In Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR) the writers present the procedure to experimentally determine LoD (Limit of Detection) based on sample replicates and also a novel method to estimate the confidence of the LoD.
The study is based on standard statistical methods recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of the limit of detection.
There is no general guidance specifying acceptable threshold value of LoQ. At TATAA Biocenter, unless we have other guidance, we specify LoQ as the lowest concentration where replicates show a CV ≤ 35% on back calculated concentrations.
Abstract (from Biomolecular Detection and Quantification):
Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.
