We therefore assume that within the range 1-150 target seed content per lot of 3000 seeds, randomly distributed in 20 aliquots, there will be an aliquot containing exactly one target seed or/and a pair of aliquots differing by exactly one target seed (with the probability not less than 0.95). The minimum increment is also exactly one seed. Seed lot is a discrete system, which means that the target seed content (in the seed count mode) cannot be fractional and the minimum possible content being exactly one seed.
Thus, we assume it is possible to make exact quantitative analysis once the exact number of target seeds in at least one aliquot or exact increment between a pair of aliquots is known. However, real-time PCR provides exact figures for threshold cycle as the well-established function of the concentration of the tested object. The lot is then randomly distributed into 20 aliquots (subsamples) and the presence of target seeds is estimated basing on the number of positive aliquots (aliquots that gave positive result for the presence of target seeds). One of SeedCalc testing plans implies that a seed lot (sample) of 3000 seeds including unknown number of target seeds (seeds, that are subject to detection and differ in detected traits from the main bulk).
ISTA (International Seed Testing Association) has developed a statistical tool (SeedCalc) 3 using to assess seed purity/impurity, including level of GMO presence in conventional seed lots with qualitative testing plans using a Bayesian approach, so the results are statistical approximation. Therefore, a method providing for exact quantitation of seed traits without using reference standards can be highly demanded. However, certified reference materials may be quite expensive and not easily available, if available at all. 1 There are recent developments on using digital PCR with direct DNA copy counting, 2 it also requires CRM though.
The common and globally approved method of quantitative analysis of biotechnology traits/genetically modified organisms (GMO) is PCR in real time mode when the value of threshold cycle (C t) of tested sample is compared with C t values obtained through sequential dilution of the corresponding certified (standard) reference material (CRM) presented in the form of linear regression in semi-logarithmic co-ordinates C t vs log.
Keywords: Biotechnology seeds, seed testing, quantitative analysis, hypergeometric distribution, statistical algorythm, real-time PCR, certified reference materials, self-reference Abbreviations The approach can be of significant value in the issues of adventitious/technically unavoidable presence (AP), labeling, low level presence (LLP) and thus significantly simplify and decrease the cost of seeds breeding and trade. The approach may be applied not to PCR techniques only but to seed testing with whatever physical method provide the observed signal is obtained in exact values and the correlation between the signal and concentration of traced seeds is known. The approach has been proven theoretically and supported experimentally with the analysis of the presence of seeds of soybean lines A2704-12, A5547-127 and GTS 40-3-2 in seed lots with the use of PCR in real-time mode tracing the transformation events. An approach to the exact quantitative analysis of seed traits without using reference material is proposed basing on the quantum (discreet) nature of seed distribution is seed lots.
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