Polymerase Chain Reaction (PCR), the amplification of DNA, is an established process used in molecular biology. PCR technology has made strides in medical and infectious disease diagnoses, forensics and research applications. Decisions about treatment options and clinical efficacy are often based on DNA and RNA analysis of patient cells. Ensuring proper contamination control guarantees that clinicians get the best possible results.
PCR污染的最常见原因：
- Incorrect lab set-up and improper workflow
- Operator contamination
- Sample separation failure
- 实验室设备灭菌不当
Laboratory Set-Up for Optimum PCR Workflow
创建适当的实验室环境并建立单向工作流是防止PCR污染的两种方法。为了确保适当的设施控制，质量保证人员将建立针对实验室空间和可用设备规模的有记录的标准化协议。人员应仔细培训，并提供书面程序的访问权限。
This unidirectional workflow involves the following 5 best practices:
- 为每个PCR反应步骤建立单独的工作区域或房间
- NEVER return previously processed tubes to preceding work area or room
- Master-mix should be prepared and aliquoted in the reaction vessel and moved to an adjacent and segregated area
- DNA template must NEVER be present in the area where the master-mix is prepared
- Reaction tubes from completed amplifications should NEVER be stored with un-amplified samples or unused reagents
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
任何实验室中污染的最大贡献者是操作员。为了减轻这种情况，重要的是，操作员要观察已建立的单向工作流并维护适当的实验室装备。请按照以下技巧消除操作员污染：
- Personnel must always wear gloves, and change them regularly
- 操作员必须在保持干净的工作表面上练习尽职调查
- 操作员必须观察既定的单向工作流程协议
PCR引擎盖，灭菌和操作员安全
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反应应在单独的PCR反应引擎盖中进行
- The entire hood work surface, as well as pipettes, should be sterilized with a 1:10 or 1:20 dilution of bleach followed by a rinse with 70% ethanol
- Each PCR-reaction hood should contain dedicated pieces of equipment (pipettes, centrifuges, plastic labware) that should not be removed and/or transferred to another hood
PCR引擎盖中的推荐移液器
Choose positive-displacement pipettes with barrier tips to prevent carry-over from one sample to the next. These pipettes are ideal for working with volatile liquids and highly viscous samples because of their disposable micro-syringe tips. Positive displacement pipettes not only provide more accuracy, they also give superior sample protection compared to air-displacement pipettes.
结论
Contamination in PCR reactions can be controlled 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.
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Photos by杰梅里奇,
