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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
Table of contents
I.
Description...................................................................................2
II.
Principle.......................................................................................2
III. Kit Components............................................................................3
IV. Storage..........................................................................................4
V. Precautions for Operation.....................................................4
VI. Protocol............................................................................................4
VII. Application Example........................................................................8
VIII.
References................................................................................12
IX.
Related products...........................................................................12
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
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 (Polymerase Chain Reaction) process is recognized as one of the more useful methods for such food-related quality control
applications.
PCR comprises a technique in which minute amounts of DNA can be used as a template for amplifying only the desired DNA fragments. A single cycle of this method comprises three incubation steps;
DNA Thermal denaturation, primer annealing, and DNA polymerase chain reaction. Amplification by
a factor of 1 million can be achieved in relatively short periods of time by repeating this process.
The Bacteria Screening PCR kit is designed to use 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 cereus bacteria, and genus
Staphylococcus , including Staphylococcus aureus strains. (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. Principle:
The PCR (Polymerase Chain Reaction) process comprises the following three steps:
1. DNA denaturation step
2. Primer annealing step
3. Polymerase chain reaction extension step
In vitro DNA amplification is achieved by repeating these steps (See figure below). This method
makes it possible to amplify target DNA by a factor of 1 million in a period of several hours
1 Cycle
Step 1
Step 2
Temperature (℃ )
ステップ１ ステップ 2
94
Step 3
ステップ 3
Denatruration
温
度 72
（℃）
ステップ1∼4を
35 Cycles
25∼30回繰り返す
ステップ
Step 4 4
目的DNA断片を
106-fold amplification
of
target DNA fragment
10万倍に増幅
熱変性
相補鎖の合成
Synthesis
of
cDNA
55
プライマーの
Primer
アニーリング
Annealing
22
１
Step1: Step2:
Step3: Step4: TAKARA BIO INC.
2
3
Rime時間
(min.)
(min)
4
5
Denature the target double-stranded DNA fragment in the
reaction mixture containing primer, dNTP, and polymerase.
Anneal primer to obtained single-stranded DNA.
Synthesize cDNA with DNA polymerase.
Return to Step 1 - to denature the amplified double-stranded DNA again to yield single-stranded DNA.
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
Steps 1 to 4 are repeated as a single cycle. Note that the conditions that produce maximum amplification for a given DNA fragment will vary, and so must be set individually for each fragment.
III. Kit Components (50 μ l x 50 reactions for ENT and BS primers):
1. 2X Premix Solution*1
2X conc.250 μ l X 10 tubes (100 reactions)
2. Primer Mix ENT
5 μ M each 125 μ l (50 reactions)
3. Primer Mix BS
5 μ M each 125 μ l (50 reactions)
4. Positive Control ENT
25 μ l (10 reactions)
5. Positive Control BS
25 μ l (10 reactions)
6. dH2O
1 ml X 3 tubes
Ⓡ
2
7. 10% Chelex Solution* 12 ml - 2 vials
*1 Contents of 2X Premix Solution
TaKaRa Ex Taq TM HS
: 0.1 units/ μ l
Opitimized Buffer
: 2X conc.
(Mg2+ Concentration
: 4 mM)
dNTP Mixture
: 0.4 mM each
*2 Chelex Ⓡ is registered trademark of Bio-Rad Laboratories.
Reagents and Instruments Required but Not Supplied in this product
[Reagents]
1. Agarose L03 (TaKaRa Code 5003) or NuSieve Ⓡ 3:1 Agarose (Lonza Biosciences)
2. Electrophoresis buffer [TBE powder (TaKaRa Cat.# T905), etc.]
3. DNA size marker [ϕX174 Hae III digest (TaKaRa Cat.# 3405A/B), 100bp DNA Ladder (TaKaRa Cat.#
3407A/B), etc.]
4. Loading buffer [6X: 36% glycerol, 0.05% bromophenol blue, 30 mM EDTA, 0.05% xylene cyanol]
(Note: Included with the item "3. DNA molecular marker" above.)
5. DNA Stain [SYBR Ⓡ Green I, GelStar Ⓡ Nucleic Acid Stain or ethidium bromide]
[Instruments]
1. Thermal Cyclers [TaKaRa PCR Thermal Cycler Dice (TaKaRa Cat.# TP600/TP650)]
2. Electrophoresis apparatus
3. Power supply for electrophoresis apparatus
4. Ultraviolet transilluminator (capable of approximately 300 nm wavelength)
5. Instruments capable of photographing electrophoresis gel, such as Polaroid camera
6. Heating block (capable of heating up to 100℃ )
7. Refrigerated centrifuge compatible with 1.5 ml tubes
[Other Items]
1. 0.2 ml PCR tubes [TaKaRa Micro PCR tube (TaKaRa Cat.# 9047)]
2. Micropippettes
3. Tips for micropipettes (with hydrophobic filter)
4. Polaroid film
5. Tray for staining agarose gel (Be sure to use polypropylene trays when utilizing SYBR Ⓡ Green I
and GelStar Ⓡ )
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Bacteria Screening PCR Kit
IV. Storage:
Cat.# RR114A
v.0703
The 10% Chelex Ⓡ solution would be transported at room temperature but must be stored
at 4℃ . All other components must be both transported and stored at -20℃ .
V. Precautions for Operation:
The "2X Premix Solution" contains enzymes, so intensive mixing procedures should be
avoided. Repeated freezing and thawing will also result in deterioration of active elements.
It is advisable to thaw only the required number of tubes of solution, to dispense 25- μ l
portions into the designated PCR tubes and to store these at -20℃ .
VI. Protocol:
1. Preparation of PCR Sample Solutions
A. DNA Extraction (In the case when large amount of bacteria can be prepared.)
1) Take 20 to 100 μ l of bacterial culture media that has been incubated overnight
in BHI medium*, SCD medium** or another suitable medium and centrifuge at
12,000 rpm (13,000 X g) for 3 to 5 minutes. (Use of 1.5 ml micro test tube with
screw cap is recommended.)
[In cases where higher sensitivity is required, wash and suspend the separated
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 a volume of pre-mixed 10% Chelex
Solution equivalent to 2 to 10 times the volume of the separated bacteria (typically
50 to 200 μ l) using a truncated tip (end of the truncated tip should be sterilized)
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) Extract a minute quantity of the bacteria from the colony and suspended in a 50
to 200 μ l volume of 10 % Chelex Ⓡ solution. (Use of 1.5 ml micro test tube 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 sample from Food Sample combined with culture technique
1) Dilute the sample to be tested (food sample) by 5 to 20 times in BHI medium*, SCD
medium** or another suitable medium, and crush using a bacteriological examination homogenizer (Stomacher, etc.). Allow to set for between 4 and 7 minutes
or culture with agitation.
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.
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
3) Add 200 μ l 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.
In cases where the greater sensitivity is required, use the procedures described in either [Option 1]
or [Option 2] below.
Options for Preparation of Bacterial DNA
[Option 1] (Use of 1.5 ml micro test tube with screw cap is recommended.)
1) Wash the pellet containing the separated bacteria (see step 2 in section C above) using
sterilized water and TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) then suspend in a
200 μ l volume 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 one minute.
4) Use the centrifuged supernatant as the DNA Sample Solution for PCR.
[Option 2] (Use of 1.5 ml micro test tube with screw cap is recommended.)
1) Suspend the pellet containing the centrifuged bacteria (see step 2 in section C above)
in 100 μ l of acromopeptidase*** (250 U/ml, in TE buffer) and incubate for 10 to 15
minutes at 37℃ to 55℃ . (This enables enzymes to break down the cell walls of grampositive Staphylococcus aureus bacteria. This, in turn, increases the volume of recoverable DNA. Although this process is not required for Enterobacteriaceae and Vibrionaceae
families of bacteria, it has no adverse effects on their detection sensitivity.)
2) Add a 100 μ l volume 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 centrifuged supernatant as the DNA Sample Solution for the PCR.
Note 1: Some processed foods (particularly heavily spiced ones) may disturb the effectiveness
of PCR. In that case, additional DNA exaction methods, such as the GENERATION Capture Column Kit (Gentra Systems) and DNeasy Plant Kit (QIAGEN) may be required.
Note 2: With food products containing high volumes of dead bacteria, there are cases where
dead bacteria will be detected, even when combinations of culture techniques are applied.
Note 3: The BS primer 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* and SCD**
medium.
* BHI medium: Brain heart infusion broth
** SCD medium: Tripto soya broth
*** Acromopeptidase: Acromopeptidase (TBL-1) and less-refined products
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
2. Preparation of PCR Reaction Mixture
The following reaction solutions are mixed together on ice in a 0.2 ml PCR tube.
Solution volume 2X Premix Solution
25.0 μ l
Primer Mix ENT or BS (5 μ M each) 2.5 μ l
DNA Sample Solution* 2.5 μ l**
dH2O
up to 50.0 μ l
Final conc.
1X
0.25 μ M each
* A mixture containing dH2O instead of DNA sample solution is used for PCR as a negative control. The positive control mixture is created by adding 2.5 μ l of positive control ENT or BS.
** DNA sample solution can be added in volumes up to 5 μ l. Exceeding this volume may result
in inhibited reactions.
3. PCR Conditions
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.
4. Preparation of Agarose Gel
1) Dispense electrophoresis buffer into a triangular flask, and then add 1.8% (W/V) Agarose L03
or 3% NuSieve Ⓡ 3:1 Agarose gradually as the flask contents are mixed.
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) Prepare the gel tray.
4) Once the agarose gel cools (to 50 to 60℃ ), pour into the tray, insert the comb to create slots
for applying samples and allow gel to set at room temperature for 30 to 60 minutes.
(When Pre-Staining Using Ethidium Bromide)
After the agarose gel cools (to 50 to 60℃ ), add ethidium bromide solution to a final concentration of 0.5 μ g/ml. Mix thoroughly and pour into the gel tray. Allow gel to set at room
temperature for 30 to 60 minutes.
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
5) After the gel has set, place it in an electrophoresis vat and add electrophoresis buffer to until
the gel is completely immersed.
6) Gently remove the comb, taking care not to break the gel.
5. Electrophoresis
1) Connect the electrophoresis apparatus, taking care to connect the anode and cathcde poles
correctly. (Note: DNA amplified by PCR are negatively charged and will move from cathode
to anode.)
2) Add 1.0 μ l of 6X loading buffer to the 2 to 5 μ l quantities of PCR reactant and mix. Gently
dispense into the lanes in the gel using micropippettes. (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. Checking Stain Bands (only step 3) required for ethidium bromide pre-staining
1) Create 1 μ g/ml ethidium bromide solution, SYBR Ⓡ Green I solution or GelStar Ⓡ *(diluted by
a factor of 10,000 using TBE buffer or electrophoresis buffer) in sufficient quantity to completely immerse the gel and dispense into the agarose staining tray.
2) Place the gel on which electrophoresis has been performed into the tray and allow to set for
20 to 30 minutes.
3) Move the gel to the ultraviolet transilluminator, take photographs as required and compare
the lanes containing sample solutions with those containing DNA markers to confirm the
presence and size of nucleotide bands.
* Make sure to use the designated filter when using SYBR Ⓡ Green I or GelStar Ⓡ .
Precautions for operation
Always ensure to use gloves to avoid any contact when working with ethidium bromide, SYBR Ⓡ
Green I, GelStar Ⓡ or handling gel stained with DNA stains.
7. Assessing Results
A band of approximately 420 bp (419 to 425 bp; 424 bp with E. coli ) can be detected with
ENT primer when the PCR sample contains Enterobacteriaceae family strains, including E. coli
and salmonella bacteria. A band of approximately 380 bp (380 to 382 bp; 381 bp with Bacillus
cereus ) can be detected when using BS primer when the PCR sample contains genus Bacillus
strains, including Bacillus cereus , or genus Staphylococcus strains, including Staphylococcus
aureus. (For lists of bacilli detected using the ENT and BS primers, refer to Lists of Applied Data
1 and 2 in section VII.)
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.
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Bacteria Screening PCR Kit
Sample electrophoresis results
Band of approx. 420 bp detected by
ENT Primer
Band of approx. 380 bp detected by
BS Primer
ENT Primer failed to detect any bands
BS Primer failed to detect and bands
No band detected in Negative Control
lane
420-bp or 380-bp band detected in
Negative Control lane
Cat.# RR114A
v.0703
Assesment
PCR sample contains Enterobacteriaceae family strains,
such as E.coli or salmonella bacteria.
PCR sample contains genus Bacillus strains, such as
Bacillus cereus, or genus Staphylococcus strains, such as
Staphylococcus aureus.
Enterobacteriaceae family strains in the PCR sample are
below the detection limit when 424-bp band is detected in
Positive Control ENT lane. If 424-bp band is not appeared in the
Positive Control ENT lane, something may be wrong with
PCR detection. Retry PCR detection.
Genus Bacillus strains or genus Staphylococcus strains in PCR
sample are beyond the detectable range when 381-bp band is
detected in Positive Control BS lane.* If 381-bp band
not detected in the Positive Control BS lane, something may
be wrong with PCR detection. Retry PCR detection.
Indicates no contamination.
Indicates possibility of contamination. Perform decontamination
of reaction solution mixing area and equipment used. Retry PCR
detection.
* To improve sensitivity, use the techniques described in options 1 and 2 of the section
"1. Preparation of PCR Sample Solutions" as necessary.
VII. Application Example:
Studies of Specificity of Primers ENT and BS to various bacteria
[Methods]
PCR detection was performed with both the ENT and BS primers using 50 pg of genomic DNA
(equivalent to approxi mately 104 copies) as a template.
PCR Conditions:
95℃60 sec
95℃30 sec
59℃30 sec
72℃30 sec
30 cycles*
72℃60 sec
4℃
Agarose electrophoresis analysis was performed on 2 μ l volumes of reaction solution after
PCR reaction.
[Result]
Figure 1 shows sample results obtained from agarose gel electrophoresis analysis. Table 1
shows the reaction characteristics expressed as positive or negative (+, -).
(Primer ENT was used for reactions involving Enterobacteriaceae (excluding Proteus mirabilis)
and Vibrionaceae family strains of bacteria. Primer BS was used for reactions involving genus
Bacillus , genus Staphylococcus and genus Aerococcus bacteria.)
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Bacteria Screening PCR Kit
Cat.# RR114A
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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
(1) ENT Primer Results
(2)BS Primer Results
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
Table 1 Assayed Bacteria and Reaction Characteristics
Family
Rhizobiaceae
Alcaligenaceae
Neisseriaceae
Xanthomonadaceae
Legionellaceae
Pseudomonadaceae
Moraxellaceae
Vibrionaceae
Enterobacteriaceae
Pasteurellaceae
Campylobacteraceae
Helicobacteraceae
Clostridiaceae
Eubacteriaceae
Acidaminococcaceae
Bacillaceae
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 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 10 TAKARA BIO INC.
Source of strains
ATCC 19358T
T
JCM 1474 JCM 1249T
ATCC 13077T
JCM 1975T
JCM 7571T
ATCC 27843T
T
JCM 5963 JCM 9532T
JCM 6841T
JCM 3725T
ATCC 17802T
T
ATCC 25521 ATCC 7966T
JCM 1236T
JCM 1232T
JCM 1657T
JCM 1649T
(2)
(2)
(3)
JCM 1666T
JCM 1662T
ATCC 14029T
JCM 1669T
IFo 13245
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
ENT　BS
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+/-
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
(Continued)
Family
Genus, Species
Source of strains　ENT
BS
Listeriaceae
Staphylococcaceae
Lactobacillaceae
Aerococcaceae
Enterococcaceae
Streptococcaceae
Coriobacteriaceae
Actinomycetaceae
Micrococcaceae
Microbacteriaceae
Corynebacteriaceae
Propionibacteriaceae
Bifidobacteriaceae
Bacteroidaceae
Porphyromonadaceae
Prevotellaceae
Flavobacteriaceae
Flexibacteraceae
Fusobacteriaceae
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 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
ATCC 25586T
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+/+
+
+
+
+
-
-
Assessment Results
+
: PCR amplified material detected.
+/-
: Minute quantities of PCR amplified materials detected.
-
: PCR amplified material not detected.
Note:
(1) While the genus Aeromonas currently belongs to the Vibrionaceae family, there is currently some discussion
on 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. (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 is isolated and identified by Nisshin Foods.
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Bacteria Screening PCR Kit
Cat.# RR114A
v.0703
VIII. References:
Rupf, S., Merte, K. and Eschrich, K. (1999) J. Dent. Res ., 78, 850-856.
Wang, R.-F., Cao, W.-W. and Cerriglia, C.E. (1997) J. Appl. Microbiol., 83, 727-736.
IX. Related products:
O-157(Verocytotoxin Genes) PCR Screening Set (TaKaRa Cat.#RR100)
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 TAKARA BIO INC.
If you require licenses for other use, please call at +81 77 543 7247 or contact from our website at www.takara-bio.com .
NOTICE TO PURCHASER: LIMITED LICENSE
This product is sold under licensing arrangements with F.Hoffmann-La Roche Ltd, Roche Molecular Systems, Inc. and
Applied Biosystems.
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.
Chelex Ⓡ is trade mark of Bio-Rad Laboratories, Inc. SYBR Ⓡ is a registered trademark of Molecular Probes, Inc. GelStar Ⓡ
is a trademark of Cambrex Corporation.
This product is manufactured and sold by Takara Bio under license from Nisshin Foods, Inc.
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