UNIVERSAL format

OXVIKP-U OXVIKP-G v.2

1 Intended use
Component
RealCycler OXVIKP-U / OXVIKP-G is an in vitro diagnostic kit of
reagents which allows real-time PCR qualitative detection of
carbapenemases blaOXA gene, metallobetalactamases blaVIM gene
and carbapenemases blaKPC genes simultaneously in clinical
samples.
The system includes an internal control CHIC (Competitive
Heterologous Internal Control) to prevent false negatives due to
reaction inhibition.

Vials

Volume

OXVIKP-U

OXVIKP-G

AmpliMix

430 L

Positive Control

60 L

Number of determinations: RealCycler OXVIKP-U allows to

perform 30 tests (24 for SmartCycler). RealCycler OXVIKP-G allows
to perform 60 tests (48 for SmartCycler).

2 Principle of the test

5 Stability and storage

The polymerase chain reaction (PCR) is based on the amplification

of a specific region of the DNA/RNA by using complementary primers
to the target sequence. Real-time PCR uses marked probes with
fluorophores that emit fluorescence in the case of amplification. The
cycle of the PCR protocol in which appears significant fluorescence
is proportional to the DNA/RNA quantity present in the sample. This
value is called Cycle Threshold (Ct) or Cycle Quantification (Cq).

All components of the RealCycler OXVIKP-U / OXVIKP-G kit should

be stored at -20 C. All reagents of the kit are stable at -20 C until
expiration date that is included in the external label.

6 Additional materials and equipment

required and not supplied

The amplification of blaOXA gene is detected in the corresponding

channel of FAM fluorophore, CHIC is detected in the corresponding
channel of Alx532 or HEX fluorophore, blaVIM gene is detected in
the corresponding channel of TxR fluorophore and blaKPC genes is
detected in the corresponding channel of Alx647 or Cy5 fluorophore.

- Real-time PCR instrument.

- DNA purification system.
- Disposable gloves.
- Calibrated pipettes.
- Pipette tips with filter.
- Freezer (-20 C).
- Negative control (DNA from negative samples, water or elution
buffer).

3 Technical specifications
Sensitivity
blaOXA-48: 100 copies/L.
blaVIM: 100 copies/L.
blaKPC: 1000 copies/L.

7 Warnings and Precautions

The analytical sensitivity has been determined by limit dilution. This

sensitivity has been showed in repeated assays with reproducibility
over 95%.

- All components of the kit must be kept cold while you are working.
- Load tubes in the SmartCycler immediately after adding DNA.
- Do not expose tubes with AmpliMix to light for a long time.
- Repeated freezing and thawing of the reagents can decrease the
sensitivity of the kit.
- Use disposable gloves.
- Use adequate and calibrated pipettes and pipette tips with filter.
- The tests must be carried out by qualified personnel and following
good laboratory practices.
- It is recommended to carry out both positive control and negative
control whenever an analysis is realized.
- Do not use the kit after the expiry date.
- The presence of polymorphisms in the binding sequences of probes
or primers to pathogen DNA/RNA can lead to erroneous results in a
sample. If discordances appear between results and clinical
observations it is recommended to check the results obtained using
alternative methods.
- The results obtained with this diagnostic kit should be used and
interpreted within the context of the clinical history of the patient.
Clinical decisions should not be made using solely the results of this
kit.
- For in vitro diagnostic use.
- Cepheid and SmartCycler are trademarks of Cepheid
Corporation.
- CFX96 is a trademark of Bio-Rad.
- QIAamp is a trademark of QIAGEN Group.
- Maxwell is a trademark of Promega Corporation.
- NucliSENS easyMAG is a trademark of bioMrieux.

Specificity
blaOXA: blaOXA genes (blaOXA-48, blaOXA-162, blaOXA-163,
blaOXA-244, blaOXA-245, blaOXA-247 and blaOXA-370 genes and
eventually, other sequences of blaOXA group).
blaVIM: blaVIM gene.
blaKPC: blaKPC genes.
Specificity validation has been performed according to experimental
assays and BLAST analysis (www.ncbi.nlm.nih.gov/blast).

4 Contents
RealCycler OXVIKP-U / OXVIKP-G includes the AmpliMix and an
OXVIKP DNA Positive Control, which contains a mixture of
blaOXA-48, blaVIM and blaKPC DNA.
All reagents are ready to use without adding or rebuilding any
component.

RealCycler OXVIKP-U OXVIKP-G v.2

- Prepare the necessary amplification tubes for samples and controls.

- Pipette 14 L of AmpliMix into each amplification tube.
- Add 6 L of DNA sample or control to each reaction tube.
- Load tubes in the Real-time PCR instrument.
- File > New > Plate > Unknown > Select the fluorophores (FAM,
HEX, TxR and Cy5) > OK > Save.
- Select OXVIKP protocol.
- Start Run > Close lid > Start Run.

8 Clinical samples
- Collect samples in sterile tubes.
- Storage and transportation frozen at -20 C until use.
- The kit is compatible with any sample in which the pathogen is
present and high quality DNA can be extracted from.
Validated clinical samples:
blaOXA-48: culture and bronchoalveolar lavage.
blaVIM: culture and bronchoalveolar lavage.
blaKPC: culture and bronchoalveolar lavage.

c.2) For instruments requiring a reaction volume of 25 L (e.g.:

SmartCycler (Cepheid)).
- Thaw AmpliMix and Positive Control. Use as negative control
DNA from negative samples, water or DNA elution buffer (not
supplied).
- Prepare the necessary amplification tubes for samples and controls.
- Pipette 17,5 L of AmpliMix into each amplification tube.
- Add 7,5 L of DNA sample or control to each reaction tube.
- Load tubes in the Real-time PCR instrument.
- Select the OXVIKP protocol.
- Start Run.

9 Procedure
a) Nucleic acids purification
DNA should be purificated from the clinical sample using an
appropriate procedure. There are many nucleic acids purification
systems available in the market. Please carry out the purification
according to the manufacturers instructions and using the
recommended volume.

d) Adjust the fluorescence threshold

Validated purification systems:

d.1) CFX96 de Bio-Rad

QIAamp DNA Blood Mini kit (references 51104, 51106). QIAGEN.

Maxwell 16 Cell LEV DNA Purification kit (reference AS1140).
Promega Corporation.

Adjust the fluorescence threshold for each channel:

- FAM: Settings > Baseline Threshold > User defined > 400 > OK.
- HEX: Settings > Baseline Threshold > User defined > 250 > OK.
- TxR: Settings > Baseline Threshold > User defined > 250 > OK.
- Cy5: Settings > Baseline Threshold > User defined > 250 > OK.

Compatible purification systems:

BioRobot EZ1. QIAGEN.
QIAcube. QIAGEN.
NucliSENS easyMAG. bioMrieux.

d.2) SmartCycler de Cepheid

b) Protocol

- Analysis settings > Manual Thresh Fluor Units > 30.0.

Program the OXVIKP amplification protocol according to the

following specifications:

e) Control results interpretation

- Valid control results

Time

Temperature

Cycles

Fluorescence

15:00

95 C

OFF

0:15

95 C

0:30

60 C

0:30

72 C

Channels

45

Ch1

Ch2

Ch3

Ch4

FAM

Alx532
CHIC

TxR

Alx647

POS

POS

Indifferent

POS

POS

VALID

NEG

NEG

POS

NEG

NEG

VALID

Control

OFF
ON
OFF

Interpr.

Fluorophores selection:
- FAM: detects blaOXA gene.
- Alx532/HEX: detects CHIC.
- TxR: detects blaVIM gene.
- Alx647/Cy5: detects blaKPC genes.

- Invalid control results

In case of obtaining in any channel of the Positive Control
(excepting for CHIC) a negative result, it is considered as an invalid
control. The results obtained in the samples included in the working
series must be discarded (not assessable).

c) PCR reaction set-up

c.1) For instruments requiring a reaction volume of 20 L (e.g.:
CFX96 (Bio-Rad)).

In case of obtaining in any channel of the negative control (excepting

for CHIC) a value of Ct > 0, the results obtained in the samples
included in the working series must be discarded (not assessable).

- Thaw AmpliMix and Positive Control. Use as negative control

DNA from negative samples, water or DNA elution buffer (not
supplied).

RealCycler OXVIKP-U OXVIKP-G v.2

f) Sample results interpretation

Interpret the result obtained in each sample by the combination of
signals indicated in the following table:
Channels
Ch1

Ch2

Ch3

Ch4

FAM

Alx532
CHIC

TxR

Alx647

POS

Indifferent

NEG

NEG

POSITIVE
blaOXA

NEG

Indifferent

POS

NEG

POSITIVE
blaVIM

Interpretation

NEG

Indifferent

NEG

POS

POSITIVE
blaKPC

POS

Indifferent

POS

NEG

POSITIVE
blaOXA and blaVIM

POS

Indifferent

NEG

POS

POSITIVE
blaOXA and blaKPC

NEG

Indifferent

POS

POS

POSITIVE
blaVIM and blaKPC

POS

Indifferent

POS

POS

POSITIVE
blaOXA, blaVIM
and blaKPC

NEG

POS

NEG

NEG

NOT DETECTED

NEG

NEG

NEG

NEG

NOT ASSESSABLE

Figure 2 (Channel HEX: CHIC): Result obtained from the

amplification of a negative control and a positive control. Negative
control: amplification signal is observed. Positive control:
amplification signal is observed.

Figure 3 (Channel TxR: blaVIM): Result obtained from the

amplification of a negative control and a positive control. Negative
control: not detected. Positive control: amplification signal is
observed.

g) Example result
g.1) CFX96 (Bio-Rad)

Figure 4 (Channel Cy5: blaKPC): Result obtained from the

amplification of a negative control and a positive control. Negative
control: not detected. Positive control: amplification signal is
observed.

Figure 1 (Channel FAM: blaOXA-48): Result obtained from the

amplification of a negative control and a positive control. Negative
control: not detected. Positive control: amplification signal is
observed.

RealCycler OXVIKP-U OXVIKP-G v.2

g.2) SmartCycler (Cepheid)

Figure 4 (Channel Alx647: blaKPC): Result obtained from the

amplification of a negative control, a blaOXA-48 positive control, a
blaVIM positive control and a blaKPC positive control. Negative
control (A1 blue): not detected. blaOXA-48 positive control (A2 grey):
not detected. blaVIM positive control (A3 orange): not detected.
blaKPC positive control (A4 green): Ct=26,38.

Figure 1 (Channel FAM: blaOXA-48): Result obtained from the

amplification of a negative control, a blaOXA-48 positive control, a
blaVIM positive control and a blaKPC positive control. Negative
control (A1 blue): not detected. blaOXA-48 positive control (A2 grey):
Ct=31,07. blaVIM positive control (A3 orange): not detected. blaKPC
positive control (A4 green): not detected.

10 Results interpretation using the software

Visor RealCycler
a) Export results
a.1) From CFX96 (Bio-Rad) instrument
- Export > open the exportation window > Export all data sheets
Excel 2007 (*.xlsx).

a.2) From SmartCycler Dx instrument

- Export > open the window Export data > Export (leave the default
option Export Optics Data, Export Results Tables and Export Assay
Settings).

Figure 2 (Channel Alx532: CHIC): Result obtained from the

amplification of a negative control, a blaOXA-48 positive control, a
blaVIM positive control and a blaKPC positive control. Negative
control (A1 blue): Ct=30,74. blaOXA-48 positive control (A2 grey):
Ct=30,78. blaVIM positive control (A3 orange): Ct=31,35. blaKPC
positive control (A4 green): Ct=30,94.

- Save (optionally change the file name).

Figure 3 (Channel TxR: blaVIM): Result obtained from the

amplification of a negative control, a blaOXA-48 positive control, a
blaVIM positive control and a blaKPC positive control. Negative
control (A1 blue): not detected. blaOXA-48 positive control (A2 grey):
not detected. blaVIM positive control (A3 orange): Ct=30,92. blaKPC
positive control (A4 green): not detected.

RealCycler OXVIKP-U OXVIKP-G v.2

a.3) From SmartCycler R+D instrument

c.2) From SmartCycler instrument (Dx and R+D)

- Export > open the window Export data > Export (leave the default
option Export Optics Data and Export Results Table and Analysis
Settings).

- Data > Import Run Data > select CSV file.

- Select Use Preanalytical Data from SmartCycler (this option will
delete all the samples data from the Visor) or choose Use
Preanalytical Data from Visor (this option will not import the sample
identification from SmartCycler).
- Import data.
- On the Sample column optionally identify the controls and the
samples of the series.

d) Generate report
- File > Reports > Select the samples to print.

- Save (optionally change the file name).

e) Results interpretation
- Pathogen detection
The software Visor RealCycler will indicate on which channel a signal
is detected or not. The signal obtained in the controls and the
samples can be valid (VAL) or invalid (INV). In the case of CHIC the
signal can be VAL (negative samples for all the pathogens) or VAL*
(positive samples for some of the pathogens). If a signal is obtained,
it will be considered positive for this pathogen as long as the sample
status is valid.

b) Visor RealCycler set-up

- Open the Visor RealCycler and enter username and password.
- Select the used instrument > Options > Setup.
- Data > Lot input > select AmpliMix OXVIKP-U / OXVIKP-G.
- Enter the lot value specified in the external label of the kit
RealCycler OXVIKP-U / OXVIKP-G.
- AmpliMix column > deploy with the right button mouse and select
AmpliMix OXVIKP-U / OXVIKP-G. The name of the detected
pathogens in each channel will appear automatically.
- Lot column > select AmpliMix lot.

- Sample status
STATUS

Interpretation

CHIC

Positive
Control

Negative
Control

VALID

POS

POS

Ct=0

WARNING

POS

Missing

Ct=0

WARNING

POS

POS

Missing

WARNING

POS

Missing

Missing

INVALID

NEG

Valid

Valid

INVALID

POS

Invalid

Valid

INVALID

POS

Valid

Invalid

INVALID

POS

Invalid

Invalid

c) Import results
c.1) From CFX96 (Bio-Rad) instrument
- Data > Import Run Data > select *.xlsx Amplification data
(fluorescence) and Sample Data files.
- Select Use Preanalytical Data from CFX96 (this option will delete
all the samples data entered on the Visor) or choose Use
Preanalytical Data from Visor (this option will not import the sample
identification from CFX96).
- Import data.
- On the Sample column optionally identify the controls and the
samples of the series.

RealCycler OXVIKP-U OXVIKP-G v.2

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If the curves that surpass the threshold are abnormal (not sigmoid) or
lineals, they are not suitable for the analysis and the obtained results
must be discarded. Visor RealCycler performs an analysis to detect
them, in which case it will classify them as invalid. However, the user
should value the curves individually and decide if they are suitable
for the analysis or not, and discarded those abnormal (not sigmoid)
or lineals.

11 Quality control
Every lot of RealCycler OXVIKP-U / OXVIKP-G kit has been tested
according to the specifications of the real-time PCR using the
SmartCycler instrument (Cepheid).

12 Observations
a) Fluorophores compatibility table
Used
fluorophore

Emmision
(nm)

FAM

519

HEX

556

JOE, VIC, CAL Fluor

Orange 560, Alexa 532

Texas Red

610

ROX, LC Red 610, CAL

Fluor Red 610

ATTO 647N

669

Cy5, Alexa 647, LC Red

670, Quasar 670, Oyster
645

Alternative fluorophores

b) Lineal signals

In some cases lineal signals on the CFX96 (Bio-Rad) instrument

appears. The lineal curves are results not valuables that should be
discarded.

Date of publication: September, 2015.

Progenie Molecular
Edificio Progenie. Valle de la Ballestera 56. 46015. Spain
T: +34 902 91 05 05 F: +34 902 91 05 06
www.progenie-molecular.com

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RealCycler OXVIKP-U OXVIKP-G v.2

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