The powerful exponential amplification achieved by the nucleic acid amplification technology also results in a potential risk of false positive signals because of contamination. Since up to 10¹² copies of a specific target sequence can be generated in a single PCR, even minimal amounts of aerosol can contain thousands of DNA copies.
The essential factor in avoiding cross contamination is to physically separate the pre-PCR and the post-PCR work areas—ideally in two separate buildings. In a routine clinical laboratory this is not practical, but the "golden standard" (Level 3) for a PCR laboratory performing in-house PCR should be considered: four separate rooms (Figure 1) with unidirectional workflow (from laboratory 1 through 4) and unidirectional airflow (from laboratory 1 through 4) if individual airflow cannot be installed.
Each room should be separated from any of the other rooms by at least two doors, and, if possible, a positive air pressure in laboratories 1, 2, and 3 should be maintained. Laboratories 1, 2, and 3 should have a laminar airflow bench – or a safety bench if positive air pressure is maintained. In laboratory 1, no DNA is permitted.
This laboratory is used for production of mastermixes and setup of the individual PCR analysis except addition of sample DNA. Laboratory 2 is used for extraction of clinical samples and in laboratory 3, the nucleic acids extracted from the clinical samples are added to the premade PCR mixes. In laboratory 4, the thermal cyclers are placed, and postamplification procedures such as detection can be performed in this laboratory. Level 2: If the carry-over prevention system is included in the in-house analysis, laboratory 3 can be omitted.
Extraction of the clinical material and addition of the extracted material to the premade PCR mixes are performed in laboratory 2 - preferably in two laminar airflow benches. Level 1: If only commercial PCR kits are used, patient sample extraction and analysis setup (pre-amplification procedures) can be performed in two laminar airflow benches in laboratory 1. The amplification and postamplification procedures are performed in laboratory 4.
Besides the recommendations regarding laboratory design, some general guidelines should also be observed: the use of dedicated pipeting devices in each laboratory, the use of gloves during all laboratory procedures, the use of filtertips in the preamplification areas and the use of containers with Clorox or other DNA-denaturing products for minimizing potential aerosol problems during disposal of pipettips containing DNA.
Furthermore, the use of aliqouted reagents and the use of a low-copy-number positive control (no more than 100 copies) are recommended. Because of the potential problems with the nucleic acid amplification technologies, especially if an in-house analysis is performed, it is essential to ensure that there is a high level of motivation, education, and information with the personnel performing these analyses.
Schematics of a Level 3 PCR Laboratory
(Laboratory 4 is ideally separated from Laboratory 1-3 – e.g. placed in another building or floor)