I. Description...2. Kit Components...2. Storage...2. Reagents not supplied in the kit...3. V. Protocols...3. A. Detection example...

Transcription

1 Table of contents I. Description...2 II. III. IV Kit Components...2 Storage...2 Reagents not supplied in the kit...3 V. Protocols...3 A. Detection example...3 B. PCR Reaction...4 C. Preparation of agarose gel...4 D. Gel electrophoresis...4 E. Verification of stained band...5 VI. VII. Judgement...6 Reference...7

2 I. Description: Bacillus cereus (B. cereus) is a spore-forming bacillus that is distributed widely in natural environments such as soil, air, and water, as well as in crops and feed. This spore resists high temperature, germinates in an eutrophic environment, becomes a trophozoite to proliferate, and produces toxins. Two toxins, emetic toxin (cereulide) and diarrheal toxin (enterotoxin), are involved in food poisoning. Cereulide is heat-resistant and thus causes food poisoning even after heating when cereulide-producing strains of B. cereus massively proliferate in food. Cereulide has been detected by bioassay and LC-MS analysis conventionally. However, these technologies involve complicated procedures and a considerable amount of time. For this reason, an easy and rapid specific detection method of cereulide-producing strains of B. cereus has been anticipated. The joint study by the School of Medicine, Nagoya University and the Nagoya City Public Health Research Institute revealed that cereulide-nonproducing strains lack a portion of the cereulide synthetic enzyme (CRS) gene, and identified the sequence of the missing portion. The cereulide producing stain can be identified by detecting the amplified product of this missing region through PCR utilizing primers which are designed based on the missing portion of CRS. One hundred of clinical strain of emetic food poisoning by B. cereus were selected at random, and they were tested with this PCR method.the result shows the PCR products were obtained at 100% probability and these amplified products were verified as cereulide-producing. And non-cereulide producing strains were not detected. Accordingly, this method was proved to allow specific detection of cereulide-producing strains. This Kit is designed for PCR to detect cereulide-producing strains of B. cereus, B. cereus and the related bacteria (B. thuringiensis and B. anthracis) rapidly and specifically obtained from isolated culture medium. The cereulide synthetic enzyme gene (CRS) possessed by cereulide-producing strains of B. cereus and the lecithinase gene (LE) possessed by B. cereus and the by related bacteria (B. thuringiensis and B. anthracis) are amplified simultaneously in a tube using CRS and LE primers, respectively. The amplified products are detected by agarose electrophoresis. This Kit includes the enzyme for the hot start enzyme TaKaRa Ex Taq TM HS, preventing mispriming and primer dimerrelated non-specific amplification, and thus, it achieves detection at high sensitivity. In addition, this Kit is able to identify a false negative reaction through the use of internal control. This kit was designed by TaKaRa through collaboration with Dr. Norio Agata of Nagoya City Public Health Research Institute. II. Kit Components (25 μ l X 50 reactions): 1. 5 x PCR Premix * 500 μ l 2. CRS Primer Mixture 50 μ l (for detecting cereulide synthetic enzyme gene) 3. LE Primer Mixture 50 μ l (for detecting lecithinase gene) 4. I.C. Primer Mixture 50 μ l (for detecting internal control) 5. CRS Positive Control Template 10 μ l 6. LE Positive Control Template 10 μ l * Includes dntp Mixture, internal control and TaKaRa Ex Taq TM HS III. Storage: -20 (for shipping and storage) 2

3 IV. Reagents not supplied in the kit 1. Sterilized distilled water 2. NuSieve R 3:1 Agarose (Lonza Biosciences) 3. Electrophoresis buffer (TBE powder (Cat.#T905)) 4. DNA size marker phy Marker (Cat.#3404A/B) φ X174 Hinc II digest (Cat.#3406A/B) 00 bp DNA ladder (Cat.#3407A/B) 5. Loading buffer (6x : 30% glycerol, 0.03% bromophenol blue, 0.03% xylene cyanol, 30mM EDTA, this loading buffer is supplied with Takara's DNA size marker) 6. DNA stainer (Ethidium bromide, or SYBR R Green I (Lonza Biosciences) or GelStar R Nucleic Acid Stain (Lonza Biosciences)) Required Equipment 1. Heating block (applicable at 95 ) 2. Refrigerate centrifuge, compatible with 1.5 ml tubes 3. Authorized thermal cycler TaKaRa PCR Thermal Cycler Dice TM (Cat.#TP600,TP650) 4. Electrophoresis apparatus 5. Power supply 6. Ultraviolet transilluminator 7. Polaroid camera to photograph stained gel (When using SYBR R Green I or GelStar R Nucleic Acid Stain for staining a gel, a filter designated for use with SYBR Green I or GelStar R Nucleic Acid Stain should be used.) ml or 0.2 ml PCR tube (TaKaRa Micro PCR tube (Cat.#9047)) 9. Micropipettes for 20 μ l and for 200 μ l 10. Micropipette tips 11. Polaroid film 12. Tray for staining agarose gel V. Protocol: A. Detection example from heat-extracted bacteria sample 1-1: In case of direct application of bacteria sample to PCR 1.) Take a tiny volume of bacteria from colonies on the plate (eg. NGKG medium*) by using a sterilized tip of micropipette. Suspend it in 100 μ l of sterilized water. 2.) Heat at 95 for 5 minutes. 3.) Apply 1 μ l for PCR reaction. 1-2: In case of PCR reaction by collecting bacteria from food, in combination with culture method 1.) Add 10 times volume of SCD medium** added with polymyxin into a sample material, and grindle and mix with a homogenizer for bacteria testing (e.g. stocker). Incubate at 35 for 5-6 hours or overnight. 2.) Centrifuge 1.3 ml of the culture liquid at 1000 rpm (approx. 100 x g) for 1 min. Take the supernatant in ml into a fresh tube and centrifuge at 12,000 rpm (approx. 13,000 x g) for 3 min. 3.) Remove supernatant and suspend the pellet in 100 μ l of TE buffer (containing 10 mm Tris-HCI, 0.1 mm EDTA, ph8.0). Heat at 95 for 5 minutes, and apply 1 μ l to the subsequenct PCR reaction. If the inhibitation is observed in PCR reaction, further dilute the sample with TE buffer and apply it to PCR reaction. *NGKG medium: Isolation medium for Bacillus cereus (Nissui Pharmaceutical Co., Ltd.) **SCD medium: Tryptosoya bouillon (Nissui Pharmaceutical Co., Ltd.) 3

4 B. PCR reaction 1) Prepare the following reaction mixture in a fresh PCR tube. 5 x PCR Premix 10 μ l CRS Primer mixture 1 μ l LE Primer mixture 1 μ l I.C.Primer Mixture 1 μ l dh 2O 36 μ l 49 μ l 2) Add 1 μ l of the heat-extracted sample or Positive control template. Prepare a negative control by adding 1 μ l of sterilized distilled water instead of the sample. 3) Secure tightly a cap of each tube and set in a thermal cycler. Perform PCR under the following condition. 94, 30 sec. 55, 30 sec. 40 cycles 72, 30 sec. The reaction completes in about 2 hours. The PCR reactants can be stored at -20. C. Preparation of agarose gel 1) Dispense electrophoresis buffer into a triangle flask and slowly add NuSieve 3:1 agarose (Lonza Biosciences) to the concentration of 3% (w/v) with mixing. 2) Heat for 2-3 min. in a microwave. After heating, mix well and confirm that the agarose is uniformly solved. Heat the slurry again for the minimum time required to allow all of the grains of agarose to dissolve. 3) Set up the gel board. 4) After the agarose gel solution cools to 50-60, pour the solution into the gel board and insert a comb to generate slots. Leave for 30 min to 1 hour at room temperature and harden the gel. *When staining the gel with ethidium bromide before applying samples. Cool the solution to and add ethidium bromide solution in a final concentration of 0.5 μ g/ml and mix gently to be dissolved uniformly. Pour the solution into the gel board. Leave for 30 min to 1hour at room temprature and harden the gel. 5) After the gel hardens enough, mount the gel in a electrophoresis tank. 6) Pour the electrophoresis buffer into the electrophoresis tank so that the gel is completely immersed. 7) Remove the comb carefully not to break the gel. D. Gel electrophoresis 1) Connect the electrical leads carefully not to mistake the electrodes, between anode and cathode. As the DNA amplified by PCR is charged with negative, it migrates from cathode to anode. 2) Add 2.0 μ l of 6 x loading buffer to each tube containing 10 μ l of PCR reactant and mix. Slowly load the mixture into the slots of the submerged gel using a micropipette. Marker DNAs of known size should be loaded into slots on both the right and left sides of the gel. 3) Apply a constant voltage of V and run the gel until the bromophenol blue* have migrated 2-3 cm in front of the comb. *Bromophenol blue migrates faster. 4

5 E. Verification of stained band* *When the gel has been prestained with ethidium bromide, perform only 3). 1) Prepare 1 μ g/ml ethidium bromide solution, or SYBR R Green I* (Lonza Biosciences), or solution or GelStar R solution (pre-diluted by fold with TBE buffer or electrophoresis buffer) in the amount enough to submerge the gel, and keep it in a tray for staining agarose gel. 2) Put the gel in the tray and leave it without moving for min. 3) Set the gel on an ultraviolet transilluminator and photogragh the gel.* Verify the size of the bands of reactants comparing wiht Marker DNAs. *When using SYBR R Green I (Lonza Biosciences) or GelStar R for staining, please use the filter designated for use with SYBR R Green I should be used.) CAUTION: Gloves should be worn in handling Ethidium bromide, SYBR R Green I / GelStar R, or the stained gel. 5

6 VI. Judgement The following shows the size of amplified products by PCR reaction. 1) Cereulide synthetic enzyme (CRS) gene: 426 bp 2) Lecithinase (LE) gene: 227bp 3) Internal Control (I.C.): 106 bp 1) 2) 3) The following three patterns (A-C) are expected as an electrophoresis result of sample. Molecular Weight (M.W.) Marker used: 100 bp DNA ladder M.W. Marker A B C 3) 1) 2) 3) 2) 3) <---1) 426 bp <---2) 227 bp <---3) 106 bp CRS: - LE: - I.C. : + CRS: + CRS: - LE: + LE: + I.C.: + I.C.: + Pattern A means that Bacillus cereus or the similar strains to Bacillus cereus are contaminated in less than detection limit, or that the other bacteria might be contaminated. Pattern B means that PCR sample contains Bacillus cereus cereulide-producing strain. Pattern C means that PCR sample contains Bacillus cereus cereulide-nonproducing strain or the other strain similar to Bacillus cereus (B.thuringiensis, B. anthracis). - Negative control reaction shows the pattern A. - If Negative control shows the pattern B or C, the contamination is suspected during the reaction. Accordingly, disinfect the places and instruments used for reaction and for mixture preparation. then proceed the reaction again. - When no amplified products were obtained in negative control reaction, PCR reaction did not proceed correctly. Several causes can be suspected, such as deterioration of used reagent, used instrumens were out of order, or any inhibiting substance were contaminated in the sample. It is recommended to prepare the sample again, and repeat the reaction with that newly prepared sample. Note 1: The 106 bp band, the amplified product derived from Internal Control template, may not be verified in the pattern of B or C, depending on an used sample. Note 2: In the reaction with CRS Positive Control Template, the bands of 426 bp from CRS gene and of 106 bp from Internal Control are obtained. When using LE Positive Control Template, the bands of 227 bp from CRS gene and 106 bp from Internal Control are obtained. 6

7 Application example: PCR detection was performed using heat-extracted samples prepared from strains of Bacillus cereus. M M. 100 bp ladder marker 1. Negative control 2. emetic toxin (cereulide) producing 3. emetic toxin (cereulide) nonproducing <---1) 426 bp <---2) 227 bp <---3) 106 bp VII. Reference: 1. Agata.N. (2002) International Journal Of Food Microbiology Agata.N.,et al. (1994) FEMS Microbiol. Lett Schraft.H.,et al. (1995) Applied and Enviromental Microbiology NOTE: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from If you require licenses for other use, please call at or contact from our website at 7

8 NOTICE TO PURCHASER: LIMITED LICENSE This product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Environmental Testing, Food Testing, Industrial Microbiology, including reporting results of purchaser's activities for a fee or other commercial consideration, and also for the purchaser's own internal research. No right under any other patent claim (such as the patented 5' Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972, and the dsdna-binding dye process claims in US Patents Nos 5,994,056 and 6,171,785) is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Licensed under U.S. Patent 5,338,671 and 5,587,287 and corresponding patents in other countries. U.S.Patent 5,436,149 for LA Technology is owned by 8 Phone: FAX: