Light Cycler System

for Dengue Detection

Dengue is a single-stranded RNA virus which belongs to the family of Flavi viruses.
Aedes mosqutoes are responsible for transmission to humans and thus the disease
affects mostly urban regions with tropical or subtropical climate. About 80% of all
Dengue infections are asymptomatic while most others have only fever, headaches and
skin rush and the disease may take a few weeks. However, a second infection with a
different type can be fatal, making the Dengue virus dangerous.
Real-Time PCR detection and typing of Dengue virus usually addresses the partially
conserved 3-UTR region of the genome. Due to the typical RNA-viral sequence
variations it is difficult to identify one particular probe to be strictly related to one type
or which could be used for a melting curve based analysis. In contract array detection
provides the possibility to use different probes at the same time.
The LightMix Kit Dengue 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). The resulting PCR product can be applied directly on a Chipron DNA-Array to
determine the type of Dengue virus.
This LightMix Kit is tested on the LightCycler 2.0 Instrument with Roche Diagnostics
Lightcycler FastStart DNA Master HybProbe.

LightMix Kit Dengue Virus

Simple, speedy, secure

 Detection of Dengue Virus (type 1-4) cDNA using the Roche Diagnostics LightCycler 2.0 Instruments
 A 164 to 187 bp (depending on the virus type) fragment of Dengue Virus 3-UTR region cDNA copy is
amplified with specific primers.
 The resulting PCR fragment is analyzed with hybridization rpobes labeled with LightCycler Red 640
(detected in channel 640 (F2))
 The PCR reaction is monitored by an additional PCR product of 349 bp from the internal control (IC).
This control does not interfere with Dengue Virus specific reactions. The hybridization probes are
labeled with LC 690 which is recorded in channel 705 (F3)
 The IC is supplied separately to allow running the assay in the presence or absence of the IC
 The use of color compensation file: LightCycler Multicolor Demo Set or Light Cycler Color
Compensation Set is prerequisite to run the duplex reaction

Reagents Information

Biological Sample: Serum

Biological sample extraction kit
High Pure Viral Nucleic Acid Kit (Cat. No. 11 858 874 001)
Reverse Transcriptase
Transcriptor First Strand cDNA Synthesis Kit (Cat. No. 04 379 012 001)
Thermal Cycler: Light Cycler type 2.0
Amplification Specific Reagents and PCR protocols
LightMix Kit Dengue Virus (Cat. No. 40-0439-16)
LightCycler FastStart DNA Master HybProbe (Cat. No. 03 003 248 001)
LightCycler Multicolor Demo Set, or (Cat. No. 03 624 854 001)
LightCycler Color Compensation Set (Cat. No. 12 158 850 001)
Disposables: LightCycler Capillaries (20 l) (Cat. No. 04 929 292 001)

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 Qualitative Dengue Detection WorkFlow
Serum Manual sample Reverse Transcription cDNA Real Time PCR for
preparation Dengue Detection

HighPure Trasncriptor First

Viral Nucleic Strand cDNA
Acid Kit Synthesis Kit

PCR Workflow

Add DNA Template

or standard
- Water, PCR Grade
- Mg2+ sol. 25 mM
- Reagent Mix
(LightMix Dengue
Virus) Reaction Capillary
- IC Mix on LightCycler 2.0
- Roche Master
and analysis

Results

PT Roche Indonesia Tel. 21 30413000

Diagnostics Division Fax. 21 51400090
Artha Graha Lt.21-SCBD Lot 25 Email: lianita.salim@roche.com
Jl. Jend.Sudirman Kav.52-53 ratna_agung_aman.taufani@roche.com
Jakarta 12190

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