Manual FTD Tropical fever core

Transcription

1 Manual FTD Tropical fever core 32 reactions (catalog no. FTD-36-32) 64 reactions (catalog no. FTD-36-64) 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 FTD-36-32, FTD Fast Track Diagnostics Luxembourg S.à.r.l.; 29, rue Henri Koch; L-4354 Esch-sur-Alzette; Luxembourg FTD 36 32_64 MANUAL- v3 2015_09 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 36 32_64 MANUAL- v3 2015_09 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 FTD Tropical fever core Category: Multiplex Real-time PCR for detection of dengue virus, chikungunya virus, Salmonella spp., West Nile virus, Plasmodium spp., Rickettsia spp., Leptospira spp. 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 36 32_64 MANUAL- v3 2015_09 EN - 3 -

4 3. Intended use The FTD Tropical fever core is an in vitro test for the qualitative detection of bacterial, parasitic and viral nucleic acid in whole blood or urine samples as an aid to the evaluation of infections with dengue virus, chikungunya virus, Salmonella spp., West Nile virus, Plasmodium spp., Rickettsia spp. and Leptospira spp. 4. Pathogen information Dengue virus (DENV) is a positive-strand RNA virus of the genus Flavivirus. DENV is transmitted to humans by the bite of infected Aedes mosquitoes, mainly Aedes aegypti. After an incubation period of 2-7 days, dengue infection is asymptomatic in the majority of cases or may result in a wide spectrum of clinical symptoms, ranging from a mild flu-like syndrome such as fever in combination with severe headache, retro-orbital pain, myalgia and arthralgia (known as dengue fever) to the most severe forms of the disease, which are characterized by leukopenia, thrombocytopenia, increased vascular fragility, and permeability (dengue hemorrhagic fever). The latter may progress to hypovolemic shock (dengue shock syndrome). DENV infection is a major cause of disease in tropical and subtropical areas, with million infections occurring each year. Chikungunya virus (ChikV) is a single-stranded RNA virus of the genus Alphavirus, first identified in Tanzania in The transmission of this virus occurs by the bite of infected mosquitoes of the genus Aedes. Also blood borne transmission is possible. The incubation period ranges from 3 to 12 days. The onset is usually abrupt and the acute stage is characterized by sudden high fever, incapacitating arthralgia, myalgias, and skin rash. Chronic arthritis develops in about 15% of the patients and is associated with fever, asthenia, exacerbation of arthralgias, inflammatory polyarthritis, and stiffness. Neurological, ocular, and mucocutaneous manifestations have also been described. FTD 36 32_64 MANUAL- v3 2015_09 EN - 4 -

5 Rickettsia are obligate intracellular, Gram-negative bacteria of the genera Rickettsia, Orientia, Ehrlichia, Neorickettsia and Anaplasma. They are differentiated into the typhus group, spotted fever group and scrub typhus group. Rickettsiae are transmitted throughvia saliva or dust of dried feces of infected ticks,mites,fleas and lice. They are spread via lymphatic vessels to the regional lymph nodes and via blood vessels to endothelium throughout the body. Symptoms include fever, headache, muscle pain, cough and gastrointestinal problems. Rash, eschar, splenomegaly and lymphadenopathies are also common. Infected people may suffer pneumonitis, encephalitis and myocarditis at a later stage. Leptospira are obligate aerobic Gram-neg spirochete bacteria which are transmitted by direct contact with urine or fluids from infected animals or contaminated water. The infection occurs through cuts in the skin or through the mucous membranes, rarely through human-to-human contact or through animal bites and causes leptospirosis (Weil s disease). The incubation period is 2 days to 3 weeks. The majority of patients manifest a mild, anicteric febrile illness, but a minority of patients develops a severe form with multiorgan involvement,. Weil's disease is characterized by high fever, significant jaundice, renal failure, hepatic necrosis, pulmonary involvement, cardiovascular collapse, neurologic changes and hemorrhagic diathesis. West Nile virus (WNV) is an arthropod-borne positive-stranded virus of the genus Flavivirus. It is mostly transmitted by the bite of an infected mosquito (Aedes spp or Culex spp) or through contact with infected animals. It can also be transmitted by blood transfusion. Wild birds are the natural reservoir for WNV. The incubation period is 2-8 days. About 80% of human infections are apparently asymptomatic. Of those persons in whom symptoms develop, most have self-limited West Nile fever, characterized by the acute onset of fever, headache, fatigue, malaise, muscle pain, gastrointestinal symptoms and rarely a transient macular rash on the trunk and extremities. Neuroinvasive disease, termed West Nile encephalitis or meningitis occurs only in 1% of WNV infected persons and is characterized by a decreased level of consciousness and extrapyramidal disorders. FTD 36 32_64 MANUAL- v3 2015_09 EN - 5 -

6 Plasmodium is a parasitic protozoon of the genus Plasmodium. Four species are traditionally regarded as causing malaria in humans: Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium falciparum. P. knowlesi has recently been described as another causative agent. Human malaria occurs by transmission of Plasmodium sporozoites via a bite from an infected anopheline mosquito. The most severe form of Malaria (Malaria tropica) is caused by Plasmodium falciparum while the disease caused by P. vivax, P. ovale, P. malariae and P. knowlesi is rarely fatal. In the severe forms mental confusion, kidney failure, acute respiratory disease syndrome, coma and death may occur. The incubation time takes 7 days or more after the first exposure. Salmonella is a genius of rod-shaped, gram-negative, non-spore forming, predominantly motile enterobacteria. Salmonella are closely related to the Escherichia genus and are found worldwide in warm- and cold-blooded animals, in humans, and in nonliving habitats. They cause illnesses in humans and many animals, such as typhoid fever, paratyphoid fever, and the food-borne illness salmonellosis. Salmonella can survive several weeks in a dry environment and several months in water; thus, they are frequently found in polluted water; contamination from the excrement of carrier animals being particularly important. Aquatic vertebrates, notably birds and reptiles, are important vectors of Salmonella. Poultry, cattle, and sheep being frequently agents of contamination, Salmonella can be found in food, particularly meats and raw eggs. FTD 36 32_64 MANUAL- v3 2015_09 EN - 6 -

7 5. Contents Table 2: TF1 PP TF2 PP PC Table of contents: PP = primer and probe, IC = internal control, PC = positive control, NC = negative control. Contents Primer/Probe mix for dengue virus, Rickettsia spp., Salmonella spp. and West Nile virus Primer/Probe mix for Plasmodium spp., chikungunya virus, Leptospira spp. and internal control (S.equi) Positive control containing plasmids for the detection of dengue virus, Rickettsia spp., Salmonella spp, West Nile virus, Plasmodium spp., chikungunya virus and Leptospira spp. FTD FTD x 48 µl 2 x 48 µl 1 x 48 µl 2 x 48 µl 1 x 300 µl 2 x 300 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 36 32_64 MANUAL- v3 2015_09 EN - 7 -

8 6. Precautions and warnings 6.1 Safety information Warning notice: the negative control contains lysis buffer. Hazard description: Xn Harmful Risk phrases: R 22 Harmful if swallowed. R 36/38 Irritating to eyes and skin. Safety phrases: S 13 Keep away from food, drink and animal feeding stuffs. S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S 36 Wear suitable protective clothing. S 46 If swallowed, seek medical advice immediately and show this container or label. 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 36 32_64 MANUAL- v3 2015_09 EN - 8 -

9 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 viral RNA is transcribed into cdna using a specific primer-mediated reverse transcription step followed immediately in the same tube by polymerase chain reaction. The presence of specific pathogen 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 Streptococcus equi (Sequi) as an extraction control- the internal control (IC)- which is introduced into each sample and the negative control at the lysis buffer stage of the extraction process. FTD 36 32_64 MANUAL- v3 2015_09 EN - 9 -

10 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 whole blood or urine samples of human origin. For long term storage FTD recommends to store all samples at -20 C until extraction. Attention: Samples of heparinised patients must not be used as Heparin is a PCR-interfering substance. FTD 36 32_64 MANUAL- v3 2015_09 EN

11 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 36 32_64 MANUAL- v3 2015_09 EN

12 Note: If typhoid fever is expected 5 ml of whole blood should be pre-cultured in Tryptic Soy Broth (TSB) 10% oxgall medium for 5h at 37 C. Afterwards the extraction can be done with 10 ml of the culture medium using the QIAamp DNA blood midi/maxi (Qiagen) extraction kit according to the manufacturer's instruction. The elution has to be done in 2 steps by re-loading the column. For more detailed information please contact info@ftd-ltd.com Main PCR setup procedure Preparation of PCR with Fast-track mastermix: 1. Thaw reagents for the reaction: TF1 PP and TF2 PP, the positive controls (PC) and 2x RT-PCR buffer (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 (orange cap) of Fast-track mastermix in a freezer or on a cooling block at all times. 2. Pipette the required amount (see Table 3 below) of 2x RT-PCR buffer (Fast-track mastermix) to the according amount of TF1 PP and TF2 PP. Take care to change the tips after each pipetting step. 3. Pipette the required amount (see Table 3 below) of 25x RT-PCR enzyme (Fast-track mastermix) to TF1 PP and TF2 PP with 2x RT-PCR buffer (Fasttrack mastermix). Take care to change the tips after each pipetting step. Vortex the complete master mixes briefly and spin them down. If you use the SmartCycler please add per reaction 4µl of FTD smartmix to the TF1 PP and TF2 PP with the buffer and enzyme. 4. Make sure to refreeze the remaining volumes of PP, PC and 2x RT-PCR buffer (Fast-track mastermix) right after usage. FTD 36 32_64 MANUAL- v3 2015_09 EN

13 Table 3: FTD-36-32: FTD-36-64: Shown are the amounts of reagents that are needed for 1, 15, 32 and 64 wells. 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. 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-36-32/64 PPmix 1.5 µl 22.5 µl 48 µl 96 µl Buffer 12.5 µl µl 400 µl 800 µl Enzyme 1 µl 15 µl 32 µl 64 µl Total 15 µl 225 µl 480 µl 960 µl FTD 36 32_64 MANUAL- v3 2015_09 EN

14 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 TF1 PP with buffer and enzyme in the wells. 3. Pipette 15 µl of the TF2 PP with buffer and enzyme in the wells. 4. Add 10 µl of the extracted samples, the extracted negative control and the positive controls (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. Centrifuge briefly. 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 36 32_64 MANUAL- v3 2015_09 EN

15 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 = mastermix and samples 1-12 PC= mastermix and positive control (C1, D1) NC= mastermix and negative control (C2, D2) yellown background = mastermix with TF1 PP (A1-12, C1-2) green background = mastermix with TF2 PP (B1-12, D1-2) FTD 36 32_64 MANUAL- v3 2015_09 EN

16 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) * Dengue virus green 520 TF1 Rickettsia spp. yellow 550 West Nile virus orange 610 Salmonella spp. red 670 Plasmodium spp. green 520 TF2 Chikungunya virus Yellow 550 Leptospira spp. orange 610 S. equi (IC) red 670 *The mentioned detection wavelengths are from the ABI They can be slightly different on other machines. PCR programme: Fast-track mastermix 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 Detailed information on programming of the thermocyclers is provided in the instruction manuals of the cyclers which can be downloaded from our homepage FTD 36 32_64 MANUAL- v3 2015_09 EN

17 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. The reagents for FTD color compensation are supplied by FTD. 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 (Fast-track diagnostics). If you want to use different enzymes please refer to FTD. FTD 36 32_64 MANUAL- v3 2015_09 EN

18 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 36 32_64 MANUAL- v3 2015_09 EN

19 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 36 32_64 MANUAL- v3 2015_09 EN

20 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 36 32_64 MANUAL- v3 2015_09 EN

21 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 shows an exponential fluorescence trace at a wavelength ~520 (green channel) with TF1 PP, it contains dengue virus nucleic acid. FTD 36 32_64 MANUAL- v3 2015_09 EN

22 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 36 32_64 MANUAL- v3 2015_09 EN

23 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 36 32_64 MANUAL- v3 2015_09 EN