AffiPCR® Dengue Real Time PCR

Target Disease Type: Dangerous Infections

Specific Application: Dengue Virus

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Dengue RT-PCR V63-S-50FRT DataSheet

INTRODUCTION

Dengue is one of the most important arthropod-borne viral diseases with large global burden. The disease is caused by dengue virus (DENV), a member of Flaviviridae family. DENV is transmitted to human by Aedes mosquitoes as vector. Dengue clinical manifestations vary from asymptomatic or mild flu-like syndrome known as classic Dengue Fever (DF) to more severe form known as Dengue Hemorrhagic Fever (DHF) and the potentially fatal Dengue Shock Syndrome (DSS) .

Similar to other RNA viruses, DENV possess diverse genetic characteristics as shown by the presence of various genotypes within serotypes. Estimates of the global incidence of dengue infections per year have ranged between 50 million and 200 million; however, recent estimates using cartographic approaches suggest this number is closer to almost 400 million.

The expansion of dengue is expected to increase due to factors such as the modern dynamics of climate change, globalization, travel, trade, socioeconomics, settlement and also viral evolution. No vaccine or specific antiviral therapy currently exists to address the growing threat of dengue. Acute infection with dengue virus is confirmed when the virus is isolated from serum or autopsy

tissue specimens, or the specific dengue virus genome is identified by reverse transcription-polymerase chain reaction (RT–PCR) from serum or plasma, cerebrospinal fluid, or autopsy tissue specimens during an acute febrile illness. Methods such as one-step, real time RT–PCR are now widely used to detect dengue viral genes in acute-phase serum samples, approximately the first 5 days of symptoms. This detection coincides with the viremia and the febrile phase of illness onset.

INTENDED USE

DENGUE Real-TM PCR kit is an in vitro nucleic acid amplification test for qualitative detection of Dengue virus RNA in the biological material (blood plasma, histological (autopsy) material (brain and visceral tissues) ) using real-time hybridization-fluorescence detection of amplified products. The PCR kit is used for studying the biological material, taken from the persons suspected of dengue fever without distinction of form and presence of manifestation.

PRINCIPLE OF ASSAY

Dengue virus detection by the polymerase chain reaction (PCR) is based on the RNA extraction form the test material with the Internal Control (IC) and simultaneous carrying out of reverse transcription reaction and amplification of Dengue virus cDNA fragments and Internal Control (IC) cDNA with hybridization-fluorescence detection. The Internal Control must be used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition.

The RNA obtained at the RNA extraction stage is reverse transcribed by using the M-MLV Revertase, and amplified in to cDNA fragments by using specific primers and the enzyme Hot Start TaqF polymerase. In the real-time PCR, the amplified product is detected with the use of fluorescent dyes. These dyes are linked to oligonucleotide probes, which bind specifically to the amplified product during thermocycling. The real-time monitoring of fluorescence intensities during

the real-time PCR allows the detection of accumulating product without re-opening the reaction tubes after the PCR run. DENGUE Real-TM PCR kit uses “hot-start”, which greatly reduces the frequency of nonspecifically primed reactions. “Hot-start” is guaranteed by the separation of nucleotides and TaqF polymerase by chemically modified polymerase (TaqF) . The chemically modified polymerase (TaqF) is activated by heating at 95 °C for 15 min.

At the RT-PCR stage, 2 reactions are carried out simultaneously in one tube – amplification of the Dengue virus cDNA and the Internal Control (IC) cDNA sequences. The amplification results of Dengue virus cDNA and Internal Control (IC) cDNA are detected in 2 different fluorescence detection channels:

• the DENGUE virus cDNA is detected in the JOE/HEX/Yellow channel;
• the IC cDNA is detected in the FAM/Green channel