Clara® 抗抑制探针一步法预混液Separate-Rox

​产品概述​

Clara® 抑制剂耐受性探针法预混液和一步法预混液是广谱抑制剂耐受性的 4× 预混液，分别用于探针法 qPCR 和一步法 RT-qPCR。即使存在 PCR 抑制剂，使用 Clara® 抑制剂耐受性探针法预混液也能实现尖端的 DNA 和 RNA 检测。这些广谱、抑制剂耐受性的 qPCR 预混液经过专业设计，能够克服来自实验室、环境和临床样本中的各种挑战性抑制剂，确保 DNA 检测结果可靠并简化工作流程。对于希望扩增 RNA 的终端用户，我们推荐 Clara® 抑制剂耐受性探针法一步法预混液。

什么是Clara®抑制剂耐受性探针法预混液？​​

PCR和qPCR可受多种化学物质抑制，从而影响qPCR工作流程和诊断过程。Clara®抑制剂耐受性探针法预混液是一种4×即用型预混液，包含DNA聚合酶、缓冲液、镁离子、dNTPs以及成功进行探针法qPCR所需的其他组分，均为单管形式。终端用户可将此预混液与引物、探针和模板混合，用于单重或多重检测。该预混液通用性强，兼容所有探针化学（如TaqMan、Scorpions、分子信标等），并兼容所有商业化的实时PCR仪。Clara®抑制剂耐受性探针法预混液专为体外诊断试剂盒开发以及需要高效、可靠qPCR反应的各种研究应用而设计。

​什么是Clara®抑制剂耐受性探针法一步法预混液？​​

Clara®抑制剂耐受性探针法一步法预混液是一种4×即用型预混液，包含DNA聚合酶、逆转录酶、RNase抑制剂、缓冲液、镁离子、dNTPs以及成功进行探针法（RT-）qPCR所需的其他组分，均为单管形式。终端用户可将此预混液与引物、探针和模板混合，用于单重或多重检测。该预混液通用性强，兼容所有探针化学，并兼容所有商业化的实时PCR仪。Clara®抑制剂耐受性探针法一步法预混液专为体外诊断试剂盒开发而设计，也可用于需要高效、可靠（RT-）qPCR反应的各种研究应用。

​抑制剂耐受性测试​

Clara® 抑制剂耐受性探针法预混液已针对以下物质进行了广泛测试：

• 粗唾液

• 粗制血液

• 实验室化学品

• 临床抑制剂

• 植物和环境抑制剂

​产品特点​

• ​广谱抑制剂耐受性：​​ 有效对抗多种PCR抑制剂。

• ​4× 浓缩配方：​​ 适合高通量和高度多重检测。

• ​优异的RNA/DNA检测能力：​​ 对RNA和DNA靶标均有高检测灵敏度。

• ​低模板定量可靠：​​ 可准确定量低丰度模板。

• ​抗体介导的热启动技术：​​ 提高反应特异性和灵敏度。

• ​广泛的仪器兼容性：​​ 兼容所有实时荧光PCR平台——标准和快速循环条件。

• ​支持超快循环：​​ 逆转录可短至3分钟，PCR变性1秒，退火/延伸3秒。

• ​一步法配方：​​ 包含修饰的 UltraScript® 逆转录酶和RNase抑制剂，单管操作。

​应用领域​

• 物种丰度定量

• 基因分型

• 等位基因鉴别

• 体外诊断试剂盒开发

• 单重及多重RNA/DNA检测

• 粗唾液样本qPCR

​图1. 使用Clara®抑制剂耐受性探针法预混液检测DNA靶标。​​

使用Clara®抑制剂耐受性探针法预混液，在探针法qPCR反应中扩增γ-肌动蛋白的系列稀释液。使用了四种模板稀释度，每个靶标设三个技术重复，反应体系为20 μL。循环条件为：95°C 3分钟；随后95°C 10秒，60°C 20秒，共50个循环。Clara®抑制剂耐受性探针法预混液可成功扩增低至每反应0.25 pg/μL的cDNA靶标。

​Detection of a DNA target with Clara® Inhibitor-Tolerant Probe Mix.
Clara® Inhibitor-Tolerant Probe Mix was used to amplify a serial dilution of γ-actin in a probe-based qPCR reaction. Four template dilutions (250, 25, 25, and 0.25 pg/μL per reaction) with three technical replicates for each target were used in 20 μL reactions. Cycling conditions were: 95 °C for 3 min, followed by 50 cycles of 95 °C for 10 s, and 60 °C for 20 s. Clara® Inhibitor-Tolerant Probe Mix successfully amplifies cDNA targets from as little as 0.25 pg/μL per reaction.

图2. 在有/无唾液情况下对DNA靶标进行四重检测。​​

使用Clara®抑制剂耐受性探针法预混液，在多重qPCR检测中扩增四个DNA靶标，比较存在人唾液（紫色）和不存在人唾液（蓝色）的情况。反应体系为20 μL，每个靶标/稀释度设三个技术重复。含唾液的反应中含有5 μL用通用转运培养基1/10稀释的唾液。循环条件为：95°C 3分钟；随后95°C 10秒，60°C 20秒，共50个循环。Clara®抑制剂耐受性探针法预混液即使在存在人唾液的情况下，也能在多重检测设置中成功扩增DNA靶标。

Fourplex detection of DNA targets with and without saliva.
Four DNA targets, GEN (general MPX), β-microtubulin (B2M), CB (Congo Basin MPX), WA (West Africa MPX), were amplified, in the presence (purple) and absence (blue) of human saliva, in a multiplex qPCR assay with Clara® Inhibitor-Tolerant Probe Mix. Four template dilutions (10000, 1000, 100, and 10 copies for the monkeypox (MPX) viral targets, and 250, 25, 2.5, and 0.25 pg/ μL for B2M) with three technical replicates for each target were used in 20 μL reactions. Reactions with saliva contained 5 μL saliva diluted 1/10 in universal transport medium, corresponding to 2.5% human saliva per reaction. Cycling conditions were: 95 °C for 3 min, followed by 50 cycles of 95 °C for 10 s, and 60 °C for 20 s. Clara® Inhibitor-Tolerant Probe Mix successfully amplifies DNA targets in multiplex setup even in the presence of human saliva.

​图3. 使用Clara®抑制剂耐受性探针法一步法预混液进行五重RNA靶标检测。​​

使用Clara®抑制剂耐受性探针法一步法预混液，在多重一步法RT-qPCR反应中扩增五个RNA靶标。每个稀释度设三个技术重复，反应体系为20 μL。循环条件为：47°C 10分钟，95°C 2分钟；随后95°C 10秒，60°C 30秒，共50个循环。Clara®抑制剂耐受性探针法一步法预混液在五重一步法RT-qPCR反应设置中，于标准循环条件下显示出高灵敏度和效率。

​Pentaplex detection of RNA targets with Clara® Inhibitor-Tolerant Probe 1-Step Mix.
Five RNA targets, SARS-CoV-2 E-gene (E-gene), Influenza-A (INF-A), Influenza-B (INF-B), Respiratory Syncytial Virus (RSV) and mouse β-actin were amplified in multiplex 1-step RT-qPCR reactions with Clara® Inhibitor-Tolerant Probe 1-Step Mix. Four template dilutions (4000, 400, 40, and 4 copies, for viral targets, and 1.25 ng/μL, 125 pg/μL, 12.5 pg/μL, and 1.25 pg/μL total mouse RNA for β-Actin) with three technical replicates per dilution were used in 20 μL reactions. Cycling conditions were: 47 °C for 10 min, 95 °C for 2 min, followed by 50 cycles of 95 °C for 10 s, and 60 °C for 30 s. Clara® Inhibitor-Tolerant Probe 1-Step Mix shows high sensitivity and efficiency under standard cycling conditions in a pentaplex 1-step RT-qPCR reaction setup.

图4. 在有/无唾液情况下对病毒RNA靶标进行四重检测。​​

使用Clara®抑制剂耐受性探针法一步法预混液，在多重一步法RT-qPCR反应中扩增四个RNA靶标，比较存在人唾液（紫色）和不存在人唾液（蓝色）的情况。每个靶标设四个模板稀释度，每个稀释度三个技术重复，反应体系为20 μL。含唾液的反应中含有5 μL用通用转运培养基1/10稀释的唾液。循环条件为：47°C 10分钟，95°C 2分钟；随后95°C 10秒，60°C 30秒，共50个循环。Clara®抑制剂耐受性探针法一步法预混液即使在存在人唾液的情况下，也能在多重检测设置中成功扩增RNA靶标。

Clara®抑制剂耐受性探针法预混液适用于哪些类型的模板？​​

• Clara®抑制剂耐受性探针法预混液适用于扩增DNA模板。

• Clara®抑制剂耐受性探针法一步法预混液适用于RNA和DNA模板的扩增，在扩增混合模板时效率无损失。

Fourplex detection of viral RNA targets with and without saliva.
Four RNA targets, SARS-CoV-2 E-gene (E-gene), Respiratory Syncytial Virus (RSV), Influenza-a (INF-A), and Influenza-b (INF-B), were amplified, in the presence (purple) and absence (blue) of human saliva, in multiplex 1-step RT-qPCR reactions with Clara® Inhibitor-Tolerant Probe 1-Step Mix. Four template dilutions (4000, 400, 40, and 4 copies) with three technical replicates for each target were used in 20 μL reactions. Reactions with saliva contained 5 μL saliva diluted 1/10 in universal transport medium, corresponding to 2.5% human saliva per reaction. Cycling conditions were: 47 °C for 10 min, 95 °C for 2 min, followed by 50 cycles of 95 °C for 10 s, and 60 °C for 30 s. Clara® Inhibitor-Tolerant Probe 1-Step Mix successfully amplifies RNA targets in multiplex setup even in the presence of human saliva.

​图5. Clara®抑制剂耐受性探针法一步法预混液与竞品的比较。​​

在存在人唾液的条件下，使用四种不同的预混液（包括Clara®）对四个RNA靶标进行多重一步法RT-qPCR反应比较。反应条件同上。Clara®抑制剂耐受性探针法一步法预混液在存在人唾液的多重检测设置中，其性能匹配或优于三种竞品。

​Clara®抑制剂耐受性探针法预混液可耐受的抑制剂类型​

Clara®抑制剂耐受性探针法预混液和一步法预混液已针对多种常见PCR抑制剂进行了测试并证明具有耐受性，包括标准实验室化学品（如SDS、胍盐、乙醇）和生物样本抑制剂，例如血液样本中的抑制剂（血红素、羟高铁血红素、血红蛋白、肝素、IgG免疫球蛋白、乳铁蛋白、柠檬酸钠）、唾液、尿液（尿素）、植物和环境样本中的抑制剂（腐殖酸、儿茶素、槲皮素、单宁酸、纤维素、叶绿素）。该预混液已针对粗唾液/血液以及多种纯化学抑制剂进行了广泛验证。

Competitor comparison of Clara® Inhibitor-Tolerant Probe 1-Step Mix with competitor mixes.
Four RNA targets, SARS-CoV-2 E-gene (E-gene), Respiratory Syncytial Virus (RSV), Influenza-A (INF-A), and Influenza-B (INF-B) were amplified, in the presence of human saliva, in multiplex 1-step RT-qPCR reactions with: Clara® Inhibitor-Tolerant Probe 1-Step Mix (purple, PCRBIO), One Step PrimeScript III RT-PCR Kit from Takara (grey, Takara), Air-Dryable Direct RNA/DNA qPCR Saliva (grey, Meridian SR), and Inhibitor-Tolerant RT-qPCR Mix (grey, Meridian IT) from Meridian. Four template dilutions (4000, 400, 40, and 4 copies) with three technical replicates for each target were used in 20 μL reactions. Reactions with saliva contained 5 μL saliva diluted 1/10 in universal transport medium, corresponding to 2.5% human saliva per reaction. Cycling conditions were: 47 °C for 10 min, 95 °C for 2 min, followed by 50 cycles of 95 °C for 10 s, and 60 °C for 30 s. Clara® Inhibitor-Tolerant Probe 1-Step Mix matches or outperforms three competitor products in multiplex setup in the presence of human saliva.

图6. Clara抑制剂耐受性化学在抑制剂存在下提供稳健性能。​​

使用Clara®抑制剂耐受性探针法一步法预混液，挑战递增浓度的各种临床、植物、环境和实验室化学抑制剂。根据未抑制的对照反应（100%活性）计算各抑制剂浓度存在下的活性百分比。PCR Biosystems的抑制剂耐受性化学在大多数测试抑制剂存在下均能提供稳健的qPCR性能。*纤维素在所用水性反应条件下不溶，以悬浮液形式加入。

Clara Inhibitor-Tolerant chemistry offers robust performance in the presence of inhibitors. Clara® Inhibitor-Tolerant Probe 1-Step Mix was challenged with increasing amounts of various clinical, plant, environmental and laboratory chemical inhibitors (see axis labels for specific inhibitors). The % activity in the presence of each inhibitor concentration was calculated based on a corresponding uninhibited control reaction (100% activity). PCR Biosystems inhibitor-tolerant chemistry offers robust qPCR performance in the presence of most of the tested inhibitors. *Cellulose is not water soluble under reaction conditions used and was added in suspension.