Dengue Virus Nucleic Acid Detection Kit (Fluorescent RT-PCR) Instructions (Part 1)
【Product Name】Dengue Virus Nucleic Acid Detection Kit (Fluorescent RT-PCR)
【Packaging Specifications】50 Test / Box
【Expected Use】
This kit is used for qualitative detection of Dengue Virus nucleic acid in samples. Dengue fever is an acute infectious disease caused by dengue virus (DV), which is widespread in tropical and subtropical regions of the world and is usually transmitted by Aedes aegypti or Aedes albopictus, is a mosquito-borne viral disease. Dengue virus belongs to the Flaviviridae family of Flaviviruses. It is an RNA virus with a genome composed of single-stranded positive-stranded RNA. There are four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Recovery from infection is believed to provide lifelong immunity against that serotype. However, cross-immunity to the other serotypes after recovery is only partial, and temporary. Subsequent infections (secondary infection) by other serotypes increase the risk of developing severe dengue. Dengue has distinct epidemiological patterns, associated with the four serotypes of the virus. These can co-circulate within a region, and indeed many countries are hyper-endemic for all four serotypes. Dengue has an alarming impact on both human health and the global and national economies. DENV is frequently transported from one place to another by infected travellers; when susceptible vectors are present in these new areas, there is the potential for local transmission to be established. Dengue Virus infection causes only mild illness, but dengue virus can also cause acute flu-like illness that sometimes develops into a potentially fatal complication called severe dengue. There is currently no specific medicine for the treatment of dengue fever/severe dengue fever.
This kit detects the conserved regions of Dengue Virus with high sensitivity and specificity.
【The Principle of Inspection】
The kit uses real-time fluorescence PCR technology for detection of Dengue Virus. Probes have a fluorescent reporter and a quencher at their 5' and 3' ends, respectively. During PCR amplification, the proximity of fluorescent reporter with the quencher prevents the reporter form fluorescing. When the Taq DNA polymerase (5'→3' exonuclease activity) reaches the dual-labeled probe, its 5' →3' exonuclease activity cleaves the fluorescent reporter from the probe. The amount of free reporter accumulates as the number of PCR cycles increases. The fluorescent signal from the free reporter is measured in real time and allows qualitative of the amount of target sequence. Specific primers and probes are designed to detect the highly conservative regions of Dengue Virus, are labeled with FAM. In addition, the introduction of UNG enzyme + dUTP anti-pollution measures into the PCR detection system can effectively degrade the aerosol pollution of amplification products and avoid false positives.
【The Main Components】
Components | Ingredient | Quantity |
RT-PCR Master Mix | Contains nucleotides of triphosphate, magnesium ions, etc. | 1× 625μL |
DV Mix | primers, probes, etc. | 1× 310μL |
Enzyme Mix | Contains DNA polymerase, Reverse transcriptase, etc. | 1× 65μL |
DV PC | TE solution containing the target gene | 1× 200μL |
DV NC | DEPC H2O | 1× 200μL |
Possible Accessories: Pipette, Pipette Tips, Vortex Mixer, and mini centrifugal (instantly), RNA Extraction reagent.
Warning: DO NOT interchange, mix, or combine reagents from kits with different master lot numbers.
【Storage Conditions and Expiration Date】
1. The kits are stable when stored at -20±5°C for 12 months
2. Transportation conditions: must be sealed with dry ice and ice bags, and transported under refrigerated conditions no more than 5 days.
3. Freezing and Thawing Stability: Freezing and thawing cycle is limited to 5 times without impacting performance or reliability.
4. Date of production and duration of use: see label.
【Applicable Instruments】
This kit is suitable for Anitoa labs MQ/Z/F series fluorescent PCRs, ABI series, QuantStudio3/5, Biorad CFX96, Roche Cobas Z480, etc.
【Sample Requirements】
Sample types：Serum.
The collected specimens should be sent for testing immediately. Specimens that cannot be tested should be stored at -20±5°C for 7 days, stored at-70°C for 12 months.
Multiple freeze/thaw cycles should be avoided. Specimens should be transported in a sealed frozenpitcher with ice or in a sealed foam box with ice.
【The Test Method】
1. Reagent Preparation (Reagent Preparation Area)
Unpacking all reagents from Kit, place at room temperature for thawing, all reagents shall be vortexed briefly before use. According to the specimen number to prepare reaction buffers, and it is recommended to set negative control and positive control for each test.
Reaction Components | Volume（μL）/Test |
RT-PCR Master Mix | 12.5 |
DV Mix | 6.2 |
Enzyme Mix | 1.3 |
Total Volume | 20 |
Calculate the volume as described above, add reagents to a sterile microcentrifuge tube, mix well, and then distribute 20μL to each PCR reaction tubes, transfer all PCR tubes to Sample Preparation Area.
2.Sample Preparation（Sample Preparation Area）
2.1 Nucleic Acid Extraction
Refer to the product manual of the nucleic acid extraction or purification kit for operation.
2.2 Sampling
Add 5μL specimen, 5μL of DV NC and 5μL of DV PC to distributed reaction tubes respectively, total volume 25μL/tube, caped the tube, and centrifuge at low speed instantaneously. The experimental shall be carried out on ice as far as possible.
3. PCR Amplification (PCR Amplification Area)
Place the PCR reaction tube in the Anitoa labs MQ/Z/F series fluorescent PCR instrument and set the cycle parameters as follows:
Steps | Number of cycles | Temperature | Reaction time |
1 | 1 | 50℃ | 10min |
2 | 1 | 95℃ | 1min |
3 | 40 | 95℃ | 3s |
55℃ (collecting fluorescent) | 20s |
Fluorescent signals are collected as FAM and VIC, and the data is collected at 55℃.
Download More Information
Dengue Virus Nucleic Acid Detection Kit (Fluorescent RT-PCR).pdf
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