尼帕病毒（Nipah Virus, NiV）是副粘病毒科亨尼帕病毒属的单负链RNA病毒，以果蝠为天然宿主，可通过接触感染动物分泌物或食用污染食物传播，亦存在人际传播风险。该病毒可引发严重脑炎和呼吸系统疾病，临床表现为发热、头痛、意识障碍等症状，潜伏期通常为4-14天（最长可达45天），致死率达40%-75%。 Nipah Virus基因组是由六个转录单位及3’和5’端的非翻译区所组成，六个转录单位为N、P、M、F、G、L。N基因高度保守，可作为 PCR 诊断的靶片段。 本引物为预先设计、经过qPCR验证、预混的引物对。qPCR (Quantitative PCR)即定量PCR，也称实时荧光定量PCR或实时定量PCR (Real-time quantitative PCR)、实时PCR (Real-time PCR)，是一种在DNA扩增反应过程中，以荧光定量测定每个聚合酶链式反应(PCR)循环后产物总量的方法。qPCR常用的两种方法是SYBR Green等荧光染料法和探针法。SYBR Green等荧光染料法是使用带有荧光的、非特异的DNA结合染料SYBR Green等以检测PCR过程中积累的PCR扩增产物；而探针法(Probe method)，也常被称为TaqMan探针法，不使用荧光染料，而采用荧光基团和淬灭基团(Quencher)标记的DNA探针靶向拟通过PCR检测的目标序列。对于SYBR Green等染料法，引物至关重要。本系列引物产品采用阿拉丁开发的引物设计算法，优化了序列并经过验证，特异性佳，扩增效率高，引物二聚体形成发生率低，qPCR数据可靠。本系列引物大多数扩增产物(Amplicon)的长度约100-150 bp。本产品为预混冻干粉，每管含正向引物(Forward primer，也称上游引物)和反向引物(Reverse primer，也称下游引物)各1 nmol，共2 nmol，不含核酸酶(Nuclease-free)，只需加入400 μL超纯水溶解成2.5 μM each，即可使用。按20 μL或25 μL体系使用2 μL引物，本产品每管可以用于200次qPCR实验。
注意事项：
1. PCR扩增产物的长度可能会因基因转录后存在多种剪接形式而有所差异。
2. 虽然本系列引物产品的特异性好，但仍建议进行熔解曲线(Melt curve)分析以确定扩增反应的特异性。如果只有一个熔解曲线峰(对应的退火温度即双链DNA产物的Tm值)，说明只有一种单一产物；如果熔解曲线出现双峰、多峰或杂峰峰，可能是引物二聚体或非特异性扩增、存在基因组DNA污染、试剂及环境被污染等。建议设置不含模板的对照(No template control, NTC)，即反应体系中包含除模板以外的所有反应组分，根据样品孔和无模板对照孔熔解曲线的差异，可判断是否存在引物二聚体或其它的非特异性扩增。
3. 长期保存建议储存浓度为100 μM，保存于-20 °C 。
4. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
1. PCR反应体系的设置：
(1) 开启本产品前，3000-8000×g离心1分钟，以防开盖时引物干粉散失。每管加入400 μL超纯水，先盖好盖子颠倒混匀数次，然后离心机快速离心几秒，开盖后再轻轻吹打混匀，即得400 μL 2.5 μM each的Primer Mix。
(2) 融解并混匀PCR反应所需的各种溶液。SYBR Green qPCR Mix需完全融解并混匀后置于冰浴上或冰盒内。推荐使用 UltraSYBR One Step RT-qPCR Kit (U665567)。
(3) 参考下表在室温或冰浴上设置PCR反应体系，以96孔板和 UltraSYBR One Step RT-qPCR Kit 为例。
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 |
注：1. 通常引物的终浓度为0.2-0.5 μM each时可获得良好的检测效果，也可根据情况在0.1-1.0 μM each范围内调整引物的终浓度。
2. 96孔板的推荐反应体系为25 µL，也可以根据实际实验需求，按比例扩大或缩小反应体系。
3. 建议设置不加模板的阴性对照组。
(4) 用移液器轻轻吹打混匀或轻微Vortex混匀，室温离心数秒，使液体积聚于管底。
(5) 将设置好的PCR反应管置于荧光定量PCR仪上，开始定量PCR反应。
2. PCR反应程序：
两步法：
(1) 反转录：45ºC 10 min；
(2) 预变性：95ºC 5 min；
(3) 变性：95ºC 10 s；
(4) 退火/延伸：60ºC 45 s；
(5) 重复步骤(3)和步骤(4)，总共30-40个循环；
(6) 熔解曲线分析请以所使用的荧光定量PCR仪推荐的程序进行设定。
三步法：
(1) 反转录：45ºC 10 min；
(2) 预变性：95ºC 5 min；
(3) 变性：95ºC 15 s；
(4) 退火：56ºC -64ºC 30s；
(5) 延伸：72ºC 30 s；
(6) 重复步骤(3)至步骤(5)，总共35-40个循环；
(7) 熔解曲线分析请以所使用的荧光定量PCR仪推荐的程序进行设定。
注：以上举例为常规qPCR反应系统，仅供参考。实际反应条件因模板、引物等的结构不同而各异，需根据模板、引物、目的片段的特点设定最佳反应条件，并根据比例放大或缩小反应体系。三步法PCR扩增时，退火温度请以 56℃-64℃的范围作为设定参考。
3. 使用荧光定量PCR仪提供的软件分析结果。
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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