Download Bacteria Screening PCR Kit

Cat. #
RR114A
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Bacteria Screening PCR Kit
Product Manual
v201310Da
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
Table of Contents
I．
Description............................................................................................. 3
II．
Components.......................................................................................... 3
III． Storage..................................................................................................... 4
IV． Materials Required but not Provided........................................... 4
V．
Precautions for Operation................................................................ 4
VI． Principle ................................................................................................. 5
VII． Protocol................................................................................................... 6
VIII． Application Examples......................................................................11
IX． References............................................................................................15
X． Related Products................................................................................15
2
URL:http://www.takara-bio.com
Cat. #RR114A
Bacteria Screening PCR Kit
v201310Da
I．Description
In order to maintain consumer trust in the safety of food products, a high priority has
been placed on assuring product quality at each step of the food supply process. PCR
is recognized as one of the more useful methods for such food-related quality control
applications.
PCR is a technique in which minute amounts of DNA can be used as a template for
amplifying only a desired DNA fragment. A single cycle of this method consists of
three incubation steps; DNA thermal denaturation, primer annealing, and primer extension with DNA polymerase. Amplification by a factor of 1 million can be achieved
in relatively short periods of time by repeating these steps.
The Bacteria Screening PCR kit uses the 16S rRNA gene as a target and employs the
ENT primer for detecting strains in the Enterobacteriaceae family, including E. coli and
Salmonella bacteria, and the BS primer for detecting genus Bacillus strains, including
Bacillus cereus , and genus Staphylococcus , including Staphylococcus aureus . (Table 1
lists the reactivity characteristics of the various bacterial groups tested.)
When used in combination with an enrichment culture, the Bacteria Screening PCR
Kit can be used to rapidly detect the presence of even minute numbers of bacteria in
samples.
II．Components (50 μl x 50 reactions for ENT and BS primers)
1．2X Premix Solution ＊ 1
2．Primer Mix ENT
3．Primer Mix BS
4．Positive Control ENT
5．Positive Control BS
6．dH2O
7．10% Chelex® Solution ＊ 2
2X conc.
5 μM each
5 μM each
250 μlx 10 (100 reactions)
125 μl
(50 reactions)
125 μl
(50 reactions)
25 μl
(10 reactions)
25 μl
(10 reactions)
1 mlx 3
12 mlx 2
＊ 1：2X Premix Solution contains the followings:
TaKaRa Ex Taq HS
：0.1 units/μl
Optimized Buffer
：2X conc.
(Mg2+ concentration
：4 mM)
dNTP Mixture
：0.4 mM each
＊ 2：Chelex® is registered trademark of Bio-Rad Laboratories.
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3
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
III．Storage
10％ Chelex® solution : Stored at 4℃, shipped at room temperature
Other components :
Stored and shipped at -20℃
IV．Materials Required but not Provided
[ Reagents ]
-Agarose L03 (Cat. #5003) or NuSieve™ 3：1 Agarose (Lonza; Cat.
#50090/50091/50094)
-Electrophoresis buffer [ TBE powder (Cat. #T905) , etc. ]
-DNA marker
ϕX174 Hae III digest (Cat. #3405A/B)
100 bp DNA Ladder (Cat. #3407A/B) , etc.
-Loading buffer [ 6X：36％ glycerol, 0.05％ bromophenol blue, 30 mM EDTA, 0.05％
xylene cyanol ] (Note：Supplied with Takara DNA marker.)
-DNA Staining
Ethidium bromide
[ Equipment ]
-Thermal cyclers
TaKaRa PCR Thermal Cycler Dice Touch (Cat. #TP350)*
TaKaRa PCR Thermal Cycler Dice Gradient/Standard (Cat. #TP600/TP650)*
-Electrophoresis apparatus
-Ultraviolet transilluminator (Wavelength 300 nm)
-Polaroid camera or digital imaging system to photograph stained gel
-Heating block (applicable at 100℃)
-Refrigerated centrifuge, compatible for 1.5 ml tube
[ Other ]
-0.2 ml PCR tube
-Micropipettes and tips (with hydrophobic filter)
-Tray for staining agarose gel
* : Not available in all geographic locations. Check for availability in your region.
V．Precautions for Operation
2X Premix Solution contains enzymes; avoid vigorous mixing. Repeated freezing and
thawing will also result in decreased activity. It is advisable to thaw only the required
number of tubes of solution, dispense 25-μl aliquots into PCR tubes, and store at
-20℃.
4
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Cat. #RR114A
Bacteria Screening PCR Kit
v201310Da
VI．Principle
The PCR (Polymerase Chain Reaction) process consists of the following 3 steps：
1．DNA denaturation step
2．Primer annealing step
3．Polymerase extension step
In vitro DNA amplification is achieved by repeating these steps (see figure below) .
PCR allows amplification of target DNA by a factor of 1 million in a few hours.
1 Cycle
Temperature (℃)
Step 1
94
Step 2
Denaturation
35 Cycles
Step 3
Step 4
106-fold amplification of
target DNA fragment
72
Synthesis of
DNA
55
Primer
Annealing
22
1
2
3
Time (min.)
4
5
Step 1： Denature the target double-stranded DNA fragment in the reaction mixture
containing primers, dNTPs, and polymerase.
Step 2： Anneal primer to single-stranded DNA.
Step 3： Synthesize DNA with DNA polymerase.
Step 4： Return to Step 1 - to denature the amplified double-stranded DNA again to
yield single-stranded DNA.
Steps 1 to 4 are repeated. Note that the conditions that produce maximum amplification for a given DNA fragment will vary, and must be optimized for each target
fragment.
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Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
VII．Protocol
1．Preparation of Sample Solutions
A．DNA Extraction (In cases where large amount of bacteria can be prepared.)
1) Take 20 to 100 μl of bacterial culture media that has been incubated
overnight in BHI medium＊1, SCD medium＊2, or another suitable medium
and centrifuge at 12,000 rpm (13,000 X g ) for 3 to 5 minutes. (Use of 1.5 ml
microtube with screw cap is recommended.)
[ In cases where higher sensitivity is required, wash and suspend the collected
bacteria in 1 ml of distilled water or TE buffer (10 mM Tris HCl, 0.1 mM EDTA,
pH 8.0) , then centrifuge again. ]
2) Once the bacteria have been separated, add 2 to 10 volumes of pre-mixed
10％ Chelex Solution (typically 50 to 200 μl) using a pipet tip (sterile) that
has been cut to allow a wider opening to suspend the bacteria.
3) Heat the suspended bacteria solution at 99℃ for 5 minutes, then quickly
cool by placing on ice for 1 minute or more.
4) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute.
5) Use the centrifuged supernatant as the DNA sample solution for PCR.
B．Preparation of heat-extracted sample directly from bacteria
1) Pick up a minute quantity of the bacteria from the colony and suspend
in 50 to 200 μl of 10％ Chelex® solution. (Use of a 1.5 ml microtube with
screw cap is recommended.)
2) Heat the suspended bacteria solution at 99℃ for 5 minutes, then quickly
cool by placing on ice for 1 minute or more.
3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute.
4) Use the centrifuged supernatant as the DNA Sample Solution for PCR.
C．Preparation of DNA sample from food sample combined with culturing
1) Suspend the sample to be tested (food sample) 5 to 20 volumes of BHI medium ＊ 1, SCD medium ＊ 2 or another suitable medium, and crush using homogenizer (Stomacher, etc.). Culture with or without agitation for 4 to 7
hours.
2) Centrifuge 1.3 ml of the culture solution at 1,000 rpm (approximately 100 X g )
for 1 minute. Then centrifuge 1.0 to 1.2 ml of the supernatant at 12,000 rpm
(approx. 13,000 X g ) for 3 minutes.
3) Add 200 μl of 10％ Chelex® solution to the pellet and heat at 99℃ for 5
minutes, then quickly cool by placing on ice for 1 minute or more.
4) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute.
5) Use the centrifuged supernatant as the DNA Sample Solution for PCR.
＊ 1：BHI medium：Brain Heart Infusion medium
＊ 2：SCD medium：Soybean Casein Digest medium 6
URL:http://www.takara-bio.com
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
Note : In cases where the greater sensitivity is required, use the procedures described
in either [ Option 1 ] or [ Option 2 ] below.
[ Option 1 ] (Use of a 1.5 ml microtube with screw cap is recommended)
1) Wash the pellet containing the separated bacteria (see step 2 in section
C above) using sterile water or TE buffer (10 mM Tris-HCl, 0.1 mM EDTA,
pH8.0) then suspend in a 200 μl of Chelex® solution.
2) Heat at 99℃ for a period of 5 minutes then quickly cool by placing on ice
for 1 minute or more.
3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute.
4) Use the supernatant as the DNA Sample Solution for PCR.
[ Option 2 ] (Use of a 1.5 ml microtube with screw cap is recommended)
1) Suspend the pellet containing the centrifuged bacteria (see step 2 in section C above) in 100 μl of acromopeptidase＊3 (250 U/ml, in TE buffer) and
incubate for 10 to 15 minutes at 37 to 55℃. (This enables the enzymes to
break down the cell walls of gram-positive bacteria such as Staphylococcus
aureus . This, in turn, increases the content of recoverable DNA. Although
this process is not required for Enterobacteriaceae and Vibrionaceae bacteria families, it does not adversely affect detection.)
2) Add a 100 μl of Chelex® solution and heat at 99℃ for 5 minutes, then
quickly cool by placing on ice for 1 minute or more.
3) Centrifuge at 12,000 rpm (approx. 13,000 X g ) for 1 minute.
4) Use the supernatant as the DNA Sample Solution for PCR.
Note 1： Some processed foods (particularly heavily spiced ones) may inhibit PCR.
In such cases, purify DNA using a kit such as the Generation Capture Column Kit (QIAGEN) and DNeasy Plant Mini Kit (QIAGEN).
Note 2： With food products containing high content of dead bacteria, dead bacteria may be detected, even when combined to culture techniques.
Note 3： The Primer Mix BS may react with other bacillus types, such as those
found in fermented food products.
Note 4： In some cases, Vibionaceae family bacteria may not grow very well in
BHI＊1 and SCD＊3 medium.
＊ 3：Acromopeptidase：Acromopeptidase (TBL-1) and less-refined
products
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Cat. #RR114A
Bacteria Screening PCR Kit
v201310Da
2．Preparation of PCR Reaction Mixture
Mix the following components on ice in a 0.2 ml PCR tube.
2X Premix Solution
Primer Mix ENT or BS (5 μM each)
DNA Sample Solution ＊ 1
dH2O
Volume
25.0 μl
2.5 μl
2.5 μl ＊ 2
to 50.0 μl
Final conc.
1X
0.25μM each
＊ 1：A mixture containing dH2O instead of DNA sample solution is used as a Negative Control. The positive control mixture is produced by adding 2.5 μl of
Positive Control ENT or BS.
＊ 2：DNA sample solution can be added up to 5 μl. Exceeding this volume may
result in inhibition of the PCR reactions.
3．PCR Reaction
Ensure that the reaction tube cap is firmly tightened, and then load the tube correctly into the Thermal Cycler unit and perform reactions according to the following specifications.
95℃
↓
95℃
59℃
72℃
↓
72℃
4℃
60 sec.
30 sec.
30 sec.
30 sec.
30 cycles＊
60 sec.
＊：Perform reactions with 30 cycles. Conducting reactions with an excessive
number of cycles can result in secondary amplification products from Taq
polymerase.
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URL:http://www.takara-bio.com
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
4．Preparation of Agarose Gel
1) Dispense electrophoresis buffer into a triangular flask, and then add Agarose L03 or NuSieve™ 3：1 Agarose to a concentration of 1.8% (w/v) or 3% (w/v), respectively, with mixing.
2) Heat the mixture for 2 to 3 minutes in a microwave. Remove and mix thoroughly to ensure that the mixture has dissolved uniformly. If not, heat and mix
again.
3) Set up the gel tray.
4) Once the agarose gel cools to 50 - 60℃, pour the solution into the tray, insert
the comb to generate slots for applying samples, and leave at room temperature for 30 to 60 minutes to harden the gel.
* When pre-staining Using Ethidium Bromide.
After the agarose gel cools to 50 - 60℃, add ethidium bromide solution to a
final concentration of 0.5 μg / ml. Mix thoroughly and pour into the gel tray.
Leave at room temperature for 30 - 60 minutes and harden the gel.
5) After the gel hardens, place it in an electrophoresis tank and add electrophoresis buffer so that the gel is completely immersed.
6) Remove the comb, carefully not to break the gel.
5．Electrophoresis
1) Connect the electrophoresis apparatus, taking care to connect the anode and
cathode poles correctly. (Note : DNA is negatively charged and will move from
cathode to anode.)
2) Add 1.0 μl of 6X loading buffer to 2 to 5 μl of PCR reactant and mix. Gently
dispense into the lanes in the gel using a micropippette. (Dispense appropriate
amounts of DNA marker into the lanes at both ends.)
3) Apply fixed voltage of 50 to 150 V and perform electrophoresis.
6．Staining of Bands
* When the gel has been prestained with ethidium bromide, perform only step 3.
1) Prepare 1 μg/ml ethidium bromide solution in sufficient quantity to completely immerse the gel and dispense into the gel staining tray.
2) Place the gel into the tray and allow to leave for 20 to 30 minutes.
3) Set the gel on the ultraviolet transilluminator and photograph the gel. Compare the sample DNA bands with DNA marker to confirm the presence and size
of DNA bands.
Precautions
Always use gloves when working with ethidium bromide or handling stained gel.
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Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
7．Assessment
A band of approximately 419 - 425 bp ( 424 bp in E. coli ) can be detected with ENT
primer in the sample containing Enterobacteriaceae family, including E. coli and
Salmonella. A band of approximately 380 - 382 bp (381 bp in Bacillus cereus ) can
be detected with BS primer when the sample contains genus Bacillus, including
Bacillus cereus , or genus Staphylococcus, including Staphylococcus aureus . (For
lists of bacteria detected using the ENT and BS primers, refer to Table 1 at VIII. Application Examples.)
A band of 424 bp can be detected by the ENT primer when using the Positive Control ENT.
A band of 381 bp can be detected by the BS primer when using the Positive Control BS.
Electrophoresis results
Approx. 420 bp band detected by ENT
Primer
Approx. 380 bp band detected by BS
Primer
Assessment
Sample contains Enterobacteriaceae family strains, such
as E. coli or Salmonella bacteria.
Sample contains genus Bacillus strains, such as Bacillus
cereus , or genus Staphylococcus strains, such as Staphylococcus aureus .
ENT Primer failed to detect any bands Enterobacteriaceae family bacteria in the sample are
below the detection limit when 424-bp band is detected
in Positive Control ENT. If a 424-bp band is present in the
Positive Control ENT, something may be wrong with the
PCR detection. Repeat PCR.
BS Primer failed to detect any bands
Bacteria in Bacillus or Staphylococcus genera in sample
are below the detectable range when 381-bp band is
detected in Positive Control BS＊. If a 381-bp band is not
present in the Positive Control BS, something may be
wrong with the PCR detection. Repeat PCR.
No band detected in Negative Control Indicates no contamination.
420-bp or 380-bp band detected in
Indicates possibility of contamination. Perform deconNegative Control
tamination of reaction solution mixing area and equipment used. Repeat PCR.
＊：To improve sensitivity, use the techniques described in Options 1 and 2 of the
section "VII-1. Preparation of Sample Solutions" as necessary.
10
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Cat. #RR114A
Bacteria Screening PCR Kit
v201310Da
VIII．Application Examples
Specificity of Primers ENT and BS to various bacteria
[ Methods ]
PCR was performed with both the ENT and BS primers using 50 pg of genomic
DNA (equivalent to approximately 104 copies) as a template.
PCR Reaction：
95℃
60 sec.
↓
95℃
30 sec.
59℃
30 sec.
72℃
30 sec.
↓
72℃
60 sec.
4℃
30 cycles
Agarose electrophoresis analysis was performed on 2 μl of reaction solution
after PCR reaction.
[ Result ]
Figure 1 shows results of agarose gel electrophoresis. Table 1 shows the reaction specificity shown as positive or negative ( ＋ , － ).
(Primer Mix ENT was used for reactions involving Enterobacteriaceae (excluding Proteus mirabilis ) and Vibrionaceae family bacteria. Primer BS was used for
reactions involving genus Bacillus , Staphylococcus and Aerococcus bacteria.) Fig. 1 PCR Reaction Results for ENT and BS Primers (Electrophoresis)
Lane 1：ϕX174 Hae III digest Marker
Lane 14：Campylobacter coli DNA
Lane 2：Negative Control (No Template DNA)
Lane 15：Flavobacterium johnsoniae DNA
Lane 3：Serratia ficaria DNA
Lane 16：Streptococcus mutans DNA
Lane 4：Klebsiella pneumoniae DNA
Lane 17：Staphylococcus epidermides DNA
Lane 5：Salmonella enteritidis DNA
Lane 18：Staphylococcus aureus DNA
Lane 6：Escherichia coli DNA
Lane 19：Bacillus subtilis DNA
Lane 7：Citrobacter freundii DNA
Lane 20：Bacillus megaterium DNA
Lane 8：Yersinia enterocolitica DNA
Lane 21：Bacillus cereus DNA
Lane 9：Vibrio vulnificus DNA
Lane 22：Aerococcus viridans DNA
Lane 10：Photobacterium leiognathi DNA
Lane 23：Enterococcus faecalis DNA
Lane 11：Aeromonas hydrophila DNA
Lane 24：Lactobacillus casei DNA
Lane 12：Pseudomonas aeruginosa DNA
Lane 25：ϕX174 Hae III digest Marker
Lane 13：Alcaligenes faecalis DNA
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Bacteria Screening PCR Kit
1 2 3
4 5
6
Cat. #RR114A
v201310Da
7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25
Figure 1-1. ENT Primer Results
1 2 3
4 5
6
7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25
Figure 1-2. BS Primer Results
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URL:http://www.takara-bio.com
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
Table 1．Assayed Bacterial Species and Reaction Specificity
Family
Rhizobiaceae
Alcaligenaceae
Neisseriaceae
Xanthomonadaceae
Legionellaceae
Pseudomonadaceae
Moraxellaceae
Vibrionaceae
Enterobacteriaceae
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Genus, Species
Agrobacterium radiobacte
Alcaligenes faecalis
Chromobacterium violaceum
Neisseria meningitides
Xanthomonas maltophilia
Legionella pneumophila
Pseudomonas aeruginosa
Pseudomonas fluorescens
Moraxella antipestifer
Acinetobacter baumannii
Vibrio vulnificus
Vibrio parahaemolyticus
Photobacterium leiognathi
Aeromonas hydrophila (1)
Enterobacter agglomerans
Enterobacter cloacae
Citrobacter freundii
Escherichia coli
Escherichia coli JM109
Escherichia coli HB101
Escherichia coli 0157：H7
Hafnia alvei
Klebsiella pneumoniae
Plesiomonas shigelloides
Proteus mirabilis
Salmonella typhimurium
Source of strains ENT*
ATCC 19358T
－
JCM 1474T
－
JCM 1249T
－
ATCC 13077T
－
JCM 1975T
－
JCM 7571T
－
ATCC 27843T
－
JCM 5963T
－
JCM 9532T
－
JCM 6841T
－
JCM 3725T
＋
ATCC 17802T
＋
ATCC 25521T
＋
ATCC 7966T
＋
JCM 1236T
＋
JCM 1232T
＋
JCM 1657T
＋
JCM 1649T
＋
(2)
＋
(2)
＋
(3)
＋
JCM 1666T
＋
JCM 1662T
＋
ATCC 14029T
＋
JCM 1669T
－
IFo 13245
＋
BS*
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
－
13
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
(Continued)
Family
Pasteurellaceae
Campylobacteraceae
Helicobacteraceae
Clostridiaceae
Eubacteriaceae
Acidaminococcaceae
Bacillaceae
Listeriaceae
Staphylococcaceae
Lactobacillaceae
Aerococcaceae
Enterococcaceae
Streptococcaceae
Coriobacteriaceae
Actinomycetaceae
Micrococcaceae
Microbacteriaceae
Corynebacteriaceae
Propionibacteriaceae
Bifidobacteriaceae
Bacteroidaceae
Porphyromonadaceae
Prevotellaceae
Flavobacteriaceae
Flexibacteraceae
Fusobacteriaceae
14
Genus, Species
Salmonella typhimurium
Salmonella enteritidis
Serratia ficaria
Serratia marcescens
Shigella flexneri
Yersinia enterocolitica
Haemophilus influenzae
Campylobacter coli
Helicobacter pylori
Clostridium perfringens
Eubacterium alactolyticum (4)
Veillonella alcalescens
Bacillus cereus
Bacillus cereus
Bacillus thuringiensis
Bacillus licheniformis
Bacillus megaterium
Bacillus pumilus
Bacillus subtilis
Listeria grayi
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermides
Lactobacillus casei
Lactobacillus gasseri
Lactobacillus bulgaricus
Aerococcus sp.
Aerococcus viridans
Enterococcus faecalis
Streptococcus mutans
Streptococcus salivarius
Streptococcus agalactiae
Collinsella aerofaciens
Actinomyces naeslundii
Micrococcus luteus
Microbacterium lacticum
Corynebacterium kutscheri
Corynebacterium xerosis
Propionibacterium acnes
Bifidobacterium longum
Bifidobacterium adolescentis
Bacteroides vulgatus
Porphyromonas gingivalis
Prevotella intermedia
Flavobacterium johnsoniae
Cytophaga arvensicola
Fusobacterium nucleatum
Source of strains ENT*
BS*
IFo 14211
＋
－
IFo 3313
＋
－
JCM 1241T
＋
－
JCM 1239T
＋
－
ATCC 29903T
＋
－
ATCC 9610T
＋
－
ATCC 33391T
－
＋
ATCC 33559T
－
＋
ATCC 43504T
－
＋
JCM 1290T
－
＋
JCM 6480T
－
＋
ATCC 27215
－
＋
IFo 15305T
－
＋
ATCC 11950
－
＋
ATCC 10792T
－
＋
JCM 2505T
－
＋
JCM 2506T
－ ＋/－
JCM 2508T
－
＋
IFo 13719T
－
＋
ATCC 19120T
－ ＋/－
IFo 3060
－
＋
JCM 2874
－
＋
JCM 2414T
－
＋
JCM 1134T
－
－
JCM 1131T
－
－
JCM 1002T
－
－
MRA1 (5)
－
＋
IFo 12219T
－
＋
MRA2 (5)
－
－
JCM 5705T
－
－
JCM 5707T
－
－
JCM 5671T
－
－
JCM 10188T
－
－
JCM 8349T
－
－
JCM 1464T
－
－
JCM 1379
－
－
JCM 9385T
－
－
JCM 1971T
－
－
JCM 6425T
－
－
JCM 1217T
－
－
JCM 1275T
－
－
JCM 5826T
－
－
ATCC 33277T
－
－
ATCC 25611T
－
－
JCM 8514T
－
－
JCM 2836T
－
－
JCM 2836T
－
－
URL:http://www.takara-bio.com
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
* : Assessment
＋： PCR amplified product detected.
＋ / －： Minute quantities of PCR amplified product detected.
－： PCR amplified product not detected.
Note：
(1) While the genus Aeromonas currently belongs to the Vibrionaceae family,
there has been discussion of reclassifying it as a member of the Aeromonadaceae family. (Refer to TAXONOMIC OUTLINE OF THE PROCARYOTE GENERA
BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, SECOND EDITION Release
1.0, April 2001).
(2) Commercially Available Strains for Research.
(3) DNA purchased from Nissui Co., Ltd. (Tokyo, Japan) , which is a mixture of ATCC
43889 and ATCC 43890.
(4) Reclassified as Pseudoramibacter alactolyticym . Clin Infect Dis 1997 Sep;25
Suppl 2：S78-87
(5) MRA1and MRA2 are isolated and identified by Nisshin Foods.
IX．References
1) Rupf, S., Merte, K. and Eschrich, K. (1999) J. Dent. Res . 78: 850-856.
2) Wang, R.-F., Cao, W.-W. and Cerriglia, C.E. (1997) J. Appl. Microbiol . 83: 727-736.
X．Related Products
O-157 (Verocytotoxin Genes) PCR Screening Set (Cat. #RR100)*
*Not available in all geographic locations. Check for availability in your region.
URL:http://www.takara-bio.com
15
Bacteria Screening PCR Kit
Cat. #RR114A
v201310Da
NOTICE TO PURCHASER: LIMITED LICENSE
[A1] PCR Notice
This product is covered by one or more of the following US patents and corresponding patent
claims outside the US: 5,789,224, 5,618,711 and 6,127,155. 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 dsDNAbinding 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.
[L15] Hot Start PCR
Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other
countries.
[L33] Bacteria Screening
This product is covered by the claims of U.S. Patent No. 7,326,779 and its foreign counterpart patent
claims.
[M57] LA Technology
This product is covered by the claims 6-16 outside the U.S. corresponding to the expired U.S. Patent
No. 5,436,149.
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.
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URL:http://www.takara-bio.com