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PCR considerations

Whether adding PCR to your existing laboratory, modifying existing PCR workflows, or starting from scratch, preventing contamination is a top priority. As PCR can effectively amplify even the tiniest of quantities of DNA and RNA, the risk of amplifying a contaminant and ruining the validity of an assay is very real. Contamination typically comes from non-amplified environmental substances such as aerosols, and from carryover contamination of amplicons from earlier PCR cycles. As such, not only do best-practice processes and procedures (P&P) need to be followed (e.g., unidirectional workflow, thorough cleaning procedures, proper preparation and disposal), but also where to place PCR-related equipment must be carefully considered.

When possible, separate rooms for sample preparation, PCR setup, and post-PCR activities, each with their own airflow control, are encouraged. However, the laboratory attempting to add PCR to an already small clinical diagnostic lab may not have the luxury of having multiple rooms. In that case, a single-room setup may suffice, if the workflow areas remain demarcated or physically partitioned. Additionally, a single-room setup must also have stricter P&P and design controls to offset the space constraints. For example, the sample preparation area of the room should have a laminar flow hood with UV light that is regularly cleaned, and post-PCR analysis may need to occur later in the day after cleanup from prior steps. Of course, always maintaining unidirectional workflow—regardless of number of rooms—is also critical to minimizing contamination. For example, technicians shouldn't be transporting amplified materials into the DNA extraction area.

Although dated, Roche Diagnostics' 2006 PCR Applications Manual provides a detailed breakdown of setting up the laboratory for PCR. Das et al. and Dr. Jennifer Redig provide additional valuable insight. The World Health Organization (WHO) also provides guidance for setting up molecular testing in the lab.

Isothermal amplification considerations

Similarly, because DNA and RNR amplification is involved, contamination concerns exist with isothermal amplification techniques. Multiple pipetting steps and repeated freezing and thawing of reagents can still lead to cross-contamination, as does opening the reaction chamber after reaction is completed. However, the advent of microfluidics and lateral flow technologies in isothermal amplification processes has seen the development of "fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization." Even more cutting-edge techniques to reduce contamination such as the CUT-LAMP technique of Bao et al. or the dUTP/UDG system for COVID-19 RT-LAMP reactions of Kellner et al. hold further promise in making isothermal amplification processes in the laboratory easier to manage. That said, labs running isothermal amplification processes such as LAMP requiring analysis with agarose gel electrophoresis or a method requiring the opening of reaction vessels will preferably have a secondary area set up for analysis steps so as to minimize the chances of contamination.