为RNA病毒检测开发的分子检验设备
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By LabMedica International staff writers Posted on 19 Mar 2018 |
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图片：定量逆转录酶聚合酶链反应(qRT-PCR)热塑性芯片（图片蒙美国弗朗霍夫研究所惠赐）。
日前开发成功的一台微流控连续液流定量逆转录酶聚合酶链反应(qRT-PCR)系统可检测病毒RNA靶体，它的创造者一直致力于降低制造成本，寻找潜在的商务合作伙伴。
qRT-PCR系统一直被用来检测混有病毒的液体样本中的RNA指导的RNA聚合酶L (L基因)，它是埃博拉病毒的生物标志物。当前的目标是基于该技术开发床旁(POC)设备，在资源贫乏地区诊断传染病。
美国特拉华州纽瓦克市弗朗霍夫美国分子生物技术中心的科学家开发的微流控芯片内含注射泵、加热器和光学检测系统。科研小组用注射泵通过微流控连接头把液体样本和PCR试剂导入系统。经过提纯的RNA溶液进入混合腔，然后与预混合液混合，预混合液包含酶和执行扩增所需的其它试剂。混合液流过逆转录区，逆转录酶把RNA转化成互补DNA。
研究人员检验了两种一步式逆转录PCR (qRT-PCR)预混合液。科研小组首先使用了市售的Affymetrix VeriQuest预混合液，它由美国加利福尼亚州圣克拉拉的赛默飞世尔科技公司出品，内含逆转录酶和DNA聚合酶。科研小组还开发了一款定制的一步式预混合液，它结合了Affymetrix的VeriQuest预混合液与快速DNA聚合酶，包括在Affymetrix的USB快速定量PCR探针预混合液中。
新形成的互补DNA与试剂流过95°C的区域，那里的DNA聚合酶被激活，双链DNA融化。然后样本流到热循环区，在95 °C区和62 °C区之间经历40个循环。DNA在每次循环的高温区融化，在低温区退火和聚合。芯片通道的总容积是25微升，试剂消耗少，因此减少了耗材花费。
然后可以用光学检测系统测量荧光并进行实时DNA扩增。科研小组证明，该系统执行qRT-PCR需时30分钟，而传统的台式PCR仪需时80分钟。虽然他们肯定他们的自定义预混合液生成的结果一般更好，但是他们也用市售的混合液确定了该试剂的检测限，因为它很容易转向市场。科研小组以市售混合液和埃博拉病毒RNA为靶体，达到了每微升10个RNA拷贝的检测限。
将来，作者们计划重点攻关样本预处理，以便从血浆、尿液和唾液中提取RNA，因为提取高质量RNA对于生成成功的qRT-PCR结果起着至关重要的作用。科研小组给该设备定的目标市场价是2,000美元，可抛型测试芯片是0.50美元。
Related Links:
弗朗霍夫美国分子生物技术中心
Affymetrix
qRT-PCR系统一直被用来检测混有病毒的液体样本中的RNA指导的RNA聚合酶L (L基因)，它是埃博拉病毒的生物标志物。当前的目标是基于该技术开发床旁(POC)设备，在资源贫乏地区诊断传染病。
美国特拉华州纽瓦克市弗朗霍夫美国分子生物技术中心的科学家开发的微流控芯片内含注射泵、加热器和光学检测系统。科研小组用注射泵通过微流控连接头把液体样本和PCR试剂导入系统。经过提纯的RNA溶液进入混合腔，然后与预混合液混合，预混合液包含酶和执行扩增所需的其它试剂。混合液流过逆转录区，逆转录酶把RNA转化成互补DNA。
研究人员检验了两种一步式逆转录PCR (qRT-PCR)预混合液。科研小组首先使用了市售的Affymetrix VeriQuest预混合液，它由美国加利福尼亚州圣克拉拉的赛默飞世尔科技公司出品，内含逆转录酶和DNA聚合酶。科研小组还开发了一款定制的一步式预混合液，它结合了Affymetrix的VeriQuest预混合液与快速DNA聚合酶，包括在Affymetrix的USB快速定量PCR探针预混合液中。
新形成的互补DNA与试剂流过95°C的区域，那里的DNA聚合酶被激活，双链DNA融化。然后样本流到热循环区，在95 °C区和62 °C区之间经历40个循环。DNA在每次循环的高温区融化，在低温区退火和聚合。芯片通道的总容积是25微升，试剂消耗少，因此减少了耗材花费。
然后可以用光学检测系统测量荧光并进行实时DNA扩增。科研小组证明，该系统执行qRT-PCR需时30分钟，而传统的台式PCR仪需时80分钟。虽然他们肯定他们的自定义预混合液生成的结果一般更好，但是他们也用市售的混合液确定了该试剂的检测限，因为它很容易转向市场。科研小组以市售混合液和埃博拉病毒RNA为靶体，达到了每微升10个RNA拷贝的检测限。
将来，作者们计划重点攻关样本预处理，以便从血浆、尿液和唾液中提取RNA，因为提取高质量RNA对于生成成功的qRT-PCR结果起着至关重要的作用。科研小组给该设备定的目标市场价是2,000美元，可抛型测试芯片是0.50美元。
Related Links:
弗朗霍夫美国分子生物技术中心
Affymetrix
Gold Member
Immunochromatographic Assay
CRYPTO Cassette
Sample Transportation System
Tempus1800 Necto
Gold Member
Radial Immunodiffusion Assay
Radial Immunodifusion - C3 ID
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