INNO-LiPA MYCOBACTERIA v2 Amp

Transcription

1 KEY-CODE: FRI INNO-LiPA MYCOBACTERIA v2 Amp v p 1/8 English INNO-LiPA MYCOBACTERIA v2 Amp Manufactured by: Fujirebio Europe N.V. Technologiepark Gent Belgium Tel BTW BE RPR Gent Distributed by: Fujirebio Europe N.V. Tel Fax customer.support@fujirebio-europe.com Fujirebio Germany GmbH Tel Fax germany@fujirebio-europe.com Fujirebio France SARL Tel Fax france@fujirebio-europe.com Fujirebio Italia S.r.l. Tel Fax italy@fujirebio-europe.com Fujirebio Iberia S.L. Tel Fax spain@fujirebio-europe.com Note changes highlighted EUROPE GR IS LT RO SK TR LI MT EE non-europe US CA AR, BR, CO, UY, AU, NZ, RU :00 17:00 GMT+1 M T W T F S S Fujirebio Europe N.V.

2 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 2/8 TABLE OF CONTENTS Symbols used... 2 Intended use... 2 Test principle... 3 Reagents... 3 Description, preparation for use and recommended storage conditions... 3 Materials required but not provided... 4 Sample decontamination... 4 Sample preparation... 4 Amplification... 4 Safety and environment... 4 Specimen, collection preparation and handling... 5 Remarks and precautions... 5 Test procedure... 6 Results... 7 Visualization... 7 Quality control... 8 Limitations of the procedure... 8 Test performance... 8 Licenses... 8 Trademarks... 8 Symbols used Amplification kit Manufacturer In vitro diagnostic medical device Batch code Catalogue number Use by Consult instructions for use Temperature limitation Amplification buffer Primer solution Intended use The INNO-LiPA MYCOBACTERIA v2 Amp kit, for in vitro use, generates biotinylated amplified material from the 16S-23S rrna spacer region by using biotinylated oligonucleotide primers. This amplified material is subsequently used in the INNO-LiPA MYCOBACTERIA v2 assay for the detection and identification of the genus Mycobacterium and 16 different mycobacterial species.

3 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 3/8 Test principle The first step is the isolation of the bacterial DNA from full grown cultured samples (liquid or solid culture). The amplification is based on the polymerase chain reaction (PCR).The DNA sample to be amplified is introduced in a reagent mixture containing essential components for amplification, including deoxyribonucleoside 5'-triphosphates (dntps), biotinylated primers, and thermostable DNA polymerase. The primers used in this test amplify the 16S - 23S ribosomal RNA (rrna) spacer region of Mycobacterium species, and are therefore called "primers". By heating, the two strands of the DNA helix are separated (denaturation) in order to expose the target sequences to the biotinylated primers. These oligonucleotide primers are complementary to the regions flanking the probe-target sequence. Therefore, upon cooling to a specified temperature, the primers will bind to their specific sequence (annealing). At an elevated temperature, in the presence of dntps, the thermostable DNA polymerase will extend the annealed primers along the target template (extension). A biotinylated exact copy of the template sequence is produced after one cycle of denaturation, annealing, and extension. The process is repeated for 40 cycles, thus yielding a multifold amplified biotinylated target sequence. Reagents Description, preparation for use and recommended storage conditions - If kept at -25/-15 C and stored in the original vials, the reagents are stable until the expiry date of the kit. Do not use the reagents beyond the expiry date of the kit. Aliquoting the reagents after first use is recommended (4 freeze/thaw cycles allowed). - All reagents should be brought to room temperature (20-25 C) approximately 30 minutes before use and should be returned to the freezer immediately after use. All vials should be closed immediately after use. - The reagents should be stored isolated from any source of contaminating DNA, especially amplified DNA products. Avoid microbial contamination of reagents. Reagents supplied: Component Quantity Ref. Description Amplification buffer Primer solution 0.3 ml Containing all dntp's and 0.05% NaN 3 as preservative (transparent capped tube). 0.3 ml Containing biotinylated primers, MgCl 2, and 0.05% NaN 3 as preservative (green capped tube).

4 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 4/8 Materials required but not provided Sample decontamination - NALC-NaOH (3% NaOH, 0.5% N-acetyl L-cysteine, 50 mm Na 3 citrate). A: Na 3 citrate 2H 2 O (100 mm). B: NaOH (6%). Mix an equal volume of A and B together and divide this solution into 50 ml aliquots. Before using, add 0.25 g NALC. Use within 24 hours as NALC loses mucolytic activity in solution. - Phosphate buffer (33.5 mm Na 2 HPO4, 33.5 mm KH 2 PO4, ph 6.8) ml screw-capped centrifuge tubes (e.g. Falcon tubes). - Vortex. - Centrifuge (up to 3000 g). Sample preparation - TE buffer (10 mm Tris-HCl, 1 mm EDTA, ph 8.0). - Sterile screw-capped microcentrifuge tubes (1.5 ml). - Water bath or dry heating block. - Microtube centrifuge. - Vortex. - Disposable gloves. - Disposable sterile pipette tips (preferably cotton-plugged). Amplification - Pipettes adjustable to deliver 1-20 µl, µl, and µl. - Disposable gloves. - Disposable sterile pipette tips (preferably cotton-plugged). - Microtube racks. - Sterile microtubes. - Microtube centrifuge. - DNA thermal cycler and equipment. - Thermostable DNA polymerase (5 U/µL) (AmpliTaq DNA polymerase was used for assay optimization. When using other commercially available DNA polymerases, validate the test performance in your laboratory). - Vortex. - Autoclaved distilled water. Safety and environment Please refer to Safety Data Sheet (SDS) and product labelling for information on potentially hazardous components. The most recent SDS version is available on the website - Only adequately trained personnel should be permitted to perform the test procedure. - Specimens should always be handled as potentially infectious. Therefore, all blood components and biological materials should be considered as being potentially infectious and should be handled as such.

5 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 5/8 - All blood components and biological materials should be disposed of in accordance with established safety procedures. Autoclave for at least 15 minutes at 121 C. Incinerate disposable material. Mix liquid waste with sodium hypochlorite so that the final concentration is ± 1% sodium hypochlorite. Allow to stand overnight before disposal. CAUTION: Neutralize liquid waste that contains acid before adding sodium hypochlorite. - Use of personal protective equipment is necessary: gloves and safety spectacles when manipulating dangerous or infectious agents. - Waste should be handled according to the institution's waste disposal guidelines. All federal, state, and local environmental regulations should also be observed. Specimen, collection preparation and handling NOTE: Reagents are not supplied. IMPORTANT: - It is essential that all reagents and materials used for DNA extraction are free of amplification product contamination. Prepare all reagents in an amplified productfree environment. Wear disposable gloves to prevent contamination. Use autoclaved materials and disposable sterile pipette tips (preferably cotton-plugged). All specimen handling should be carried out as recommended by the Centers for Disease Control (CDC; Atlanta, GA). A. Solid medium - Culture mycobacteria on a solid medium (Löwenstein-Jensen). - Suspend a small amount from numerous colonies from the slant in 1.5 ml TE buffer (10 mm Tris-HCl, 1 mm EDTA, ph 8.0) in a screw-capped microcentrifuge tube. - Heat inactivate at 95 C for 10 minutes. - Centrifuge at g for 5 minutes. - Use 2 µl of the supernatant in a total reaction volume of 50 µl. B. Liquid medium - Collect 0.2 ml liquid medium in a screw-capped microcentrifuge tube. - Concentrate 0.2 ml liquid medium by centrifugation (15 minutes, g). - Discard supernatant according to safe laboratory practice. - Perform an additional centrifugation to remove any liquid still adhering to the tube walls. - Re-suspend the pellet in 20 µl TE buffer. - Heat inactivate at 95 C for 30 minutes. - Centrifuge at g for 10 seconds. - Freeze at -20 C for 30 minutes. - Vortex and centrifuge at g for 10 seconds. - Use 10 µl of the supernatant in a total reaction volume of 50 µl. Remarks and precautions - Briefly vortex and centrifuge the reagents before opening the vials. Close all vials immediately after use.

6 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 6/8 - Do not mix reagents between kits, unless the components have identical lot numbers. - All pipette tips and tubes used for the amplification process should be autoclaved. Use a new sterile pipette tip for each aliquoted specimen. Pipette tips with cotton plugs are recommended. - Specimens should be handled as potentially infectious. - In order to avoid DNA contamination, a maximal physical separation between the pre- and post-amplification steps is recommended. Test procedure The amplification protocol for the amplification of the 16S-23S rrna spacer region of Mycobacterium species was designed for optimal amplification using GeneAmp PCR tubes (0.5 ml) and PE-480 thermal cyclers, or MicroAmp PCR tubes (0.2 ml) and GeneAmp PCR System 9700 thermal cyclers and using thermostable DNA polymerase - see Material required but not provided. The protocol can be used for most commercial types of thermal cyclers, but may require some modifications depending on the type of thermal cycler. Prior to use, determine whether the protocol is compatible with the thermal cycler in use at your laboratory. Ensure that the thermal cycler is calibrated prior to use. Take the following necessary precautions to avoid aspecific amplification and primerdimer formation, which may impair the results: - Perform the subsequent pipetting steps on ice and without delay. - Preheat the heating block of the thermal cycler to 95 C before inserting the samples, and immediately start the cycling process. - Briefly centrifuge the reagents before opening the vials. A. Solid medium 1. Determine the number of "N": N = (Number of samples to be amplified) + (one negative control) + 1. Using a cotton-plugged pipette tip, prepare a master mix in an autoclaved 1.5 ml tube: (N x 27.7 µl) autoclaved distilled water. + (N x 10 µl) Amplification buffer (transparent-capped). + (N x 10 µl) Primer solution (green-capped). + (N x 0.3 µl) Taq polymerase (N x 1.5 U) (5 U/µL). The total volume of this amplification mix is now (N x 48 µl). Vortex briefly and aliquot 48 µl of this master mix into (N-1) autoclaved amplification tubes. 2. Add 2 drops (40 µl) of mineral oil into each tube if required (e.g. when using the PE-480). Pipette 2 µl of the DNA preparation (through the mineral oil if required). Add 2 µl of TE buffer to the negative control tube. NOTE: Do not vortex or mix! Go to 3.

7 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 7/8 B. Liquid medium 1. Determine the number of "N". N = (Number of samples to be amplified) + (one negative control) + 1. Using a cotton-plugged pipette tip, prepare a master mix in an autoclaved 1.5 ml tube: (N x 19.7 µl) autoclaved distilled water. + (N x 10 µl) Amplification buffer (transparent-capped). + (N x 10 µl) Primer solution (green-capped). + (N x 0.3 µl) Taq polymerase (N x 1.5 U) (5 U/µL). The total volume of this amplification mix is now (N x 40 µl). Vortex briefly and aliquot 40 µl of this master mix into (N-1) autoclaved amplification tubes. 2. Add 2 drops (40 µl) of mineral oil into each tube if required (e.g. when using the PE-480). Pipette 10 µl of the DNA preparation (through the mineral oil if required). Add 10 µl of TE buffer to the negative control tube. NOTE: Do not vortex or mix! 3. Place the samples into the pre-heated and calibrated thermal cycler (see instructions indicated by the manufacturer of the available thermal cycler). Start the amplification program designed for the amplification of the 16S-23S rrna spacer region of Mycobacterium spp. Amplification Profile: Cycler type PE-480, PE-2400, PE-9600, PE-9700 Step Temp Time 1. Denature 95 C 1 min. 2. Denature 95 C 30 sec. repeat cycle 3. Anneal primers 62 C 30 sec. step Extend primers 72 C 30 sec. 40 times 5. Hold at 4 C - 4. After the amplification process, use immediately in INNO-LiPA MYCOBACTERIA v2 or store the samples at -20 C. NOTE: Do not store the amplified DNA products together with unused amplification reagents. Results Visualization - The presence of the amplified product can be checked on a 2% agarose gel. Load 10 µl of amplified product per slot. The amplicon should appear as a single band with a length of 400 to 550 bp. - Please note that some amplification of non-mycobacterium species DNA may occur with the primers in the kit and that amplified products may be visible when analyzed on an agarose gel. - Due to the high specificity of the probes, these non-mycobacterial amplicons will not react on the INNO-LiPA MYCOBACTERIA v2 strip.

8 80347 INNO-LiPA MYCOBACTERIA v2 Amp / v2 / FRI95476 p 8/8 Quality control - Include at least one positive and one negative control each time an amplification test is performed. - As with any new laboratory procedure, the inclusion of additional positive and negative controls should be considered until a high degree of confidence is reached in the correct performance of the test procedure. - If the inclusion of an additional positive control is desirable, use a known positive sample, but take care to avoid contamination. - If no band is obtained on the gel for the positive control, the entire run should be discarded and the complete procedure should be repeated. If a band, as mentioned in Visualisation, is obtained on gel for the negative control, the negative control should be tested on the INNO-LiPA MYCOBACTERIA v2 strip. If the negative control does not react on strip, all amplicons of the run may be tested on strip. Otherwise, the entire run should be discarded and the complete procedure should be repeated. Limitations of the procedure - Use of this product should be limited only to personnel well trained in the techniques of amplification and hybridization. - Good laboratory practice as well as careful performance of the procedures specified will allow specific amplification. - Polymerase inhibition might be the reason for complete failure of the assay. - Powder from disposable gloves and sodium hypochlorite have an inhibiting effect on amplification. Test performance The INNO-LiPA MYCOBACTERIA v2 Amp was evaluated at one Belgian center (Antwerp). A total of 94 cultured isolates, including 73 mycobacterial species and 21 non-mycobacterial species were tested with the INNO-LiPA MYCOBACTERIA v2 primers. All 73 mycobacterial isolates were successfully amplified after initial testing. In-house amplification of an additional 11 mycobacterial species was also found to be successful after initial testing. Licenses The purchase of this product allows the purchaser to use it for amplification of nucleic acid sequences for human in vitro diagnostics in accordance with the patented method described in the package insert. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby. Trademarks - INNO-LiPA is a trademark of Fujirebio Europe N.V., registered in US and other countries. - GeneAmp, and MicroAmp are registered trademarks of Applied Biosystems, LLC.