previous analyzes STD/positive controls Diagnostics using molecular genetic analyzes can be performed only by qualified persons for this purpose trained and technically qualified. Manufacturer is not liable for erroneous results caused by improper handling. Physical separation of spaces/areas for: Nucleic acids purification Preparation of reagents for amplification PCR analysis and evaluation 1 Rev_ , ver

2 Each area must be equipped with instruments and tools that are specifically meant for the space (room or laminar box), you must use only tools in the appropriate space and do not transfer them between the outlined areas. Extraction of nucleic acids and preparation of PCR reagents must be performed in a reserved safety cabinet and PCR laminar flow hood or other sterile place which is equipped with an UV lamp. The equipment should be emitted at least 15 minutes with UV light before the use, the use of substances degrading the DNA/RNA (thermo-dna-tor, etc.) is recommended. For storage it is recommended to physically separate reagents into individual boxes for nucleic acid extraction, preparation of PCR reactions, especially you have to put into different boxes positive controls, which are the most common source of contamination. When handling reagents and accessories it is recommended: Maintain strict physical separation of individual practices Do not use expired or otherwise depreciated products Reagents can be in a clean place aliquoted for daily use and properly stored Use RNase and DNase-free solutions and sterile filter tips Before and after analysis use chemical nucleic acid decontamination method for all surfaces, pipettes, racks and accessories, or use UV lamps. Avoid aerosol formation Use disposable gloves and change them regularly. Empty containers for biological waste after each procedure During the extraction of nucleic acids and preparation of PCR left gaps in the rack between the tubes to reduce the risk of contamination between samples. When preparing PCR pipette in following order: 1st - negative control, 2nd - analyzed samples, 3rd - positive control DISPOSAL OF THE REST OF THE KIT The individual kit components and consumables are recommended to be treated as infectious material. Waste is appropriate to place into containers for the disposal of infectious biological material. KIT CONTAINS: Amplification reagents for the preparation of 50 assays for Real-Time PCR analysis: Reagents for the preparation of NS mastermix for the detection of H. influenzae and internal controls HI PMX I (600µl), HI PMX II (24µl), HI PMX III (380µl), HI STD (55 µl) - positive control Amplification reagents for the preparation of 50 assays for Real-Time PCR analysis: Reagents for the preparation of NS mastermix for the detection of N.meningitidis and S.pneumoniae NS PMX I (600µl) NS PMX II (24µl) NS PMX III (60µl) NS H20 (350µl) NS STD (55 µl) - positive control Amplification reagents for the preparation of 50 assays for Real-Time PCR analysis: Reagents for the preparation of NS mastermix for the detection of L. monocytogenes and internal controls LM PMX I (600µl) LM PMX II (24µl) LM PMX III (380µl) LM STD (55 µl) positive control Taq Polymerase (70 µl) for all mastermixes Negative control (170 µl) - for all mastermixes 2 Rev_ , ver

3 REQUIRED REAGENTS AND CONSUMABLES, WHICH ARE NOT INCLUDED IN THE KIT Purification kit Filter tips SmartCycler/PCR tubes, eventually PCR plates Tubes about volume 1,5-2ml for preparation of Mastermix Transparent adhesive foil for PCR plates REQUIRED INSTRUMENTATION Centrifuge with minimal centrifugal acceleration of 14,000 x g Thermoblock or water bath maintained at 56 C Biohazard box, box PCR Pipettes with adjustable volume μl Vortex, chilled block Real Time Cycler (SmartCycler/Cepheid; RotorGene/Qiagen; LightCycler 480/Roche). Refrigerator, freezer BIOLOGICAL MATERIAL The recommended material is cerebrospinal fluid (CSF), whole blood (EDTA/citrate), swabs. Biological material is recommended to keep at 0 to 4 C. If necessary, the sample can be frozen and stored long term at -20 C. In the case of cerebrospinal fluid analysis it is appropriate to use at least 200μl of the sample, the recommended amount is μl sample. The method was tested for species H.influenzae, N.meningitidis, S.pneumoniae and L.monocytogenes. Specificity was verified on the above mentioned species and the related organisms, and many other bacterial strains. Estimated sensitivity: 10E1-10E2/ml. PROCEDURE FOR DNA PURIFICATION FROM CSF, SERUM AND SWAB Suggested commercial kits for the processing of the above mentioned samples: QIAamp DNA Mini Kit(50) REF: QIA / (250) REF: QIA QIAamp MinElute Virus Spin Kit(50) REF: QIA QIAamp DNA Blood Mini Kit(50) REF: QIA / (250) REF: QIA Samples are recommended to process according to the manufacturer's instructions of the particular kit. The treated sample can be directly used for amplification or stored in a refrigerator at 2 to 8 C for 24h. For longer storage temperature range -20 C to -80 C is recommended. REAL-TIME PCR ANALYSIS (reaction volume = 20 μl) After a spontaneous thawing all tubes necessary to prepare the analysis should be vortex thoroughly (It is inappropriate exhibit the solutions in higher temperatures than ambient for quicker thawing!). Subsequently, all the components in the required volume needed for a mastermix preparation for particular sample count (n+1) and one positive and one negative control are pipetted into a clean sterile minicentrifuge tubes (1.5-2 ml) according to the protocols: Preparation of the HI mastermix: Preparation of the NS Mastermixu: Preparation of the LM Mastermix PCR/1reaction (20µl): 2 l DNA sample 10 l HI PMX I 0,4 l HI PMX II 7,2 l HI PMX III 0,4 l Polymerase (5U/ l) 20 l total volume sample PCR/1reaction (20µl): 2 l DNA sample 10 l NS PMX I 0,4 l NS PMX II 1 l NS PMX III 6,2 μl NS H 2O 0,4 l Polymerase (5U/ l) 20 l total volume sample PCR/1reaction (20µl): 2 l DNA sample 10 l LM PMX I 0,4 l LM PMX II 7,2 l LM PMX III 0,4 l Polymerase (5U/ l) 20 l total volume sample 3 Rev_ , ver

4 The tubes with the prepared mastermix should be vortex thoroughly and then 18 μl aliquots of the mastermix are pipetted into the Smart/PCR tubes placed in a chilled block. Then pipette 2 μl of isolate/positive and negative controls into the appropriate tubes/wells in the plate. (It is recommended to first pipet negative controls (ddh2o or TE buffer), prepared DNA samples, and as the last positive controls). After addition of the negative control, sample and the positive control it is recommended close immediately the tubes to not increase the risk of contamination between the tubes. Then seal the plate thoroughly with a transparent foil. Subsequently, tubes should be centrifuged for about 10 seconds in the centrifuge and placed in a thermal cycler. PROTOCOL REAL-TIME PCR SET-UP IN SMARTCYCLER SOFTWARE: In the software menu new temperature-time analysis profile is created over the "New protocol" button: Subsequently, enters the number of test samples and defines their location in specific positions cycler. Despite icon "Start Run " to start the analysis. Treshold values for the individual optical channels: Channel 1 (FAM) 30 RFU Channel 3 (TxR) 10 RFU for Mastermixes HI and LM Channel 3 (TxR) 30 RFU for Mastermixe NS RESULTS EVALUATION HI Mastermix Detection of Haemophilus influenzae is based on the FAM signal detection in the first channel (Channel1). Detection of the internal control is based on the TxR signal detection in the third channel (Channel3). NS Mastermix Detection of Neisseria meningitidis is based on the FAM signal detection in the first channel (Channel1). Detection of Streptococcus pneumoniae is based on the TxR signal detection in the third channel (Channel3). LM Mastermix Detection of Listeria monocytogenes is based on the FAM signal detection in the first channel (Channel1). Detection of the internal control is based on the TxR signal detection in the third channel (Channel3). 4 Rev_ , ver

5 Haemophilus influenzae(fam) + Internal control(txr) Neisseria meningitidis(fam) + Streptococcus pneumoniae(txr) 5 Rev_ , ver

6 Listeria monocytogenes(fam) + Internal control(txr) The results are judged as valid only if the positive control gives a positive result (RFU > 30), a negative control is negative in the analysis result (RFU < 30). A sample was considered positive when fluorescence exceeded an increase threshold value Threshold 30RFU. A sample is considered negative only if the analysis shows a positive internal control signal. If the increase in fluorescence exceeded Threshold later than the 45th cycle, it is recommended to repeat the analysis. In case of positive samples Listeria monocytogenes, the high intensity of positivity can cause the inhibition of amplification of internal controls - such samples are positive despite the negative signal of internal control. PROTOCOL REAL-TIME PCR SET-UP IN ROTOR-GENE SOFTWARE: 1. Set the correct rotor, suitable sample volume, sample position, and create a temperature-time analysis profile over the New icon: for both Mastermixes. STEP TEMPERATURE ( o C) TIME NUMBER OF CYCLES Hold 95 8 min s Cycling 57 40* fluorescence detection s 6 Rev_ , ver

7 2. Set the fluorescence channels as shown: temperature 57 C and check the box Perform Optimisation Before 1st Acquisition 3. Place the PCR tubes into a device. 4. Run the amplification program. 5. Complete data analysis and interpretation of results after the measurement is complete. CHANNEL THRESHOLD Treshold value OUTLIER REMOVAL Deleting deviations SLOPE CORRECT Correction of slope curve FAM/Green 0,05 15% On TxR/ROX/Orange 0,05 0% On 7 Rev_ , ver

8 Neisseria meningitidis + Haemophilus influenzae + Listeria monocytogenes channel FAM/Green Streptococcus pneumoniae + Internal controls (HI/LM mastermixes) channel ROX/Orange 8 Rev_ , ver

9 RESULTS EVALUATION DETECTION CHANNEL FAM / GREEN ROX / ORANGE NS mastermix Neisseria meningitidis + Streptococcus pneumoniae + HI mastermix Haemophilus influenzae + Internal control + LM mastermix Listeria monocytogenes + Internal control + A sample was considered positive if the Ct value is present. The fluorescence curve has a typical sigmoid shape and crosses the treshold line in exponential growth only once. A sample was considered negative if the Ct value is not present. The fluorescence curve doesn t have a typical sigmoid shape and doesn t cross the treshold line. If the sample is positive Haemophilus influenzae or Listeria monocytogenes, may inhibit internal control, in this case the sample is considered positive. SETTING THE REAL TIME PCR REACTION IN THE LIGHTCYCLER SOFTWARE: Insert the plate into the device. Choose New Experiment, on the Run protocol tab enter the factory-specified detection format - Dual Color Hydrolysis Probe/UPL Probe. Set the reaction volume - 20μl, and the temperature profile as below: Program name Cycles Analysis Mode Hold 1 None Cycling 55 Quantification Cooling 1 None Target, o C Acquisition Mode Hold (hh:mm:ss) Ramp Rate ( o C/s) 95 None 00:08: None 00:00: Single 00:00: None 00:00: None 00:00: Through the Sample Editor field at the top left enter the type of analysis, units, set type and position of individual samples and controls. 9 Rev_ , ver

10 Go back through the Experiment field at the top left and at the bottom right click on Start Run, save this run (no save cannot run test). After the run, click on Analysis in the left-hand menu. In the new window, select Abs Quant/Fit points, then mark the samples and click on Calculate. Channel FAM( ) Neisseria meningitidis +Haemophilus influenzae + Listeria monocytogenes 10 Rev_ , ver

11 Channel Yellow( ) Streptococcus pneumoniae + Internal control (HI/LM mastermixes) TROUBLESHOOTINGS Problem Cause Suggestions 1. Problems during nucleic acid See the instructions of used See the instructions of used purification purification kit purification kit a) enzyme was not added to the reaction or is inactive Ensure that the enzyme was added to the correct tube and repeat the analysis 2. Positive samples and positive controls are negative 3. Negative controls or negative samples are positive b) the wash buffers has been not completely removed during the purification using the Qiagen kit b) positive control and positive samples were repeatedly thawed and refreezed d) positive control or positive samples were stored at improper temperature e) part or the whole kit has been stored incorrectly or is expired f) during the purification using the Qiagen kit ethanol was not added in the respective buffers a) contamination from previous PCR analysis or Make sure that the centrifugation speed was properly set during the purification, repeat the purification and subsequent steps Avoid repeated thawing, repeat the procedure with new controls/samples Samples and controls should be stored at temperature recommended by the manufacturer, repeat the analysis with new controls/samples Make sure that all the components were stored at the correct temperature and check the lifetime of the kit Make sure that ethanol was added to the appropriate buffers, repeat the purification and subsequent analysis Change the tip after each pipetting 11 Rev_ , ver

12 contamination during the analysis Use the filter tips Maintain the recommended separation of individual practices Use UV lamps and disinfectants for all desktops Change gloves regularly Handle all reagents with care, avoid unnecessary contact the individual tubes and avoid aerosol formation When pipetting, avoid the contact with the inner surface of the tube or lid Test tubes in the stand and in a centrifuge alternate with empty positions Repeat the analysis with new controls and samples 4. Lower result values than expected a) isolate was stored too long before being analyzed b) purification was too slow or inappropriately discontinued c) PCR was not performed properly due to poor handling kit Isolated samples are recommended to analyze as soon as possible after purification, repeat the purification and perform Real-Time PCR immediately Samples are recommended process continuously without unnecessary time delays, repeat the isolation and subsequent analysis Make sure the kit is stored in optimal conditions recommended by the manufacturer and repeat the analysis 12 Rev_ , ver

13 Producer: DYNEX LABORATORIES, s.r.o. Contact: Lidická 977, Buštěhrad, Czech Republic, Tel: , Fax: , Headquartes: Vodičkova 791/41, Prague 1, Czech Republic 13 Rev_ , ver