Reply to Arlinghaus et al (second letter)

Abstract

We thank Dr Arlinghaus and his co-authors for their statement and the included further information regarding their approach for negative controls. Following the question how to increase the positive detection rate they suggest that nested PCR increases the sample size by a factor of two. However, it is clear that a positive nested PCR result can only be achieved if at least a single target molecule is present within the first round reaction tube. Repeating the test in terms of the additional cycles in nested PCR contributes to the increased sensitivity of nested vs single PCR as end point measurements. In contrast and as we have stated before, real-time PCR uses a very different system to define and control the detection level, thus precluding any additional benefit of higher cycling under optimized conditions. Highest accuracy with a limited number of replicates is the aim for both strategies, taqman and nested PCR. However, quantitative accuracy should be distinguished from a qualitative scoring term like ’sensitivity’ as it depends on the defined cutoff level. We asked whether the Poisson distribution can be imitated experimentally and if so, the lack of deviations from Poisson serves as a marker for quantitative accuracy at very low copy numbers. For this purpose, we performed the experiments in eight-fold replication to obtain reproducible results. To illustrate the underlying logic: it can be reasoned that a less sensitive method would not have been able to reproducibly detect the templates that were present according to Poisson. If, for instance, the expected distribution was 0, 0, 0, 1, 1, 1, 2, 3 (expected value 1), a less sensitive method might have detected 0, 0, 0, 0, 0, 1, 2, 3 and a less precise method might have resulted in 0, 0, 0, 2, 2, 2, 4, 6. In our experiments, real-time PCR showed neither of these aberrations. With respect to practical terms, it was never intended to recommend the routine use of eight replicates in a diagnostic setting. Without disregarding the importance of the data presented by Guo et al and their preference to competitive nested PCR, recent publications in this journal underscore the potential of quantitive real-time PCR and the need to optimize this strategy.