DNA的扩增聚合酶链反应（PCR）是一种用于分子生物学的既定过程。PCR技术在医学和传染病诊断，取证和研究应用方面取得了长足的进步。有关治疗选择和临床功效的决策通常基于对患者细胞的DNA和RNA分析。确保适当的污染控制确保临床医生获得最佳结果。
The most common causes of PCR contamination:
- Incorrect lab set-up and improper workflow
- 操作员污染
- 样品分离失败
- Improper sterilization of lab equipment
最佳PCR工作流程的实验室设置
Creating a proper lab environment and establishing a unidirectional workflow are two ways to prevent PCR contamination. To ensure proper facilities control, quality assurance personnel will establish a documented standardized protocol specific to the size of their lab space and available equipment. Personnel should be carefully trained and provided with access to written procedures.
This unidirectional workflow involves the following 5 best practices:
- Establish separate work areas or rooms for each PCR reaction step
- NEVER return previously processed tubes to preceding work area or room
- 主混合应准备并在反应容器中等分，然后移动到相邻的隔离区域
- DNA模板绝不能在准备主混合的区域中存在
- 切勿使用未放大样品或未使用的试剂存储的完全放大的反应管
While large labs have the luxury of creating separate rooms within the facility for each PCR reaction, small labs use PCR hoods or similar dead-air boxes (hoods without an air blower) to create the separation needed for contamination prevention.
Avoiding Operator Contamination of PCR Workstation
The biggest contributors of contamination in any lab are operators. To mitigate this, it is important that operators observe the established unidirectional workflow and maintain proper lab gear. Follow these tips to eliminate operator contamination:
- 人员必须始终戴手套，并定期换上手套
- Operators must practice due diligence in maintaining a clean work surface
- Operators must observe established unidirectional workflow protocol
PCR Hoods, Sterilization and Operator Safety
PCR hoods can be equipped with an anti-microbial UV lamp, which is operated at set intervals to sterilize the work environment. UV-C exposure can be harmful, so workers should be out of the room while the UV lamp is engaged. If this is not possible, however, lab personnel must ALWAYS wear proper protective gear when working under these lamps. It is important that hoods equipped with UV lamps are constructed of proper UV-resistant materials.
- PCR reactions should be performed in a separate PCR-reaction hood
- 整个引擎盖的工作表面以及移液器应用1:10或1:20的漂白剂进行消毒，然后用70％乙醇冲洗
- 每个PCR反应引擎盖都应包含专用的设备（移液器，离心机，塑料实验室），不应卸下和/或转移到另一个引擎盖上
Recommended Pipettes in PCR Hoods
选择带有屏障尖端的正置放置移液器，以防止从一个样本到另一个样本的携带。这些移液器非常适合使用挥发性液体和高度粘性样品，因为它们具有一次性的微传输尖端。正位移不仅提供了更准确性，而且与空气置换移液器相比，它们还提供了优越的样品保护。
Conclusion
污染在PCR反应可以控制by following basic lab best practices and incorporating some specialized techniques specific to sensitive PCR applications. Ideally, lab equipment used for studying genetic material should not be used for other experiments; even with proper cleaning protocol, there remains a chance of cross-contamination. Improve efficiency and accuracy by following detailed, documented protocol.
Read more about Terra Universal’s PCR Workstations and Equipment.
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