Ever found yourself scratching your head over mysterious target bands in the blank control of your conventional PCR? Or perhaps pondering the appearance of Ct values in your qPCR experiments' no-template controls (NTC)? Maybe you've experienced a frustrating bout of contaminated results despite replacing reagents and samples multiple times. If so, you're not alone. Many molecular biology laboratories grappling with PCR and qPCR have encountered such confounding situations.

But fear not, it's not necessarily a flaw in your reagents or enzymes. The likely culprit? Aerosol contamination!

You might think aerosol contamination isn't a big deal for nucleic acid experiments, but historically, major nucleic acid contamination incidents have had serious consequences.

On April 18, 2020, The Washington Post broke a major story: contamination issues in the laboratories of the Centers for Disease Control and Prevention (CDC) led to a delay in the rapid development of testing tools for the novel coronavirus, causing delays in COVID-19 testing nationwide. Reports indicated that after the CDC distributed test kits to 26 public health laboratories across the U.S., 24 of them reported "false-positive" results, prompting concerns about the test kits. It took CDC officials over a month to resolve the testing issues, exacerbating delays in the production and distribution of test kits across the country.

Clearly, aerosol contamination can have serious and sometimes uncontrollable consequences for nucleic acid testing experiments. This is especially true for highly sensitive nucleic acid detection assays like RT-qPCR, where even slight contamination can affect the accuracy of results.

So, what exactly is aerosol contamination, and how does it affect PCR experiments?

Aerosol contamination refers to the dispersion and suspension of solid or liquid particles in a gas medium, potentially including biological agents like bacteria or viruses. In laboratories where sensitive nucleic acid detection assays like RT-qPCR are commonplace, aerosol contamination poses a significant threat to the accuracy of results. Even minor contamination can skew Ct values, leading to erroneous assessments of gene content in samples.

But how does aerosol contamination occur in the first place?

Friction between gases and liquids can generate aerosols, and in busy labs where multiple individuals handle the same reagents and equipment, the risk of contamination skyrockets. For instance, using shared equipment like pipettes and centrifuges, conducting PCR procedures in close proximity, or even simply mixing samples in the same area can introduce contaminants. This can manifest as false positives in blank controls during gel electrophoresis or unexpected bands in qPCR assays.

So, how can laboratories mitigate the risk of aerosol contamination?

• Utilize UDG contamination prevention reagents: Aerosols may harbor copy numbers ranging from 10^4 to 10^6 copies. Hence, even minute traces of contamination can potentially yield false-positive outcomes in experiments. Nevertheless, by employing a qPCR premix equipped with a UDG contamination prevention system, such as Probe qPCR Mix (2X, UDG), the likelihood of contamination stemming from your amplification products can be greatly mitigated.
• Implement standard laboratory layout setups: Many laboratories often perform fluorescence quantitative detection experiments without adequately considering the necessity for proper nucleic acid amplification area divisions. A well-thought-out laboratory layout can significantly decrease the risk of nucleic acid contamination. Essentially, a testing laboratory should incorporate the following designated areas: a reagent storage and preparation area, a sample preparation area, and an amplification and product analysis area, each physically separated from one another. Pass-through windows should adhere to sealing standards and must prevent direct airflow between areas.
• Enforce unidirectional flow for personnel and items: In theory, the movement of personnel and materials should adhere to a unidirectional flow, starting from the reagent storage and preparation area, through the sample preparation area, amplification area, and finally to the product analysis area. This arrangement minimizes the potential for sample cross-contamination. However, in crowded laboratory settings unable to maintain this ideal flow, instruments and consumables such as pipettes, centrifuges, nucleic acid extractors, PCR machines, opened reagent boxes, and tip boxes require regular treatment with nucleic acid decontaminants to eradicate any residual nucleic acid contamination.
• Adopt standardized storage practices: To mitigate the risk of reagents being opened repeatedly, potentially leading to contamination with the genes under examination and compromising the efficacy of subsequent reagent use, it's advisable to divide reagents into smaller aliquots for storage. This practice minimizes the likelihood of aerosol contamination resulting from frequent reagent openings. Particularly for positive samples, ensuring tight sealing of caps is crucial, thereby reducing the possibility of sample exposure to the environment.
• Standardize experimental procedures: PCR products undergo multiple rounds of amplification, resulting in copy numbers far exceeding the detection limit of PCR. Therefore, during operations, movements should be slow and gentle to minimize the risk of aerosol contamination. When aspirating samples using a pipette, it's imperative to strictly adhere to the principle of slow aspiration and slow dispensing. After centrifuging to mix samples, it's advisable to wait a moment before opening the lid to prevent the gas in the reaction tube from carrying sample genes into the air. Prior to gel electrophoresis after PCR, if there are indications of loosened caps on the reaction tubes within the strip or if the liquid level in the tubes decreases post-PCR, it's crucial to recognize that cross-contamination may have occurred between tubes or within the PCR machine itself. This scenario often manifests in reaction tubes positioned near the edges of the strip.

The aforementioned sources of nucleic acid contamination can all contribute to the dispersion of aerosols into the air or direct adsorption onto surfaces. Heavy contamination may yield false-positive Ct values around 24, while moderate contamination could lead to values around 30, and light contamination may result in values around 33. Therefore, rather than attributing blame solely to reagents, it is imperative to concentrate on minimizing aerosol contamination to guarantee precise and dependable outcomes in our PCR endeavors!