PCR Bacteria Test Kit

Transcription

1 PCR Bacteria Test Kit

2 2 3 Contents Reagents and Materials 3 Application and Test Principle 4 Precautions 5 Test Protocol 5 Appendix 11 Ordering Information 12 Reagents and Materials Kit Components Bacteria Mix red cap; lyophilized Containing primer set, deoxynucleotide triphosphates datp, dctp, dgtp and dutp, hot-start Taq polymerase and internal amplification control; aliquoted for 25 reactions, lyophilized; 1 vial (PK-CA ) or 2 vials (PK-CA ) Rehydration Buffer blue cap; 1.3 ml Positive Control DNA green cap DNA-fragment of Bacillus subtilis genome, prepared by PCR, noninfectious, lyophilized; 1 vial PCR-grade water white cap; 2 ml Stability and Storage Kit components are stable during shipping. Upon receipt, store at +2 to +8 C. After rehydration of the Bacteria Mix and the Positive Control DNA, store below 18 C and avoid repeated freezing and thawing. For repeated testing of low sample numbers Bacteria Mix and Positive Control DNA should be aliquoted after rehydration. By following these recommendations, the kit is stable until the expiration date stated on the label. Supplemental Requirements PCR thermal cycler mineral oil, if required for the particular thermal cycler used PCR reaction tubes for the specific PCR device 1.5 ml reaction tubes (DNA and RNA-free) agarose gel electrophoresis apparatus microcentrifuge for 1.5 ml reaction tubes, micropipettes (10, 100 and 1000 μl) and filter tips

3 4 5 Application and Test Principle This kit utilizes the polymerase chain reaction (PCR), thereby providing the highest sensitivity for the detection of bacterial contamination in cell cultures and other cell culture derived biologicals. Detection requires as little as 52 fg of bacterial DNA corresponding to 12 bacterial genomes per sample volume. The primer set is specific to a highly conserved portion of the 16S rrna coding region in the bacterial genome. The amplified PCR products have a size of bp (except Micrococcus luteus with 447 bp) and can be analyzed by agarose gel electrophoresis. This allows for detection of common airborne contaminants in cell cultures including: Pseudomonas, Actinomyces, Escherichia, Serratia, Porphyromonas, Fusobacteria, Staphylococcus, Streptococcus, Lactobacillus, Micrococcus, Bacillus, Klebsiella, Salmonella, Enterococcus, Mycobacterium, Legionella, Prevotella, Peptostreptococcus. Eukaryotic DNA is not amplified using this kit. Only one protocol is needed for the detection of bacteria and can be performed within 3 hours. The kit also provides internal control DNA already included in the Bacteria Mix. When running the PCR with the internal control DNA, a successful PCR is indicated by a 210 bp band on the agarose gel. The lyophilized Bacteria Mix also includes hot-start Taq polymerase as well as datp, dctp, dgtp and dutp (instead of dttp; this provides the option to degrade amplicons from previous analyses using uracil- DNA glycosylase [UNG]* in order to minimize false-positive results). * UNG is not part of the kit. This kit is intended for research use only. Not for clinical diagnostics or testing of human samples. Precautions Cross contamination may lead to false-positive results. The test should be performed according to good laboratory practice: Clean work surface with plenty of water Always wear gloves Use freshly laundered overalls and protective sleeves Use a facemask and avoid talking during PCR setup Always use new boxes of certified, DNA-free PCR-clean consumables (tubes, filter tips) Do not autoclave PCR consumables (tubes, filter tips) in autoclaves that are also used for autoclaving waste Clean pipettes regularly with plenty of water or DNA-removing cleansers Do not work at work benches with recirculated air (laminar flow) or badly maintained filters and avoid drafts (e.g., passing colleagues) during pipetting Test Protocol Preparation of Sample Material 1. Antibiotics can maintain the bacterial contamination at a level beneath the detection limit of the test (2.4 x 10 3 bacteria/ml). Therefore, prior to testing, the cells should be pre-cultured in the absence of antibiotics for at least one passage. 2. DNA extraction with a commercially available DNA extraction kit is always advisable when preparing the samples in order to remove inhibitors of the PCR safely and to lyse bacteria completely. The obtained DNA extract can be used directly for the test avoiding inhibition. 3. The sample should not contain more than 300 μg/ml DNA. 4. DNA extracts and heat-inactivated samples can be kept at 2-8 C for less than a week and stored at a temperature of at least 18 C for a longer period (~1 year; avoid repeated freezing and thawing!).

4 The sample should not contain more than 300 μg/ml DNA. 6. To avoid false positive results, we strongly recommend the use of deionized, DNA-free water, aerosol-preventive filter tips and gloves. Rehydration of the Reagents 1. Centrifuge tubes with lyophilized components (5 seconds at maximum speed of the microcentrifuge) 2. Bacteria Mix: Add 520 μl of Rehydration Buffer (blue cap) and incubate for 5 minutes at room temperature. Then, vortex briefly and spin for 5 seconds 3. Positive Control DNA: Add 300 μl of PCR-grade water (white cap) and incubate for 5 minutes at room temperature. Then, vortex briefly and spin for 5 seconds Keep reagents on ice and store below -18 C after rehydration. Avoid repeated freeze-thaw cycles and store reconstituted Positive Control DNA in aliquots. 1. Bacteria Mix, red cap Positive Control DNA, green cap Spin all freeze-dried components for 5 seconds at maximum speed of the centrifuge 2. Bacteria Mix, red cap Add 520 μl Rehydration BUffer (blue cap) 3. Positive Control DNA, green cap Add 300 μl PCR grade Water (white cap) 4. Bacteria Mix, red cap Positive Control DNA, green cap 5. Bacteria Mix, red cap Positive Control DNA, green cap Incubate 5 minutes at room temperature Vortex DNA briefly and spin for 5 seconds Test should be performed with negative and positive controls and samples in duplicate. All samples and reagents must be equilibrated to 2-8 C prior to use. Loading the test tubes Total volume per reaction is 25 μl. When setting up reactions, calculations should also include positive and negative controls. Aliquot 20 μl of Bacteria Mix into each PCR reaction tube. Negative Control: Add 5 μl of PCR-grade water (white cap) to PCR reaction tube with 20 μl of Bacteria Mix as negative control. Samples: Add 5 μl of sample (cell culture supernatant or DNA extract) to PCR reaction tube with 20 μl of Bacteria Mix Positive Control: Add 5 μl of Positive Control DNA (green cap) to PCR reaction tube with 20 μl of Bacteria Mix Close all PCR tubes and spin briefly. Load PCR cycler and start cycler program. Note: The pipetting sequence should be respected and the tubes closed after each sample load. After pipetting the negative control, the tube must be sealed before proceeding with the samples. Pipetting of the samples and sealing the tubes must also be completed before proceeding with the positive control (green cap) in order to avoid cross contamination. 1. Aliquot 20 μl of Bacteria Mix to each PCR tube. 2. Negative Controls: add 5 μl PCR grade Water or elution buffer from DNA extraction kit (ref. chapter Specimen ) or (white cap). 3. Samples: add 5 μl of cell culture supernatant or DNA extract. 4. Positive Control: add 5 μl Positive Control DNA (green cap). 5. Close and spin all PCR tubes briefly, load the PCR cycler and start the program.

5 8 9 Thermal Profile Load the cycler, check each PCR tube and the cycler lid for tight fit. Program the PCR cycler or check stored temperature profile. The programming process of your cycler is explained in the manual of the instrument. Program: 1 cycle 94 C for 2 minutes 35 cycles 94 C for 30 seconds 55 C for 30 seconds 72 C for 30 seconds cool down to 4 C to 8 C Agarose Gel Run 1.5% standard agarose gel, maximal 5 mm thick, with 5 mmcomb load 5 μl of each PCR reaction, mixed with bromophenol blue loading buffer per lane (only bromophenol blue in a low concentration should be used as a run marker) stop electrophoresis after 2-3 cm run distance (depending on the electrophoresis chamber used, e.g. run for minutes at 100 V) caused by bacterial DNA loads of >5x10 6 copies/ml. The initial concentration of positive control DNA exceeds 5 x 10 6 copies/ml in order to account for DNA loss resulting from repeated freezing and thawing. Consequently, the internal control is usually not visible in the positive control reaction. Relevant amplicon sizes: Internal control: 210 bp Bacteria: bp Micrococcus luteus: 447 bp Results of a successfully performed PCR: negative control: band at 210 bp positive control/sample: band at 468 bp, possibly additional band at 210 bp negative sample inhibited sample positive sample, weak contamination positive sample, strong contamination negative control with internal control 1 kb DNA ladder Gel Evaluation If internal control DNA was used, a distinct 210 bp band should appear in every lane indicating a successfully performed PCR. This band may fade out with increased amounts of amplicons formed, No amplification of Positive Control DNA may be due to the following reasons: control DNA tubes have not been spun down before rehydration programming mistake pipetting mistake

6 10 11 Before rerun of a negative and a positive control please check thermocycler protocol and pipetting scheme. Interpretation of possible band patterns: band at 210 bp: band at approx. 467 bp and at 210 bp: strong band at approx. 467 bp: no band: Detection of bacteria band at approx. 467 bp Internal control band at 210 bp negative sample bacteria-positive sample, weak contamination bacteria-positive sample, highly contaminated PCR inhibition Interpretation positive irrelevant bacteria present in the sample negative negative PCR inhibition negative positive no bacteria detectable in the sample 2. The reagents supplied should not be mixed with reagents from different lots and used as an integral unit. 3. The reagents of the kit should not be used beyond its shelf life. 4. For each test setup, at least one negative control should be included, possibly reflecting sample preparation. Positive controls facilitate the evaluation of the test. 5. Inhibition may be caused by the sample matrix, but also by sample elution buffer of DNA extraction kits not recommended or validated. Negative controls should always be completed with the use of sample elution buffer. 6. The use of control samples is advised to secure the day-to-day validity of results. The controls should be carried out in the same manner as the samples. It is recommended to run laboratory specific control samples with a high, medial and low (e.g. 3x LOD95) level. Participation in external quality control programs is recommended. NOTES ON THE TEST PROCEDURE Rarely unspecific PCR products can be formed and become visible in the gel as faint, diffuse bands of different sizes (not 210 or 467 bp), but do not indicate positive results. These faint, diffuse bands are mainly caused by non-specific annealing due to overloading of the PCR reaction with samples containing more than 300 μg/ml of DNA. However, these unspecific PCR products do not indicate positive results. Possible primer self-annealing produces another band of bp in length, but also does not affect the precision or results of the test. If the PCR of a sample is inhibited (lower band intensity compared to negative control), PCR inhibitors can easily be removed from the sample by performing a DNA extraction with a commercially available kit and testing the sample again. 1. Read this handbook carefully and follow the recommended procedure. Any deviation from the test method can affect the results. Appendix Limited Product Warranty This warranty limits our liability for replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. PromoCell shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. Notice to Purchaser This product is optimized for use in the Polymerase Chain Reaction ( PCR ) covered by patents owned by Hoffmann-La Roche, Inc., and F. Hoffmann-La Roche Ltd. ( Roche ). No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser of this product. PromoCell does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is restricted to persons that either have a license to perform PCR or are not required to obtain a license.

7 12 13 Ordering Information Product Name Size Catalog Number PCR Bacteria Test Kit 24 assays PK-CA PCR Bacteria Test Kit 48 assays PK-CA For in vitro research use only. Not for diagnostic or therapeutic procedures. PromoCell GmbH Sickingenstr. 63/ Heidelberg Germany North America Phone: (toll free) Fax: (toll free) Deutschland Telefon: (gebührenfrei) Fax: (gebührenfrei) France Téléphone: (ligne verte) Téléfax: (ligne verte) United Kingdom Phone: (toll free) Fax: (toll free) Other Countries Phone: Fax: info@promokine.info 05/2013