BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at -20°C Ships Ice chest + Ice pads Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Product Introduction
The Nipah Virus (Nipah Virus, NiV) is a single-stranded negative-sense RNA virus belonging to the Henipavirus genus of the Paramyxoviridae family. It has fruit bats as its natural host and can be transmitted through contact with the secretions of infected animals or by consuming contaminated food. There is also a risk of human-to-human transmission. This virus can cause severe encephalitis and respiratory diseases. The clinical manifestations include fever, headache, and altered consciousness, with a typical incubation period of 4-14 days (up to 45 days). The fatality rate is approximately 40%-75%. The Nipah Virus genome consists of six transcription units and the 3' and 5' non-translated regions. The N gene is highly conserved and can be used as a target fragment for PCR diagnosis. These primers are pre-designed, verified by qPCR, and pre-mixed. qPCR (Quantitative PCR) is a method that quantitatively determines the total amount of PCR products during the amplification reaction process using fluorescence. The two common methods for qPCR are SYBR Green-based fluorescent dye method and probe method. The probe method, also known as TaqMan probe method, does not use fluorescent dyes but uses DNA probes labeled with fluorescent groups and quencher groups to target the target sequence to be detected by PCR. For the SYBR Green-based dye method, the primers are crucial. This series of primer products use the primer design algorithm developed by Aladdin, optimize the sequences and have been verified, showing excellent specificity, high amplification efficiency, and a low incidence of primer dimer formation, with reliable qPCR data. Most of the amplification products (Amplicon) of this series of primers are approximately 100-150 bp in length. This product is a pre-mixed freeze-dried powder, with 1 nmol of forward primer (also known as upstream primer) and 1 nmol of reverse primer (also known as downstream primer) in each tube, totaling 2 nmol. It does not contain nucleases (Nuclease-free), and only 400 μL of ultrapure water needs to be added to dissolve it to 2.5 μM each, ready for use. For the 20 μL or 25 μL system, 2 μL of primer is used, and this product can be used for 200 qPCR experiments per tube.
Precautions
1. The length of the PCR amplification product may vary due to the existence of multiple splicing forms after gene transcription.
2. Although the specificity of the products of this series of primers is good, it is still recommended to perform melt curve analysis to determine the specificity of the amplification reaction. If there is only one melt curve peak (corresponding to the annealing temperature, which is the Tm value of the double-stranded DNA product), it indicates that there is only one single product; if the melt curve shows double peaks, multiple peaks or overlapping peaks, it may be due to primer dimerization or non-specific amplification, presence of genomic DNA contamination, contamination of reagents and environment, etc. It is recommended to set up a no-template control (NTC), that is, the reaction system contains all reaction components except the template. Based on the differences in the melt curves of the sample wells and the no-template control wells, it can be determined whether there is primer dimerization or other non-specific amplification.
3. For long-term storage, it is recommended to store at a concentration of 100 μM at -20 °C.
4. This product is only for professional researchers' scientific research use. It shall not be used for clinical diagnosis or treatment, nor for food or medicine. It shall not be stored in ordinary residences.
5. For your safety and health, please wear laboratory coats and disposable gloves during operation.
Instructions for Use
1. Setup of the PCR reaction system:
(1) Before starting this product, centrifuge at 3000-8000×g for 1 minute to prevent the loss of primer powder when opening the cover. Add 400 μL ultrapure water to each tube. First, close the cover and invert it several times to mix well, then perform a quick centrifugation in the centrifuge for a few seconds. After opening the cover, gently shake to mix well, and you will obtain 400 μL of 2.5 μM each Primer Mix.
(2) Melt and mix all the solutions required for the PCR reaction. The SYBR Green qPCR Mix needs to be completely melted and mixed before being placed on an ice bath or in an ice box. It is recommended to use UltraSYBR One Step RT-qPCR Kit (U665567).
(3) Refer to the table below to set up the PCR reaction system at room temperature or on an ice bath. Take the 96-well plate and UltraSYBR One Step RT-qPCR Kit as an example.
| Reagent | Volume for One RT-PCR Reaction |
| 2×UltraSYBR One Step Buffer | 12.5 µL |
| UltraSYBR One Step EnzymeMix | 0.5 μL |
| qPCR Primer Pair (2.5 μM each) | 2 μL |
| RNA Template | 10 pg – 100 ng |
| RNase-Free Water | up to 25 μL |
| Total Volume | 25 µL |
Note: 1. Generally, a final concentration of 0.2 - 0.5 μM for each primer can yield good detection results. The final concentration of the primers can also be adjusted within the range of 0.1 - 1.0 μM depending on the specific situation.
2. The recommended reaction volume for the 96-well plate is 25 µL. It can also be adjusted proportionally according to the actual experimental requirements.
3. It is recommended to set up a negative control group without template.
(4) Mix gently by pipetting or vortexing, centrifuge at room temperature for a few seconds to allow the liquid to accumulate at the bottom of the tube.
(5) Place the prepared PCR reaction tubes on the fluorescence quantitative PCR instrument and start the quantitative PCR reaction.
2. PCR reaction protocol:
Two-step method:
(1) Reverse transcription: 45ºC 10 min;
(2) Pre-denaturation: 95°C 5 min;
(3) Denaturation: 95°C 10 s;
(4) Annealing/extension: 60°C 45 s;
(5) Repeat steps (3) and (4) for a total of 30-40 cycles;
(6) For melting curve analysis, please set the program as recommended by the fluorescence quantitative PCR instrument used.
Three-step method:
(1) Reverse transcription: 45°C 10 min;
(2) Pre-denaturation: 95°C 5 min;
(3) Denaturation: 95°C 15 s;
(4) Annealing: 56°C to 64°C for 30 s;
(5) Extension: 72°C 30 s;
(6) Repeat steps (3) to (5) a total of 35-40 cycles;
(7) For melting curve analysis, please set the parameters according to the recommended procedure of the fluorescence quantitative PCR instrument used.
Note: The above examples represent a conventional qPCR reaction system and are for reference only. The actual reaction conditions vary depending on the structure of the template, primers, etc. The optimal reaction conditions should be set based on the characteristics of the template, primers, and target fragment, and the reaction system should be adjusted by proportionally increasing or decreasing. For the three-step PCR amplification, the annealing temperature should be set as a reference within the range of 56℃ - 64℃.
3. Analyze the results using the software provided by the fluorescence quantitative PCR instrument.
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