Quality Standards in quantitative PCR; Specification, Validation, Controls and Standards

Abstract
Introduction: The implementation of molecular methods such as real-time PCR for food pathogen detection is desired and reasonable. Nevertheless the obstacles of precise specification and meaningful validation are not yet overcome and therefore broad range use in food testing is not yet accomplished. Specification is generally based on the determination of the detection limit, the overall efficiency of the reaction and exclusivity and inclusiveness of the assay respectively. These parameters do not provide sufficient information about the real performance of the underlying enzymatic reaction. The validation according to ISO 16140, the validation of alternative methods, has many drawbacks based on its original sense and purpose, the comparison of microbiological methods. Evaluation of real-time PCR is therefore not significant with this process due to the different nature of molecular biological methods.
Purpose: Establishment of a significant specification and validation approach in consideration of the inherent qualities of real-time PCR. Methods: A validation system including testing algorithms derived from software engineering; per se specification of the enzymatic reaction, controls covering all necessary steps and the investigation of surrounding parameters was designed. The whole approach is based on fundamental principles of systems theory and cybernetics. This alternative strategy includes every necessary detail thus leading to a maximum performance of the assay and most precise specification and validation of the whole analytical chain. Results: We present the practical application of this new approach by example of an analytical chain for the detection of L. monocytogenes, including sample preparation, DNA isolation/purification and real-time PCR detection.
Significance: New approaches for the significant specification and validation of molecular biological methods are necessary to gain confidence in such methods and furthermore support widespread implementation. The whole system approach presented herein is an equivalent attempt, which effectively supports the standard validation method ISO 16140.