TAKARA CycleavePCR VT1 User manual

1
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
Table of contents
I. Description...............................................................................2
II. Kit Components.........................................................................3
III. Reagents and Instruments Required..........................................3
IV. Storage......................................................................................3
V. Cautions....................................................................................4
VI. Protocols....................................................................................4
VI-1. Sample preparetion......................................................5
VI-2. Setting of Smart Cycler®II System...............................5
VI-3. Reaction mixture ........................................................7
VI-4. Display of results........................................................9
VI-5. Judgement..................................................................11
VII. Trouble Shooting......................................................................15
VIII. Reference.................................................................................16
IX. Related products.......................................................................17

2TAKARA BIO INC.
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
URL:http://www.takara-bio.com
I. Description: Enterohemorrhagic E. coli (EHEC) such as O157:H7 is a class of pathogenic
E. coli which
causes hemorrhagic colitis with accompanying melena and severe abdominal pain, and in
addition, hemolytic uremic syndrome. These serious symptoms are caused by verotoxin, a
cytotoxin produced by EHEC. It has been pointed out that in detection of EHEC, an assay
method of accurately and promptly determining if there are these verotoxin genes or not is
important.
This Kit is for real-time PCR assay to detect, in combination with a thermal cycler for real time
PCR, Smart Cycler®II System*1(Cepheid), verotoxin genes (VT1 and VT2 genes) which cause
EHEC-related food poisoning. PCR is a technique for amplifying specifically the fragments of
the genes of interest in a short period of time using a trace amount of DNA as template. The
cycle comprising three steps of denaturation, primer annealing and extension with DNA
polymerase are repeated, thereby amplifying the gene fragments of interest up to 106-fold in
quite a short period of time.
By utilizing Smart Cycler®System, the amplification process can be real-time monitored.
As this kit uses Takara’s PCR enzyme efficient for Hot Start PCR, Takara Ex TaqTM R-PCR,
non-specific amplification deriving from mispriming or from primer-dimers before thermal
cycling can be avoided and it achieves highly sensitive detection.
This kit employs Cycling Probe Technology(CPT)*2for detection, which is a high-sensitive
detection method utilizing a combination of chimera probe, composed of RNA and DNA, and
RNase H. The specific sequence of target gene to be amplified can be detected efficiently
during or after amplification by this method. The 5' end of the probe is labeled with a fluores-
cent substance and the 3' end is labeled with a quencher, which quenches the fluorescence
emitted from the fluorescent substance at the 5' end. As long as this probe remains intact, no
strong fluorescence can be emitted because of the quenching function. When this probe forms
a hybrid with the complementary sequence of amplified product, RNase H specifically cuts the
RNA region of this probe, resulting in emission of strong fluorescence. By measuring the
intensity of emitted fluorescence, the amount of amplified product can be monitored.
Version 2.0 achieves higher specificity by improving probes.
This Kit incorporates the FAM labeling probe for detecting VT1 and the ROX labeling probe for
detecting VT2. It also contains an internal control and the TET labeling probe for detecting the
internal control. Simultaneous monitoring of three wavelengths with Smart Cycler®II System
requires only one tube to distinguish VT1 from VT2. This Kit also is able to monitor a false
negative reaction through the use of internal control. The real-time detection with this Kit does
not require electrophoresis and the detection result can be obtained quickly (in about 30
minutes).
*1: Smart Cycler®System is a registered trademark of Cepheid.
*2: Cycling Probe Technology and DNA-RNA-DNA chimeric nucleic acid technology are
licensed by ID Biomedical Corporation.

3
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
1. TaKaRa Ex TaqTM R-PCR 5 units/µl 12.5 µl (50 reactions)
2. Tli RNase H II*1200 units/µl 25 µl (50 reactions)
3. 5 X Reaction Mixture*25 X conc. 250 µl (50 reactions)
4. VT Primer Mix*35 µM each 100 µl (50 reactions)
5. VT Chimera Probe Mix*45 X conc. 250 µl (50 reactions)
6. VT1 Positive Control 1 X 104copies/µl 10 µl (10 reactions)
7. VT2 Positive Control 1 X 104copies/µl 10 µl (10 reactions)
8. distilled water 1 ml
*1 Tli RNase H II is a thermostable RNase H derived from Thermococcus litoralis .
*2 Including dNTP Mixture and Internal Control.
*3 Primers are manufactured by SHIMADZU CORPORATION.
*4 Including probe for control reaction. Be sure to store the fluorescent labeling probes in the
light-shielding environment.
II. Kit Components (25 µl X 50 reactions):
Q
F
キメラprob
e
増幅産物
RNA
Q
F
ハ
イブリッド形成
Q
F
RNaseHによる
RNA部分の切断
Q
F
蛍光強度増大
蛍光物質
ク
エ
ンチ
ャー
RNaa
aass ssee eeHH HH
Fluorescent Quencher
substance
Chimeric probe
Amplified products
Formation of hybrid Cut RNA part by RNaseH Increase of fluorescence intensity
Principle of ALDH2 typing:
[ Probe labeling, a detection channel, and amplification size ]
Probe fluorescence substance Detection channel Amplification size
VT1 FAM and Quencher Ch1 171 bp
VT2 ROX and Quencher Ch3 171 bp
Internal control TET and Quencher Ch2 110 bp
III. Reagents and Instruments Required but Not Supplied in the Kit:
IV. Storage: -20°C (for shipping and storage)
[Reagents] Sterilized distilled water
[Equipment] 1. Smart Cycler®II System (Real time PCR instrument)
(Cepheid)
2. Special tubes for Smart Cycler®
3. Desk-top centrifuge for Smart Cycler®
4. Heat block (applicable at 95°C)
[Others] 1. Micropippets for 200 µl, 20 µl and 10µl.
2. Micropippet tips (with hydrophobic filter)

4TAKARA BIO INC.
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
URL:http://www.takara-bio.com
Throughout the experimental procedures, the following cautions should be observed:
1. When handling Smart Cycler® II System, be sure to follow the written instructions for the
device.
2. If a chimera probe or a primer is decomposed by contamination with nuclease, such
decomposition inhibits accurate detection. Sweat or saliva of an operator can cause
contamination with nuclease. Extreme caution should be exercised during operation.
3. For the specimens determined to be positive, another microbiological test should be
conducted for verification.
4. Please divide physically the operation area into the following three parts for the
procedures from preparation to detection. Do not open tubes containing amplified
products within each area.
Area 1: Preparation and dispensing of reaction mixture
Area 2: Sample preparation
Area 3: Addition of sample into a reaction mixture, and perform reaction and detection.
As this kit performs amplification and detection simultaneously through real-time PCR, there is
no need to use amplified product obtained from the reaction to subsequent process, such as
eletrophoresis, etc.
Please refrain from taking amplified products out of tubes. It can result in contamination.
V. Cautions:
VI. Protocols <Outline of protocol>
1. Sample preparation (see page 5 )
Prepare heat -extracted bacterial sample from proliferated culture solution.
2. Setting of Smart Cycler®II System (see page 5-7)
Start Smat Cycler®II System.
↓
Set the PCR conditions. [Define Protocols]
Set the graph view of the result. [Define Graphs]
↓
Set the parameters; numbe of reactions and the Protocol/Site to be used, and give a
name to Run. [Create Run]
↓
3. Preparation of reaction solution and start of reaction (see page 7-9)
Prepare the reaction solution.
↓
Transfer the prepared solution into spetial reaction tubes and add a sample.
↓
Load the spetial ubes on Smart Cycler® II System and start Run.
↓
4. Viewing of the result (see pages 9-12)
Select the graphs o be used. [Select Graphs]
↓

5
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
[Preparation of heat-extracted bacterial sample]
1) Transfer 10 µl of proliferated bacterial culture into a 1.5 ml tube.
2) Add 90 µl of sterilized water and mix.
3) Heat for 5 min. at 95°C.
4) Centrifuge at 12,000 rpm, 4°C for 10 min, and collect the supernatant. Use the obtained
supernatant as heat-extracted sample for VT1/VT2 detection.
* If the PCR reaction is inhibited with heat-extracted sample prepared through the above
method, dilute it with sterilized water by 10-fold and 100-fold and apply them for PCR
reaction.
* Proliferated culture solution should be prepared by following the standard protocol
appropriate for each food sample. Heat extracted sample can be stored at -20°C
.
(For more information on handling Smart Cycler
®
II System, see the instructions supplied with it.)
(1) Start the Smart Cycler®II System.
(2) Set the protocol.
Click the icon “Define Protocols” and then “New Protocol” button to create the protocol
by following the steps shown below. (Since the created protocol is saved, no entry is
required in subsequent reactions).
VI-1. Sample preparation (Perform in Area 2)
VI-2. Setting of Smart Cycler®II System (Perform in Area 3)
↓
Enter sample name. [Sample ID]
Set analytic parameters. [Analysis Setting]
↓
Amplification curve is viewed on the screen in real time.
↓
The reaction is terminated.
↓
5. Judgement (see page 12-16).
Define Protocols
Crick New Protocol
Create this protocol

6TAKARA BIO INC.
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
URL:http://www.takara-bio.com
(3) Set the graphs. (Since the created graphs are saved, no entry is required in subsequent
reactions).
(3)-1. Set the amplification curve (FAM) for the amplified product derived from VT1.
Click the icon “Define Graphs” and create the graphs by following the steps shown
below.
(Since the graphs have been set under a name “FAM” at initialization, no entry is
required here).
(3)-2. Set the amplification curve (TET) for the amplified product derived from Internal.
Click the icon “Define Graphs” and create the graphs by following the steps shown
below.
(Since the graphs have been set under a name “TET” at initialization, no entry is
required here).
(3)-3. Set the amplification curve (ROX) for the amplified product derived from VT2.
Click the icon “Define Graphs” and create the graphs by following the steps shown
below.
(Since the graphs have been set under a name “ROX” at initialization, no entry is
required here).

7
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
VI-3. Preparation of reaction mixture:
Enter Run Name
Select FTTC25 in
Dye Set.
Choose the site and protocol to
be used.
Click the [OK] button.
(4) Click the icon “Create Run”, enter Run Name, and select Dye-Set (FTTR25 in Smart
Cycler® or FTTC25 in Smart Cycler®II ).
Click the “Add/Remove Site” button and the “Select Protocols and Sites” screen appears.
From the menu, select the Site and Protocol to be used.
Click the Add/Romove Sites to display
the [Select Protocol and Sites] screen.
This product detects simultaneously three amplification products, VT1, VT2, and internal
control, within one reaction tube. In order to obtain the right detection result, we recommend
you to perform VT1 positive control reaction, VT2 positive control reaction, and a negative
control reaction altogether.
(1) Prepare the reaction mixture on ice shown below. (Perform in Area 1)
Prepare the mixture except adding template in the tubes of required number plus a few.
Dispense it into Smart Cycler® tubes and then add template. The required number means
sample number plus three. This additional three means the tubes which are added with
VT1 positive control or VT2 positive control, or sterilized distilled water in place of template,
respectively.

8TAKARA BIO INC.
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
URL:http://www.takara-bio.com
Per reaction final conc.
5 X Reaction Mixture 5 µl 1 X
VT Primer Mix (5 µl each) 2 µl 0.4 µM each
VT Chimera Probe Mix (5 X conc.) 5 µl 1 X
TaKaRa Ex Taq R-PCR (5 units/µl) 0.25 µl 1.25 units
Tli RNase H II (200 units/µl) 0.5 µl 100 units
Sample DNA or Positive Control 1~10 µl
Sterilize distilled water up to 25 µl
Sample (template) solution is added in the volume range of 1-10 µl. Add sterilized distilled
water to have the final volume of 25 µl. In case of adding positive control, add 1 µl as
template.
Add the components of the above reaction mixture without template into a fresh tube, by
pouring on the tube wall. Please refer to the following figure.
Close gently the lid of the tubes not so tightly and move to the Area 3.
(2) Addition of sample template (Perfom in Area 3):
Prepare one tube of negative control by adding sterilized distilled water instead of sample.
For the rest tubes, add the prepared samples into the reaction mixture prepared at (1),
and close the lid of the tube tightly.
Place the reaction mixture (15-24 µl)
into this reserver part.
Centrifuge to load down the reaction
mixture into this part of the tube.
(3) Load the reaction tubes on Smart Cycler®II System and click the "Start Run" button to start
the reaction process.

9
TAKARA BIO INC.
URL:http://www.takara-bio.com
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
VI-4. Display of results:
(1) View the View Results screen. (The same time the reaction process is started, the View
Results screen automatically appears. If another screen is open, click the icon “View
Results”).
(2) Click the “Select Graphs” button and the Select Graphs screen appears. From the menu,
select FAM (VT1 amplified curve), TET (Internal control amplified curve) ROX (VT2
amplified curve), and Temperature (the temperature chart graph).
Click and show the "Select Graphs".
When the graphs for FAM, TET, ROX, and
Temperature have been selected at the
initial setting, no entry is required here.
Choose the site and
protocol to be used
and click the [OK] button.
(3) From the Views list, select “Results Table” and enter Sample ID.
Click the
"Results Table". Enter sample ID.
(4) Click “Analysis Settings” for opening. Click “Usage” next to Ch#4 and select “Unused” from
the pull-down menu. In the Analysis Settings screen, set Manual Thresh Fluor. Units for
Ch#1, Ch#2 and Ch#3 to 100. After entering data in the cells, click the “Update Analysis”
button, and the settings are activated. This value is used as a cut-off value.

10 TAKARA BIO INC.
CycleavePCRTM O157 (VT1/VT2) Detection Kit Ver.2.0 v.0607
Cat.#CY203
URL:http://www.takara-bio.com
(5) Select the amplified curve of interest in the above Views
Amplified curve for FAM
(VTI gene)
Amplified curve for TET
(Internal control)
Amplified curve for ROX
(VT2 gene)