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Index / Liferi / Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069 / Product Detail : DD-0127-02 Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069
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#DD-0127-02 Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069

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  Price : 476   EUR
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Product name : Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069

Catalog number : DD-0127-02

Quantity: 25

Availability: Yes

Supplier name : Liferi

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About this Product :

Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069 PCR kit use certain PCR kits can be used for diagnostics. We supply CE approved PCR kits or research PCR kits. You can request more information about the use of Yersinia Enterocolitica Real Time PCR Kit *CE Marked III, IV *to see for appropriate equipment see catalogue No. MXX_000069 to info@gentaur.com
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More Details about

Yersinia Enterocolitica Real Time PCR Kit User Manual
For In Vitro Diagnostic Use Only
DD-0127-02

 

1. Intended Use


Yersinia Enterocoliticareal time PCR kit is used for the detection of Yersinia Enterocoliticareal in stool, blood,food or water samples by using real time PCR systems.


2. Principle of Real-Time PCR
The principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR
reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially (Ct) is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification.


3. Product Description


Yersinia enterocolitica is a species of gram-negative coccobacillus-shaped bacterium, belonging to the family Enterobacteriaceae. Primarily a zoonotic disease (cattle, deer, pigs, and birds), animals which recover frequently become asymptomatic carriers of the disease. Acute Y. enterocolitica infections produce severe diarrhea in humans, along with Peyer's patch necrosis, chronic lymphadenopathy, and hepatic or splenic abscesses. Additional symptoms may include entero-colitis, fever, mesenteric adenitis, erythema nodosum and acute terminal ileitis, which may be confused with appendicitis or Crohn's disease. Yersinia Enterocoliticareal time PCR kit contains a specific ready-to-use system for the detection of the Yersinia Enterocoliticausing PCR (polymerase chain reaction) in the real-time PCR system. The master contains reagents and enzyme for the specific amplification of the Yersinia EnterocoliticaDNA. Fluorescence is emitted and measured by the real time systems´ optical unit during the PCR. The detection of amplified Yersinia Enterocolitica DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1. In addition, the kit contains a system to identify possible PCR inhibition by measuring the HEX/VIC/JOE fluorescence of the internal control (IC). An external positive control defined as 1×107copies/ml is supplied which allow the determination of the gene load. For further information, please refer to section

9.3 Quantitation.


4. Kit Contents

 

Analysis sensitivity: 1×103 copies/ml; LOQ: 2×103~1×108 copies/ml


5. Storage
• All reagents should be stored at -20°C. Storage at +4°C is not recommended.
• All reagents can be used until the expiration date indicated on the kit label.
• Repeated thawing and freezing (>3x) should be avoided, as this may reduce the sensitivity of the assay.
• Cool all reagents during the working steps.
• Reaction mix should be stored in the dark.


6. Additionally Required Materials and Devices


• Biological cabinet • Real time PCR system
• Vortex mixer • Real time PCR reaction tubes/plates
• Cryo-container • Pipets (0.5μl – 1000μl)
• Sterile filter tips for micro pipets • Sterile microtubes
• Disposable gloves, powderless • Biohazard waste container
• Refrigerator and Freezer • Tube racks
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g)


7. Warnings and Precaution


• Carefully read this instruction before starting the procedure.
• For in vitro diagnostic use only.
• This assay needs to be carried out by skilled personnel.
• Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood.
• This assay needs to be run according to Good Laboratory Practice.
• Do not use the kit after its expiration date.
• Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test.
• Once the reagents have been thawed, vortex and centrifuge briefly the tubes before use.
• Quickly prepare the reaction mix on ice or in the cooling block.
• Set up two separate working areas: 1) Isolation of the RNA/ DNA and 2) Amplification/ detection of amplification products.
• Pipets, vials and other working materials should not circulate among working units.
• Use always sterile pipette tips with filters.
• Wear separate coats and gloves in each area.


8. Sample Collection, Storage and transportation


• Collect samples in sterile tubes;
• Specimens can be extracted immediately or frozen at -20°C to -80°C.
• Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents


9. Procedure


9.1 DNA-Extraction
DNA extraction buffer is supplied in the kit. Please thaw the buffer thoroughly and
spin down briefly in the centrifuge before use.
9.1.1 Blood sample
1) Take 2ml anticoagulation, and transfer the plasma layer and buffy-coat layer to another tube after
it is natural stratified.


2) Add 100μl DNA extraction buffer into the tube, and close the tube then resuspend the pellet with
vortex vigorously. Spin down briefly in a table centrifuge.

3) Incubate the tube for 10 minutes at 100°C.


4) Centrifuge the tube at 13000rpm for 5 minutes. The supernatant contains DNA extracted and can
be used for PCR template.


9.1.2 Stool or food sample
1) Take about 50mg stool or 1000mg food samples to a tube; add 1.0ml normal saline then vortex
vigorously. Centrifuge the tube at 13000rpm for 2 minutes, carefully remove and discard supernatant
from the tube without disturbing the pellet.
2) Add 100μl DNA extraction buffer, close the tube then resuspend the pellet with vortex
vigorously. Spin down briefly in a table centrifuge.
3) Incubate the tube for 10 minutes at 100°C.
4) Centrifuge the tube at 13000rpm for 5 minutes. The supernatant contains the DNA extracted and
can be used for PCR template.


9.1.3 Water sample
1) Take 3ml sample to a tube, Centrifuge the tube at 13000rpm for 2 minutes, carefully remove and
discard supernatant from the tube without disturbing the pellet.
2) Add 100μl DNA extraction buffer, close the tube then resuspend the pellet with vortex vigorously.
Spin down briefly in a table centrifuge.
3) Incubate the tube for 10 minutes at 100°C.
4) Centrifuge the tube at 13000rpm for 5 minutes. The supernatant contains the DNA extracted and
can be used for PCR template.
Attention:
A. During the incubation, make sure the tube is not open,as the vapor will
volatilize into the air and may cause contamination if the sample is positive.
B. The extraction sample should be used in 3 hours or store at -20°C for one month.
C. Different DNA extraction kits are available. You may use your own extraction systems or the
commercial kit based on the yield. For the DNA extraction, please comply with the
manufacturer’s instructions.


9.2 Internal Control
It is necessary to add internal control (IC) in the reaction mix. Internal Control (IC) allows the user to
determine and control the possibility of PCR inhibition.
Add the internal control (IC) 1μl/rxn and the result will be got in the HEX/VIC/JOE channel.


9.3 Quantitation
The kit can be used for quantitative or qualitative real-time PCR. A positive control defined as
1×107copies/ml is supplied in the kit.
For performance of quantitative real-time PCR, Standard dilutions must prepare first as
follows. Molecular Grade Water is used for dilution.
The step of dilution is not needed for performance of qualitative real-time PCR.
Take positive control (1×107copies/ml) as the starting high standard in the first tube. Respectively
pipette 36ul of Molecular Grade Water into next three tubes. Do three dilutions as the following
figures:

 

To generate a standard curve on the real-time system, all four dilution standards should be used and defined as standard with specification of the corresponding concentrations.
Attention:
A. Mix thoroughly before next transfer.
B. The positive control (1×107copies/ml) contains high concentration of the target DNA. Therefore, be careful during the dilution in order to avoid contamination.


9.4 PCR Protocol
The Master Mix volume for each reaction should be pipetted as follows:

 

※PCR system without HEX/VIC/JOE channel may be treated with 1μl Molecular Grade Water instead of 1μl IC.
1) The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples, which includes the number of the controls,standards and sample prepared. Molecular Grade Water is
used as the negative control. For reasons of unprecise pipetting, always add an extra virtual sample. Mix the master mix completely then spin down briefly in a centrifuge.
2) Pipet 36μl (22.5μl for SmartCycer II) Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate/tube. Then separately add 4μl (2.5μl for SmartCycer II) DNA sample, positive and negative controls to different reaction plate/tubes. Immediately close the plate/tubes to avoid contamination.
3) Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes.
4) Perform the following protocol in the instrument:

5) If you use ABI Prism® system, please choose “none” as passive reference and quencher.


10. Threshold setting: just above the maximum level of molecular grade water.
11.Calabration for quantitative detection: Input each concentration of standard controls at the end of run, and a standard curve will be automatically formed.
12.Quality control: Negative control, positive control, internal control and QS curve must be performed correctly, otherwise the sample results is invalid.

 

13. Data Analysis and Interpretation. The following results are possible

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