Bacteria (tuf gene) Quantitative PCR Kit

factor Tu (tuf) gene. The tuf gene has a high degree of conservation among various strains, and has a chromosomal copy number of one or two 4). Real-T...

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Cat. #

RR240A

For Research Use

Bacteria (tuf gene) Quantitative PCR Kit Product Manual

v201301Da

Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

Table of Contents I.

Description........................................................................................................... 3

II. Components........................................................................................................ 4 III. Storage................................................................................................................... 4 VI. Materials Required but not Provided......................................................... 4 V. Precautions for Use........................................................................................... 5 VI. Precautions........................................................................................................... 5 VII. Protocol.................................................................................................................. 6 VIII. Analysis................................................................................................................10 IX. Appendix.............................................................................................................12 X. Related Products..............................................................................................14 XI. References...........................................................................................................14

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URL:http://www.takara-bio.com

Bacteria (tuf gene) Quantitative PCR Kit I.

Cat. #RR240A v201301Da

Description

The Bacteria (tuf gene) Quantitative PCR Kit is intended for the quantification of bacterial number using real-time PCR. This kit can generate results more quickly than conventional methods that require 2 - 4 days of culture, and can be used for bacterial detection in the food safety and environmental research fields. Furthermore, with conventional methods, differences in optimal culture conditions for different bacteria make it necessary to use specialized culture conditions and/ or media to detect various types of bacteria present in the specimen 1), 2), 3). This kit allows the total number of a wide range of bacteria to be measured at the same time without any specialized culture conditions. Target Genes PCR detection of general bacterial strains using the 16S rRNA gene is possible, but this analysis is not suitable for precise quantification because the number of copies of 16S rRNA varies among bacterial strains. The target gene of this kit is the protein elongation factor Tu (tuf ) gene. The tuf gene has a high degree of conservation among various strains, and has a chromosomal copy number of one or two 4). Real-Time PCR Real-time PCR is a method of gene detection that monitors incorporation of fluorescent moieties in real time during the PCR amplification process. This intercalator method offers excellent speed and quantitative performance. The SYBR® Premix Ex Taq GC reagent that is included with this kit uses SYBR® Green I for detection and is capable of efficiently amplifying sequences with high GC content, making it suitable for detection of the tuf gene in a wide range of strains.

1)Heat Denaturation

F

Primer

F

F

2)Primer Annealing

Polymerase

F

F

F

Intercalator (fluorophore)

F F

F

F

3)Extension Reaction F

F

F

F

F

Figure 1. Fluorescent intercalator detection method. The intercalator fluoresces as a result of bonding to dsDNA during PCR amplification. * Drs. Bon Kimura and Hajime Takahashi of Tokyo University of Marine Science and Technology and Dr. Yuichiro Tanaka of TOYO SUISAN KAISHA, LTD. participated in the development of this kit.

URL:http://www.takara-bio.com

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

II. Components (25 μl volume, 100 Reactions) SYBR® Premix Ex Taq GC*1 2X conc. TUF Primer Mix 5X conc. dH2O ROX Reference Dye*2 50X conc. ROX Reference Dye II*2 50X conc. TUF Positive Control 1 x 105 copies/μl EASY Dilution (for Real Time PCR)

625 μl x 2 250 μl x 2 1 ml 50 μl 50 μl 100 μl 1 ml x 2

* 1 : Includes TaKaRa Ex Taq HS, dNTP Mixture, Mg2+, and SYBR® Green. * 2 : This component is to be used for analyses using a device that corrects fluorescent signals between wells, such as the real-time PCR devices by Life Technologies. Use ROX Reference Dye for the StepOnePlus™ Real-Time PCR Systems and ROX Reference Dye II for the 7500 Real-Time PCR Systems. These are not necessary with Thermal Cycler Dice Real Time System II and Thermal Cycler Dice Real Time System Lite.

III. Storage

SYBR® Premix Ex Taq GC(Package 1) 4℃ (stable for 6 months) -80℃ (for long-term storage) * Avoid storage at -20℃. Once the product is thawed, store at 4℃ and use within 6 months. * Please be sure to shield from light during storage. Furthermore, be aware of the risk of contamination when storing at 4℃. Other Components (Package 2) Store at -20℃

IV. Materials Required but not Provided Dilution solution (0.1% peptone in physiological saline, etc. based on the specimen type) Stomacher and Stomacher bag NucleoSpin® Tissue(Cat. #740952.10/.50/.250) Ethanol(> 99%) Heat block(Set to 56℃ and 70℃) Micropipette Micropipette tips (with hydrophobic filters) High-speed microcentrifuge 20 mg/ml lysozyme in 20 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100(pH8.0) Amplification equipment for real-time PCR and tubes Thermal Cycler Dice Real Time System II(Cat. #TP900/TP960)* Thermal Cycler Dice Real Time System Lite(Cat. #TP700/TP760)* Applied Biosystems 7500 Fast Real-Time PCR System(Life Technologies, Inc.) StepOnePlus™ Real-Time PCR System(Life Technologies, Inc.) Benchtop Centrifuge Micropipette Micropipette tips (with hydrophobic filters) * Not available in all geographic locations. Check for availability in your region. 4

URL:http://www.takara-bio.com

Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

V. Precautions for Use These are precautions for using this kit. Be sure to read before use. 1. Intended Use : This kit is a product for use in food and environmental testing. This is intended for research use only. 2. Results of Assay : This kit detects both viable and non-viable bacteria. If you need to detect only the viable bacteria, perform a culture test as well. (Takara Bio is not responsible for any actions taken as a result of analytical determinations made with this product.) 3. Disposal :

Samples should be handled according to regulations governing use of potentially infectious materials. Dispose of materials according to the safety regulations for your facility and in accordance with any applicable local, state, or federal regulations. Keep the work area sanitized at all times and sterilize all samples and equipment used during the experiment. Sterilization may be conducted by autoclaving at 121℃ for at least 20 minutes or by treatment with 2.5% sodium hypochlorite, followed by processing materials according to guidelines for potentially infectious waste. Process and dispose of plastic and filter paper reagent containers and instruments according to regulations concerning the handling of hazardous materials.

VI. Precautions 1. SYBR® Premix Ex Taq GC contains enzymes. Before use, make sure the reagent is evenly mixed by gently turning it upside down several times without creating bubbles; if inadequately mixed, it may not provide sufficient reactivity. Do not mix by vortexing. Please note that a white to yellowish-white precipitate may form when SYBR® Premix Ex Taq GC is stored at -80℃. This will dissolve completely when the solution is hand warmed gently or when it is left at room temperature for a short period (protect from light) and then mixed by inversion. Failure to redissolve the precipitate may result in unevenness of the reagent components; be sure to mix until homogenous before use. 2. Place reagents on ice when preparing the reaction solution. 3. SYBR® Premix Ex Taq GC includes SYBR® Green I. Do not to expose to strong light during preparation of the reaction solution. 4. We recommend that the three following areas be established in the laboratory working space and be physically separated from each other. Avoid opening/closing tubes containing amplification products in any of these areas. ○ Area 1 : reaction mixture preparation and dispensing ○ Area 2 : sample preparation ○ Area 3 : addition of samples to reaction mixtures, reaction, and detection 5. Because the amplification reaction and detection are carried out simultaneously in real-time with this kit, there is no need for electrophoresis or other procedures on the amplification product after reaction completion. Additionally, removal of the amplification product from the tube should be avoided to prevent contamination.

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

VII. Protocol Overview Sample Preparation Specimen (10 g) + Dilution Solution (90 ml) ↓ Stomacher Processing ↓ DNA Extraction Centrifuge 1 ml of the specimen solution (equivalent to 0.1 mg of the specimen) at 8,000X g for 5 minutes and remove the supernatant. ↓ DNA extraction from the precipitate using NucleoSpin® Tissue (Elution volume: 100 μl) ↓ Preparation of Real-Time PCR Reaction Solution and Start of Reaction Make serial dilutions of the TUF Positive Control and prepare standards for the standard curve. ↓ Prepare the reaction solution. ↓ Dispense the reaction solution into the reaction tubes and add the negative control (H2O), standards, and 5 μl of the specimen sample (equivalent to 0.005 g of the specimen). ↓ Set the reaction tubes in the amplification device for real-time PCR and begin the reaction. ↓ Quantitative Analysis Use the analysis software for the real-time PCR instrument to quantify tuf for the specimen sample based on the standard curve

Preparation of Sample Suspension 100 ml (Specimen : 10 g)

1/100 of sample for DNA extraction

1 ml (Equal to 0.1 g specimen)

DNA Extraction

100 μl

1/20 of sample for real-time PCR 5 μl (Equal to 0.005 g specimen) Prepare the real-time PCR reaction solution

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

VII-1. Preparation of the Sample Suspension (Work in area 2) 1. Weigh 10 g of the specimen and transfer to a Stomacher bag, etc. 2. Add 90 ml of dilution solution (such as 0.1% peptone in physiological saline) (equivalent to 9 times the specimen). 3. Homogenize as necessary using a Stomacher, etc. and use as the specimen solution for DNA extraction. VII-2. DNA Extraction (Work in area 2) Perform DNA extraction according to the NucleoSpin® Tissue protocol for bacteria (Grampositive bacteria) and elute in 100 μl. 1. Dispense 1 ml of the specimen solution into a 1.5 ml microtube. 2. Centrifuge for 5 minutes at 8,000X g and remove the supernatant. 3. Suspend the pellet in 180 μl of 20 mg/ml lysozyme in 20 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100 (pH8.0)*1 and incubate for 30 - 60 minutes at 37℃. 4. Add 25 μl of Proteinase K *2 and incubate at 56℃ for 1 - 3 hours (or overnight) until completely dissolved. 5. Agitate the sample. Add 200 μl of Buffer B3, agitate strongly, and then incubate for 10 minutes at 70℃. If undissolved material remains, centrifuge for 5 minutes at 11,000X g and transfer the supernatant to a new tube. 6. Add 210 μl of ethanol (> 99%) to the supernatant and mix well. 7. Set the NucleoSpin® Tissue Column in the collection tube. Transfer the above solution to the Column and centrifuge for 1 minute at 11,000X g . After discarding the filtrate, reset the Column in the same collection tube. 8. 1st Wash : Add 500 μl of Buffer BW to the column and centrifuge for 1 minute at 11,000X g . After discarding the filtrate, set the column in the same collection tube.

2nd Wash : Add 600 μl of Buffer B5 *3 to the column and centrifuge for 1 minute at 11,000X g . After discarding the filtrate, set the column in the same collection tube.

9. Centrifuge the column for 1 minute at 11,000X g . 10. Set the column in a 1.5 ml microtube (provided by the user). Add 100 μl of Buffer BE that has been heated to 70℃, incubate for 1 minute at room temperature, and centrifuge for 1 minute at 11,000X g . Store the eluted DNA solution at 4℃ and use as the sample for real-time PCR. For long-term storage, store at -20℃, and avoid repeated freezing and thawing. * 1 : Not included in NucleoSpin® Tissue. * 2 : Preparation of Proteinase K solution For Cat. #740952.10 : Add 260 μl of Proteinase Buffer PB to 6 mg of Proteinase K (freeze-dried) and dissolve completely. Store the prepared Proteinase K solution at -20℃ (stable for 6 months). * 3 : Preparation for Buffer B5 solution For Cat. #740952.10 : Add 16 ml of ethanol (96 - 100%) to 4 ml of Wash Buffer B5(concentrate). URL:http://www.takara-bio.com

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

VII-3. Real-Time PCR 1. Preparation of the samples for the Standard Curve (Work in area 3) Make serial dilutions of the TUF Positive Control using EASY Dilution to obtain the samples for the Standard curve. (1) 1 x 105 copies/μl (TUF Positive Control stock solution) (2) 1 x 104 copies/μl (5 μl TUF Positive Control stock solution + 45 μl EASY Dilution) (3) 1 x 103 copies/μl (5 μl of 1 x 104 copies/μl solution + 45 μl EASY Dilution) (4) 1 x 102 copies/μl (5 μl of 1 x 103 copies/μl solution + 45 μl EASY Dilution) Using these samples, it is possible to obtain quantitative values in terms of copy number of tuf gene per gram of specimen from the real-time PCR data. (Refer to the section "VIII. Analysis" for the calculation methods.) Table 1. Comparison of Copies/Reaction and Copies/g copies/reaction (Number of Copies per Reaction) 1 2 3 4

5 x 105 5 x 104 5 x 103 5 x 102

copies/g (Number of Copies per Gram of Specimen) 1 x 108 1 x 107 1 x 106 1 x 105

2. Preparation of the Reaction Solution (Work in area 1) Prepare the reaction solution on ice. Prepare enough for the required number of tubes plus some extra, dispense 20 μl into each PCR reaction tube and close the caps lightly. Add 5 μl of sterilized water in place of the DNA sample to one tube as a negative control and close the cap tightly. [For the Thermal Cycler Dice Real Time System] SYBR® Premix Ex Taq GC(2X conc.) TUF Primer Mix(5X conc.) dH2O DNA sample or standard curve sample

[For Life Technologies Real-Time PCR Instruments] SYBR® Premix Ex Taq GC(2X conc.) TUF Primer Mix(5X conc.) ROX Reference Dye or ROX Reference Dye II*1 dH2O Template

(1 Reaction) 12.5 μl 5.0 μl 2.5 μl (5.0 μl)*2 25.0 μl

(1 Reaction) 12.5 μl 5.0 μl 0.5 μl 2.0 μl (5.0 μl)*2 25.0 μl

* 1 : ROX Reference Dye is used for the StepOnePlus™, and ROX Reference Dye II is used for the 7500 Fast Real-Time PCR System. * 2 : The DNA sample and samples for standard curve should be added in step (3), NOT in this step. 8

URL:http://www.takara-bio.com

Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

[Caution] Because real-time PCR relies on the optical measurement of fluorescence, care must be taken that the tubes do not become dirty. Wear gloves when handling PCR tubes. 3. Addition of the Sample (DNA Solution) (Work in area 3) Add 5 μl of sample (the specimen DNA sample, samples for the standard curve, etc.) to the PCR reaction solution and close the caps tightly. Centrifuge briefly in a benchtop centrifuge and set in the real-time PCR amplification instrument. 4. Real-Time PCR Reaction Perform the following PCR conditions. * Please refer to the instruction manual for the real-time PCR amplification instrument for specific operating procedures. Initial Denaturation 95℃ 30 seconds 2 Step PCR 35 cycles 95℃ 5 seconds 60℃ 30 seconds (Detection of Fluorescence: FAM/SYBR®) Melt curve analysis

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

VIII. Analysis VIII-1. Principles of Quantification TUF Positive Control The TUF Positive Control included in this kit is plasmid DNA that includes the tuf gene region and is adjusted to 1 x 105 copies/μl (based on the OD260). When serially diluted, it can be used as the standard for obtaining a standard curve and then quantitative analysis can be carried out. Note : This kit detects both viable and non-viable bacteria. If you need to detect only the viable bacteria, perform a culture test as well. Calculation of the Copy Number per Gram of the Specimen When this product is used according to the instructions, an amount equivalent to 0.005 g of the specimen is used for a real-time PCR reaction, and the copies/reaction value can be multiplied by 200 to obtain the copy number of tuf per gram specimen. 10 g of the specimen + 90 ml of dilution solution ↓ Stomacher treatment 1 ml of the specimen solution (equivalent to 0.1 g of the specimen) ↓ DNA extraction 100 μl of the DNA solution (equivalent to 0.1 g of the specimen) ↓ 1/20 to real-time PCR 5 μl per reaction used in real-time PCR (equivalent to 0.005 g of the specimen) VIII-2. Relationship between the Copy Number and the Number of Bacteria Cultured Bacteria Rahnella aquatilis, Escherichia coli, and Staphylococcus aureus bacterial strains were cultured overnight in TSB media and each were serially diluted using physiological saline. Viable cell counts were determined by culturing on TSA agar plates. Additionally, 1 ml of the bacterial suspensions were analyzed by real-time PCR analysis according to instructions for this kit. 㻜 㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻥 㻝㻜

Real Time PCR (log copies/ml)

10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0

standard curve

1.0 0.0 0.0

2.0

4.0

6.0

8.0

10.0

The cell count from TSA plates (colony forming units: CFU) are plotted on the X-axis, and the copy number obtained from real-time PCR are plotted on the Y-axis. There is good correlation between the results for the TSA smear culture and real-time PCR. While there was a tendency for the copy number calculated to be somewhat high, the difference was limited to no more than one order of magnitude.

TSA (log CFU/ml)

Figure 2. Relationship between tuf gene copies and cell count for cultured cells.

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

Food Specimens 10% suspensions of 9 vegetables, 6 salads (including cut vegetables), and 8 meats were prepared. Measurement of cell count using TSA plate cultures (30℃, 48 hours) and realtime PCR analysis using this product were carried out. Table 1. Copy number and cell counts (food specimens) TSA

[log copies/g] [log cfu/g] Cabbage

1.51

0.90

Mizuna

3.38

3.20

Sprouts

4.61

3.30

Bok Choy

6.63

6.57

Cabbage

7.08

5.60

Lettuce

7.27

6.33

Mizuna

8.02

7.24

Sprouts

8.29

7.07

Mitsuba

8.65

7.10

Shredded Cabbage

2.66

2.48

Cut Lettuce

3.27

2.96

Cut Lettuce

5.09

4.41

Mixed Vegetable Salad

6.55

5.39

Green Salad

7.13

6.21

Mixed Salad

8.20

6.53

Beef

1.55

1.99

Pork

3.50

2.78

Sliced Pork

4.86

5.15

Ground Meat

5.47

4.89

Ground Chicken

5.53

4.17

Pork Loin

6.08

5.41

Chicken Thigh

6.20

4.97

Sliced Beef

6.28

6.25

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10 Real Time PCR (log copies/g)

qPCR

8 6 Vegetables

4

Salads Meats

2

Standard Curve

0 0

2

4

6

8

10

TSA (log cfu/g)

Figure 3. Relationship between tuf copies and cell count in food specimens.

The results from TSA cultures (CFU/g) are plotted on the X-axis, while the results of realtime PCR (copies/g) are plotted on the Y-axis. There is good correlation between the results from the cultures and real-time PCR. There was a tendency for the results for the copy number to be 1 - 2 orders of magnitude higher than the bacterial count. This may be due to the presence of dead cells and/or bacterial strains that are difficult to detect by culturing.

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

IX. Appendix Detectable strains Bacterial strains that have been detected with this kit are listed in Table 3. These strains showed a Ct value of 20 or less when real-time PCR was performed using 10 ng of purified genomic DNA as the template 4). Table 3. Detectable bacterial strains Strain

Source

Accession number

tuf -qPCR amplification

Ct b

FI55

Chub mackerel

AB472770

+

16.0

Flavobacterium hercynium

FI48

Spotted mackerel

AB472772

+

17.5

Flavobacterium johnsoniae

JCM 8514

AB472806

+

18.0

Bacterial strainsa

Gram negative  Bacteroidetes Chryseobacterium formosense

Sejongia antarctica

FI18

Spotted mackerel

AB472786

+

17.3

Sphingobacterium kitahiroshimence

FI23

Stone flounder

AB472789

+

16.9

AB472793

+

15.8

AB472767

+

14.3

AB472795

+

16.5

 γ-Proteobacteria Acinetobacter baumannii

JCM 6841

Acinetobacter baumannii

FI63

Aeromonas hydrophila Aeromonas molluscorum

Flatfish (meita)

JCM 1027

AB472768

+

15.0

Alteromonas macleodii

JCM 20772

AB472797

+

16.7

Citrobacter freundii

IAM 12471

AB472801

+

15.4

AB472771

+

14.1

Colwellia aestuarii

FI56

FI04

Horse mackerel

Chub mackerel

Enterobacter aerogenes

IAM 1183

AB472802

+

15.8

Erwinia carotovora

IAM 12633

AB472804

+

16.1

Escherichia coli

IAM 1137

AB472805

+

14.2

Klebsiella pneumoniae

IAM 1063

AB472808

+

15.3

AB472816

+

15.9

AB472777

+

14.7

AB472817

+

17.5

Morganella morganii Photobacterium phosphoreum Proteus mirabilis Pseudoalteromonas haloplanktis Pseudomonas fluorescens Psychrobacter immobilis Psychromonas arctica

ATCC 35200 FI59

Horse mackerel

JCM 1669 FI01

Spotted mackerel

AB472780

+

14.9

FI28

Stone flounder

AB472781

+

15.8

AB472818

+

16.8

AB472783

+

16.0

ATCC 43116 FI26

Chub mackerel

Rahnella aquatilis

JCM 1683

AB472819

+

15.9

Raoultella planticola

JCM 7251

AB472820

+

17.5

ATCC 13311

AB472822

+

15.1

AB472788

+

15.0

AB472823

+

16.2

AB472787

+

15.5

Salmonella serover Typhimurium Schineria larvae Serratia marcescens Shewanella frigidimarina

FI13

Spotted mackerel

IAM 1104 FI20

Spotted mackerel

Shewanella japonica

JCM 21433

AB472824

+

15.8

Shewanella putrefaciens

NBRC 3908

AB492873

+

14.4

AB472791

+

16.2

AB472827

+

16.3

Vibrio diazotrophicus Vibrio parahaemolyticus

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FI52 ATCC 17802

Horse mackerel

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Bacteria (tuf gene) Quantitative PCR Kit Xanthomonas euvesicatoria

Cat. #RR240A v201301Da

FI22

Chub mackerel

AB472792

+

15.5

Xanthomonas oryzae

JCM 20241

AB472828

+

19.6

Yersinia enterocolitica

ATCC 9610

AB472829

+

15.9

AB472779

+

17.0

 α-Proteobacteria Paracoccus denitrificans

FI34

Spotted mackerel

 β-Proteobacteria Alcaligenes faecalis

JCM 20522

AB472796

+

17.9

Burkholderia caledonica

JCM 21561

AB472799

+

16.3

JCM 11549

AB472794

+

18.4

IAM 1076

AB472798

+

16.8

Brochothrix thermosphacta

NBRC 12167

AB492875

+

15.3

Carnobacterium divergens

JCM 5816

AB472800

+

15.7

NBRC 15684

AB492874

+

16.2

Enterococcus faecalis

JCM 5803

AB472803

+

14.8

Lactococcus lactis

NRIC 1174

AB472811

+

18.1

Leuconostoc carnosum

JCM 9695

AB472812

+

17.9

Listeria monocytogenes

ATCC 15113

Gram positives  Bacilli Aerococcus sanguinicola Bacillus subtilis

Carnobacterium maltaromaticum

Planomicrobium chinense Staphylococcus aureus Staphylococcus pasteuri

FI41

AB472813

+

18.4

Chub mackerel

AB472778

+

16.0

AB472826

+

16.1

Flatfish (meita)

AB472790

+

16.0

AB472821

+

16.1

ATCC 12600 FI64

 Actinobacteria Rothia dentocariosa

JCM 3067

Rothia nasimurium

FI43

Horse mackerel

AB472784

+

18.2

Salinibacterium amurskyense

FI36

Flatfish (meita)

AB472785

+

17.2

a : Classification was based on the National Center for Biotechnology Information taxonomy. b : The cycle threshold value (Ct) for the reaction containing 10 ng of genomic DNA per reaction.

Note : Detection is expected to be possible for additional bacterial strains based on tuf gene sequence information. 10,855 gene sequences named ‘tuf ’ or ‘elongation factor’ were extracted from 1561 bacterial genome sequences registered in GenBank (as of November 21, 2011), and those with 100% matches for this product’ s primer sequence are listed. The list is available on the product webpage. http://www.takara-bio.co.jp/cycleave/elongation_factor.xlsx

URL:http://www.takara-bio.com

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

X. Related Products SYBR® Premix Ex Taq ™ GC(Cat. #RR071A/B)* Thermal Cycler Dice® Real Time System II(Cat. #TP900/TP960)* Thermal Cycler Dice® Real Time System Lite(Cat. #TP700/TP760)* NucleoSpin® Tissue(Cat. #740952.10/.50/.250) * Not available in all geographic regions. Check for availability in your region.

XI. References 1) Takeo Fujii. (1989) Viable Cell Measurement Methods for Aquatic Foods I: Culture Medium Components, Culture Temperature and Plating Methods. Tokai Suiken Jo. 118: 71-79. (Japanese) 2) Tsubasa Fukuda, Manabu Furushita, Tusneo Shiba. (2012) A Comparison of Viable Cell Counts in Fresh Fish with the Official 35℃ Culture Method and the 20℃ Cell Culture Method. Journal of National Fisheries University . 60:183-188. (Japanese) 3) Masataka Satomi, Kan Oikawa, Yutaka Yano: Fisheries Science Series 141: The Quality and Freshness of Seafood and Advanced Preservation Techniques (Edited by Jun'ichi Nakazoe and Hideaki Yamanaka) 6. Microbiological Quality Evaluation. (Japanese) 4) Tanaka Y, Takahashi H, Simidu U, Kimura B. (2010) Design of a new universal realtime PCR system targeting the tuf gene for the enumeration of bacterial counts in food. J Food Prot . 73: 670-679.

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URL:http://www.takara-bio.com

Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

NOTICE TO PURCHASER: LIMITED LICENSE [P5] PCR Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. 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 for the purchaser's own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. 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. [L11] SYBR® Green I This product is covered by the claims of U.S. Patent No. 5,436,134 and 5,658,751 and their foreign counterpart patent claims. Takara PCR products containing SYBR® Green I are sold under license from Molecular Probes Inc. only for the usage in Real-time PCR for internal research purpose. These products are not to be used for the purpose such as; providing medical, diagnostic, or any other testing, analysis or screening services or providing clinical information or clinical analysis in return for compensations. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [L46] SYBR®/Melting Curve Analysis The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. [L52] Rox Reference Dye (Research Field) Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,928,907. The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’ s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’ s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. 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. [M57] LA Technology This product is covered by the claims 6-16 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims.

URL:http://www.takara-bio.com

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Bacteria (tuf gene) Quantitative PCR Kit

Cat. #RR240A v201301Da

NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at +81 77 543 7247 or from our website at www.takara-bio.com. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. 16

URL:http://www.takara-bio.com