Diagnostic PCR: making internal amplification control mandatory
The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories.
However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes.
It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that end-users and reference laboratories have access to open-formula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available.
The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002b; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD-PCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003).
A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCR-based method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample.
The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002a). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization.
Scientific journals must provide the source of new PCR-based methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.