For life science research use only. Not for use in diagnostic procedures. For in vitro use only.

LightMix® Kit Chikungunya -Virus

Cat.-No. 40-0322-16

Kit with reagents for the detection of Chikungunya-Virus cDNA (CHIKV) using the Roche Diagnostics
LightCycler® 1.x / 2.0 / 480 II / Cobas® Z480 (open channel) Instruments.
Lyophilized mix of primers and probes for a total of 96 reactions with a final volume of 20 µl each.
Store protected from light at room temperature (18-25°C), do N OT freeze!

Instructions for use with the LightCycler® 1.x / 2.0 Instruments see pages 4-5
Instructions for use with the LightCycler® 480 II / Cobas® Z480 Instrument see pages 6-7

1. Introduction
Chikungunya (in the Makonde language "that which bends up") virus (CHIKV) is an insect-borne
positive ssRNA virus of the genus Alphavirus transmitted to humans by Aedes mosquitoes.
Chikungunya fever is diagnosed based on symptoms, physical findings (e.g., joint swelling), laboratory
testing, and the possibility of exposure to infected mosquitoes. There is no specific treatment for
chikungunya fever and care is based on symptoms. Chikungunya infection is not usually fatal 1.The
incubation period is in the range of 2-12 days.
Chikungunya-Virus was first isolated from the blood of a febrile patient in Tanzania in 1953, and has
since been cited as the cause of numerous human epidemics in many areas of Africa and Asia and
most recently in limited areas of Europe.
Diagnosis is carried out by RT-PCR, using several Chikungunya-specific genes from whole blood 2.
1
Chikungunya Fact Sheet, CDC, online, 2008
2
“Laboratory Diagnosis of Chikungunya Fevers", WHO-online, retrieved on 2008-07-11

The LightMix® Kit Chikungunya-Virus provides a fast, easy and accurate system to identify this target in
a nucleic acid extract. A control amplification reaction acts as internal control (IC).
This LightMix® Kit is tested on the LightCycler® 1.x / 2.0 / 480 II Instruments with Roche Diagnostics
‘LightCycler® FastStart DNA Master HybProbe’.

2. Description
A 181 bp fragment of the Chikungunya-Virus E1 gene is amplified with specific primers. The resulting
PCR fragment is analyzed with hybridization probes labeled with LightCycler® Red 640 (detected in
channel 640).
The PCR reaction is monitored by an additional PCR fragment of 278 bp, formed from the internal
control. This control does not interfere with the Chikungunya-Virus specific reactions. The amplification
will usually fail in the presence of higher concentrated Chikungunya-Virus DNA samples (1,000 copies
or higher) while displaying an amplification signal in negative and low-concentrated samples. The
hybridization probes are labeled with LightCycler® Red 690 (recorded in channel 705). The IC is
supplied separately to allow running the assay in the presence or absence of the IC.
The use of a color compensation file generated with the ColorCompensation kit HybProbe 40-0318 is
a prerequisite to run the duplex reaction.
The supplied standard row allows to determine the linear range of the reaction and to estimate the
quantity of the target sequence in unknown samples.

For use in LightCycler® 1.x Instruments with software version 3.5.3 read channel F2 instead of channel
640, channel F3 instead of channel 705 and channel F1 instead of channel 530 for detection. We
recommend upgrading LightCycler® 1.x Instruments to software version 4.1.

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 1/8

3. Set Contents
6 Vials with blue cap containing lyophilized primers and probes for 16 PCR reactions CHIKV.
6 Vials with white cap containing the internal control (IC)
1 Standard row with 6 lyophilized plasmid standards from 101 to 106 target equivalents per rxn
1 Sealing foil for the standard row

4. Additional Reagents and items required

Roche Diagnostics

LightMix® Kit ColorCompensation HybProbe 40-0318-00 Cat.-No. 05 997 704 001

LightCycler® FastStart DNA Master HybProbe Cat.-No. 03 003 248 001
High Pure PCR Template Preparation Kit Cat.-No. 11 796 828 001
High Pure RNA Isolation Kit Cat.-No. 11 828 665 001
High Pure Viral Nucleic Acid Kit Cat.-No. 11 858 874 001
Transcriptor First Strand cDNA Synthesis Kit Cat.-No. 04 379 012 001
LightCycler® Capillaries (20 µl) (LightCycler® 1.x / 2.0 Instruments) Cat.-No. 04 929 292 001
® ®
LightCycler 480 Multiwell Plate 384, white (LightCycler 480 Instrument) Cat.-No. 04 729 749 001
or LightCycler® 480 Multiwell Plate 96, white (LightCycler® 480 Instrument) Cat.-No. 04 729 692 001

5. Product Characteristics
PCR results are obtained within 50 minutes (50 cycles and melting curve) with the LightCycler® 1.x / 2.0
Instruments and within 80 minutes (50 cycles and melting curve) with the LightCycler® 480 II Instrument.
Sensitivity
These reagents detect 10 copies of Chikungunya-Virus cDNA using the Roche ‘LightCycler® FastStart
DNA Master HybProbe’ with the LightCycler® 1.x / 2.0 / 480 II Instruments (in an exemplary system,
using cloned targets as reference).
Measuring range
The linear measuring range of the assay is 102 to 106 copies of Chikungunya-Virus cDNA using the
Roche ‘LightCycler® FastStart DNA Master HybProbe’ with the LightCycler® 1.x / 2.0 / 480 II
Instruments.
Storage and Stability
• Lyophilized reagents are stable for at least 6 months after shipment when stored protected from light
at room temperature (18-25°C). See expiry date o n the product label.
• Do not freeze lyophilized reagents.
• Dissolved reagents are stable for at least 10 days when stored protected from light and refrigerated (4°C).

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 2/8

6. Experimental Protocol
The following procedure was developed for use with the LightCycler® 1.x / 2.0 / 480 II Instruments.
Start programming before preparing the solutions. See the Instrument operator’s manual for details.
Sample material: Use aqueous nucleic acid preparations (e.g. Roche Diagnostics ‘High Pure RNA
Isolation Kit’ combined with Roche Diagnostics ‘Transcriptor First Strand cDNA Synthesis Kit’).
Negative control: Always run at least one no-template control (NTC) - replace the template DNA with water.
Positive control: Run a positive control - replace the template DNA with the provided control DNA.
For quantification always use a fresh solution prepared from the provided standard row (single use only).

6.1. Preparation of parameter-specific reagents and reagents for the IC (16 reactions):
One reagent vial with a blue cap contains primers and probes to run 16 reactions for CHIKV.
One reagent vial with a white cap contains primers, probes and DNA to run 16 reactions for the IC.
Add 66 µl PCR-grade water to each reagent vial, mix the solution (vortex) and spin down.
►Use 4 µl reagent for a 20 µl PCR reaction.
This solution is stable at least five days when stored refrigerated at 4°C. Avoid prolonged exposure t o light.

6.2. Preparation of the standard row

The target DNA is provided in 6 different quantities to yield from 101 to106
target molecules in 5 µl once resolved. Start with the lowest concentrated
standard (first tube from the extended lip). Use the pipette tip to punch a hole
through the sealing foil. Add 40 µl PCR-grade water to each vial of the row. Mix the target DNA by
pipetting the solution up and down 10 times.

►Use 5 µl standard for a 20 µl PCR reaction.

This standard solution is not long-term stable and will lose sensitivity under prolonged storage. Use only fresh prepared
solutions as quantification references. Older solutions can be used as a qualitative standard (positive control. After adding the
target DNA to the LightCycler reaction mix use the provided sealing foil to close the vials in order to avoid changes in the
concentration due to evaporation.
Please note that opening these vials may cause contaminations of the work-space (aerosol).

6.3. Preparation of the LightCycler® reaction mix

In a cooled reaction tube, prepare the reaction mix by multiplying each volume for a single reaction by
the number of reactions to be cycled plus one additional reaction.

For use with the Roche FastStart Master

Single
Component
reaction
2.6 µl water, PCR-grade (colorless cap, provided with the Roche Master kit)
2+
2.4 µl Mg solution 25 mM (blue cap, provided with the Roche FastStart kit)
4.0 µl reagent mix (parameter specific reagents containing primers and probes, see 6.1.)
4.0 µl IC mix (IC reagents containing primers, probes and DNA, see 6.1.)
2.0 µl Roche Master (red cap, for preparation see Roche manual)

15.0 µl Volume of reaction mix

Table 1

To include the internal control add 4 µl of the IC reagent per reaction to the reaction mix.
To run the assay without the internal control substitute the 4 µl of IC with 4 µl PCR-grade water.
Mix gently, spin down and transfer 15 µl each of the reaction mix to a LightCycler® capillary
(LightCycler® 1.x / 2.0 Instrument) or to a multiwell plate (LightCycler® 480 II Instrument).
Add 5 µl of sample or standard to each capillary or well for a final reaction volume of 20 µl.
Start run.

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 3/8

7. LightCycler® 1.x / 2.0 Instruments
7.1. Programming
The protocol consists of four program steps
• 1: Denaturation: sample denaturation and enzyme activation
• 2: Cycling: PCR-amplification of the target DNA
• 3: Melting: melting curve analysis for identification of the PCR product derived from the target DNA
• 4: Cooling: cooling the instrument

Program Step: Denaturation Cycling Melting Cooling

Parameter
Analysis Mode None Quantification mode Melting Curves mode None
Cycles 1 50 1 1
Target [°C] 95 95 62 72 95 40 85 40
Hold [hh:mm:ss] 00:10:00 00:00:05 00:00:05 00:00:15 00:00:20 00:00:20 00:00:00 00:00:30
Ramp Rate [°C/s] 20 20 20 20 20 20 0.2 20
Sec Target [°C] - - 55 - - - - -
Step Size [°C] - - 0.5 - - - - -
Step Delay (Cycles) - - 1 - - - - -
Acquisition Mode None None Single None None None Cont None
(Melting not relevant for detection) Table 2

7.2. Data Analysis

For use in LightCycler® 1.x Instruments with software version 3.5.3 read channel F2 instead of channel
640, channel F3 instead of channel 705 and channel F1 instead of channel 530 for detection. We
recommend upgrading LightCycler® 1.x instruments to software version 4.1.
Switch the color compensation mode on. If this mode is not enabled run the color compensation
program. Follow the instructions in the manual of the TIB ColorCompensation HybProbe. Perform data
analysis, as described in the LightCycler® Instrument operator’s manual.
Perform data analysis, as described in the LightCycler® Instrument operator’s manual.
We recommend using the Second Derivative Maximum method (Automated (F'' max)). The cycle
number of the Crossing Point (Cp) of each sample is calculated automatically. The Fit Points method
is more-error prone due to the user's influence.
View Chikungunya-Virus data in channel 640 Quantification mode. The negative control (NTC) must
show no signal.
If the internal control (IC) is used view IC data in channel 705, Quantification mode. The negative
control and the low-concentrated Chikungunya-Virus DNA samples (10 to 1,000 copies) should show
an amplification curve for the IC with a Cp at approximately cycle 30.
The provided standard row of cloned and purified DNA with concentrations in the range from 106
copies/rxn to 101 copies/rxn of Chikungunya-Virus should have Cp values between cycles 18 and 34 .

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 4/8

7.3. Sample Data – Typical Results

®
LightCycler
2.0 Instrument
Roche Master:
Fast Start

Sample data for

Chikungunya-
Virus

*Channel 640

1E01: Cp 33-34
1E02: Cp 30-32
1E03: Cp 27-29
1E04: Cp 24-26
1E05: Cp 21-23
1E06: Cp 18-20

®
LightCycler
2.0 Instrument
Roche Master:
Fast Start

Sample data
for the IC

*Channel 705

typical
Cp 30-33

®
Fig.1. LightCycler 2.0 sample data for the Chikungunya-Virus detection system.
Upper panels: Left panel channel 640 quantification mode (Second Derivative Maximum) with amplification curves for
Chikungunya-Virus. Right panel channel 640 melting analysis for Chikungunya-Virus (not relevant for detection).
Lower panels: Left panel channel 705 quantification mode (Second Derivative Maximum) for the IC. Right panel channel 705
melting analysis for the IC (not relevant for detection).

* Note: Fluorescence levels depend on instrument settings and may vary. The characteristics of the curve (shape and trend of
fluorescence levels) should be similar to the curve in the manual. The fluorescent curves over cycles (quantification mode) must
be smooth and not zigzag shaped. The Cp values will vary from instrument to instrument by up to 2 cycles, while the distance
between two dilution steps should be relative constant (delta Cp). The Cp values described in this manual (chart text) have been
obtained using the supplied standard row; the entire set of curves can be horizontal shifted.

7.4. Interpretation of Data

Negative results mean that the virus is not detectable. The virus could be present in amounts lower
than the detection limit or could be lost during the extraction process. Relevant PCR inhibition can be
excluded by the inclusion of the control PCR (IC) which must be detectable for negative samples.

Sample 640 Sample 705 Channel 640 Channel 640 Result

CHIKV Int Control Positive Control Negative Control (warnings)
No amplification detectable amplification negative Negative (not detectable)

Cp < 37 not relevant amplification negative Positive for CHIK Virus

no amplification not detectable amplification not relevant PCR failure, repeat experiment

not relevant not relevant no amplification not relevant PCR failure, repeat experiment

not relevant not relevant not relevant positive Contamination, repeat experiment
®
Table 3. Typical analysis results (LightCycler 2.0 Instrument, Roche Master: Fast Start)

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 5/8

8. LightCycler® 480 II / Cobas® Z 480 Instruments
8.1. Programming
The protocol consists of four program steps
• 1: Denaturation: sample denaturation and enzyme activation
• 2: Cycling: PCR-amplification of the target DNA
• 3: Melting: melting curve analysis for identification of the PCR product derived from the target DNA
• 4: Cooling: cooling the instrument
Detection Format:
LightCycler® 480 II Instrument: 465-510, 498-640, 498-660
Cobas® Z480 Instrument: 465-510, 498-645, 498-700

Program Step: Denaturation Cycling Melting Cooling

Parameter
Analysis Mode None Quantification mode Melting Curves mode None
Cycles 1 50 1 1
Target [°C] 95 95 62 72 95 40 85 40
Hold [hh:mm:ss] 00:10:00 00:00:05 00:00:05 00:00:15 00:00:30 00:02:00 00:00:00 00:00:30
Ramp Rate [°C/s] 96 4.4 4.4 2.2 4.4 4.4 1.5 - 1.5
Ramp Rate [°C/s] 384 4.6 4.6 2.4 4.6 4.6 2.0 - 2.0
Sec Target [°C] - - 55 - - - - -
Step Size [°C] - - 0.5 - - - - -
Step Delay (Cycles) - - 1 - - - - -
Acquisition Mode None None Single None None None Continuous None
Acquisitions [per °C] - - - - - - 3* -
(Melting not relevant for detection) Table 4

8.2. Data Analysis

Note: Cobas® Z480 Instruments signal levels are about 50% compared to LightCycler® 480 II results.
Switch the color compensation mode on. If this mode is not enabled run the color compensation
program. Follow the instructions in the manual of the ‘LightMix® Kit Color Compensation HybProbe.
Perform data analysis, as described in the LightCycler® Instrument operator’s manual.
We recommend using the Second Derivative Maximum method (Automated (F'' max)). The cycle
number of the Crossing Point (Cp) of each sample is calculated automatically. The Fit Points method
is more-error prone due to the user's influence.
View Chikungunya-Virus data with Filter Combination 498-640 with Filter Combination. The negative
control (NTC) must show no signal.
If the internal control is used view data with Filter Combination 498-660, Quantification mode. The
negative control and the low-concentrated Chikungunya-Virus DNA samples (10 to 1,000 copies)
should show an amplification curve for the IC with a Cp at approximately cycle 30.
The provided standard row of cloned and purified DNA with concentrations in the range from 106
copies/rxn to 101 copies/rxn of Chikungunya-Virus should have Cp values between cycles 18 and 35
(Cp values calculated with Second Derivative Maximum method).

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 6/8

8.3. Sample Data – Typical Results

®
LightCycler
480 II Instrument
Roche Master:
Fast Start

Sample data for

Chikungunya-
Virus

*Filter Combin.
498-640

1E01: Cp 33-35
1E02: Cp 30-32
1E03: Cp 27-29
1E04: Cp 25-26
1E05: Cp 21-23
1E06: Cp 18-20
®
LightCycler
480 II Instrument
Roche Master:
Fast Start

Sample data
for the IC

*Filter Combin.
498-660

typical
Cp 30-33

®
Fig.1. LightCycler 480 II Sample data for the Chikungunya-Virus detection system.
Upper panels: Left panel Filter Combination 483-640 quantification mode (Second Derivative Maximum) with amplification
curves for CHIKV Right panel Filter Combination 483-640 melting analysis for CHIKV (not relevant for detection).
Lower panels: Left panel Filter Combination 498-660 quantification mode (Second Derivative Maximum) for the IC. Right panel
Filter Combination 498-660 melting analysis for the IC (not relevant for detection).

* Note: Fluorescence levels depend on instrument settings and may vary. The characteristics of the curve (shape and trend of
fluorescence levels) should be similar to the curve in the manual. The fluorescent curves over cycles (quantification mode) must
be smooth and not zigzag shaped. The Cp values will vary from instrument to instrument by up to 2 cycles, while the distance
between two dilution steps should be relative constant (delta Cp). The Cp values described in this manual (chart text) have been
obtained using the supplied standard row; the entire set of curves can be horizontal shifted.

8.4. Interpretation of Data

Negative results mean that the virus is not detectable. The virus could be present in amounts lower
than the detection limit or could be lost during the extraction process. Relevant PCR inhibition can be
excluded by the inclusion of the control PCR (IC) which must be detectable for negative samples.

Sample 640 Sample 660 Channel 640 Channel 640 Result

CHIKV Int Control Positive Control Negative Control (warnings)
No amplification detectable amplification negative Negative (not detectable)

Cp < 37 not relevant amplification negative Positive for CHIK Virus

no amplification not detectable amplification not relevant PCR failure, repeat experiment

not relevant not relevant no amplification not relevant PCR failure, repeat experiment

not relevant not relevant not relevant positive Contamination, repeat experiment
®
Table 5. Typical analysis results (LightCycler 480 II Instrument, Roche Master: Fast Start)

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 7/8

 9. Conversion Factor
The amount of virus per sample (Viral Load) is usually reported in copies / ml while PCR reports
copies per reaction. The conversion factor between both depends on sample and extraction volumes.
Extraction starts usually from less than one milliliter and PCR does not use the total extracted volume.
The viral load (VL) can be calculated using the following general formula:
VL [copies/ml] = MV x EVF x SF
where:
VL = Viral Load
MV = Measured Value [copy number per reaction]
EVF = Extraction Volume Factor [Final extraction volume / PCR sample volume]
SF = Sample Factor [1,000 µl / extracted volume of clinical sample]
For example, extracting from 200µl of clinical sample results in a correction factor of 5. Using 5 µl
extract from a total eluation volume of 100 µl results in a correction factor of x 20, resulting in a
conversion factor of 100.
VL [copies/ml] = Measured Value [copy number per reaction] X 100

10. Material Safety Data

According to OSHA 29CFR1910.1200, Commonwealth of Australia [NOHSC:1005, 1008 (1999)] and
the European Union Directives 67/548/EC and 1999/45/EC any products which not contain more than
1% of a component classified as hazardous or classified as carcinogenic do not require a Material
Safety Data Sheet (MSDS).
Product is not hazardous, not toxic, not IATA-restricted. Product is not from human, animal or plant
origin. Product contains synthetic oligonucleotide primers and probes.

11. Version History Notes in red mark events require to change procedures

V080611 First version (2008)

V100823 Last released version (2010)
V130813 Conversion Factor, MSDS and Version History included
Cut-off values (recommendation)

Roche SAP order n° 05945143001

Notice to Purchaser

A license under U.S. Patents 4,683,202, 4,683,195 and 4,965,188 or their foreign counterparts, owned by Hoffmann-La Roche
Inc. and F. Hoffmann-La Roche Ltd (“Roche”), has an up-front fee component and a running-royalty component. The purchase
price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the
product to practice the Polymerase Chain Reaction (“PCR”) and related processes described in said patents solely for the
research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use
is covered by the up-front fee component. Rights to the up-front fee component must be obtained by the end user in order to
have a complete license. These rights under the upfront fee component may be purchased from Perkin-Elmer or obtained by
purchasing an authorized thermal cycler. No right to perform or offer commercial services of any kind using PCR, including
4260159331592

without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by
implication or estoppel. Further information on purchasing licenses to practice the PCR process for research applications may
be obtained by contacting the Director of Licensing at The Perkin-Elmer Corporation, 850 Lincoln Center Drive, Foster City,
California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. The purchase of this
product does not convey any right for its use in clinical diagnostic applications. No rights for TaqMan technology under U.S.
Patents 5,210,015 and 5,487,972 are hereby conveyed.

These reagents were developed and manufactured by TIB MOLBIOL GmbH, Berlin, Germany.
®
LightCycler hybridization probes produced under license from Roche Diagnostics GmbH.

LightMix Kit Chikungunya-Virus Version 130813  2013 TIB MOLBIOL 8/8