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荧光定量PCR应用之污染篇

发布: 2018-11-28 654阅读 199评论

概述
实时荧光定量PCR （Real-time quantitative Polymerase Chain Reaction，Q-PCR）是指在PCR反应体系中加入荧光基团，利用荧光信号累积实时监测整个PCR进程，最后通过标准曲线对反应体系中未知模板进行量化分析的方法。该方法具有专属性强、灵敏度高、准确度高等优点，因而被广泛应用于药物DNA残留及病原学定性检测等领域。在荧光定量PCR应用过程中，最让人棘手头疼的问题便是污染，污染轻则导致实验失败，重则瘫痪整个实验室，可以说污染得以控制荧光定量PCR实验便成功了一半。

污染
污染的表现形式：

1. 扩增效率偏高

上图中标准曲线最低浓度点被外源DNA污染，导致提前扩增，扩增效率偏高。

思考：标曲最低浓度点对污染的耐受性比较低，原因是什么？为什么污染后扩增效率变高了？

2. 精密度不合格。
污染源多是气溶胶导致样品间的交叉污染。气溶胶是由固体或液体小质点分散悬浮在气体介质中形成的胶体分散体系。荧光定量PCR反应体系经过不断升降温，液体蒸发又冷凝，PCR管内很容易形成携带有大量DNA扩增片段的气溶胶，气溶胶容易泄露扩散（如打开扩增管盖、扩增管破裂等）而污染实验室

上图中标准曲线扩增正常，线性相关系数R=1，扩增效率Eff%=98.67，说明实验操作无问题，但孔间精密度偏差较大，为DNA气溶胶污染。

3. 阴性有扩增，实验专属性不合格

上图中标曲线性相关系数（R≥0.99）及扩增效率（90-105%）均符合要求，但阴性扩增CT值＜35，实验专属性不合格。

预防措施
实验室分区：试剂配置区、样品处理区、标曲及阳性样品制备区、反应体系配置区、扩增区等。

1. 试剂配置区：配置缓冲液、引物、探针等，存放试剂仪器有：Tris、EDTA、pH计、电子天平、磁力搅拌器等；该区域气压为正压模式，防止外源性DNA污染。

2. 样品处理区：处理Q-PCR扩增检测样品，如药物DNA残留剂量检测供试品溶液、样品DNA提取加标实验等；该区域气压为正压模式，防止外源性DNA污染。

3. 标曲及阳性样品制备区：稀释制备标准曲线溶液及阳性对照溶液。在配置标准曲线溶液过程中，标准品梯度稀释溶液在混匀时应避免剧烈涡旋震荡，防止溶液与管内空气剧烈摩擦而形成气溶胶，造成不同浓度标准曲线样品溶液之间交叉污染。另外，该区域内的移液枪要专用，不可与样品处理区及反应体系制备区的移液枪混用。完成该区域内实验，要及时更换手套，不可将手套带入反应体系制备区。该区域气压为负压模式，防止DNA扩散污染。

4. 反应体系制备区：配置Q-PCR扩增反应体系及阴性样品制备。该区域内的移液枪要专用，该区域气压为正压模式。

5. 扩增区：扩增待测样品，该区域保持通风，气压为负压模式。

6. 扩增后处理区：反应后PCR管应当丢离实验室区域，不可将反应PCR管丢在实验室垃圾桶内，切不可在实验区域内开启。

另外，上述分区实验人员在实验中要保持单向性，即实验由试剂配置区开始，经样品处理区、标曲及阳性样品制备区等，到扩增后处理区结束，不可反向而行，如实验人员不可由扩增区返回反应体系制备区，防止DNA人为带入到反应体系制备区。实验所用冰箱要专用，不可将DNA标准品与Q-PCR扩增所用的试剂（如引物、探针、DNA聚合酶等）放在同一冰箱内。

污染监控

1. 监测上述实验分区是否存在气溶胶污染，监控方法为将去离子水放置实验区域2-3h后，将该去离子水作为模板扩增，去离子水可在实验室多点放置。

2. 监测仪器（如离心机、移液枪把手等）表面是否被污染，用无菌棉签蘸取去离子水擦拭仪器及试验台表面，取少量液体做为模板进行扩增。

3. 监测移液枪是否被污染，用移液枪反复吹打无污染的去离子水，将该去离子水作为模板扩增。

4. 监测试剂是否被污染，将试剂做为阴性模板进行扩增。

污染处理

1. 通风：通风是去除实验室气溶胶污染的最好方式。

2. 紫外照射法：紫外线波长一般选择254nm，照射1h即可。

3. 生物消毒技术：喷射过氧化氢液或10%（V/V）次氯酸钠溶液成雾状液滴，弥散在需去除污染的区域，对其空气和表面污染物进行彻底清除。

4. 酸处理：对可疑器具用1M盐酸擦拭或浸泡，使残余的核酸脱嘌呤。

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