Multiplex pyrosequencing quantitative detection combined with universal primer-multiplex-PCR for genetically modified organisms.

Abstract A multiplex pyrosequencing quantitative detection technique combined with universal primer-multiplex-PCR (UP-M-PCR) was established. In this study, a pyrosequencing results analysis software was first self-compiled, which realized the DNA sequences degeneration, and converted the pyrosequencing results and base composition of the target sequences into mathematic relations. Five calculation models were put forward based on the actual situation, which adjusted the values smaller than zero or the detection limit. By applying this method, samples containing five genetically modified (GM) lines mixed in random ratio were quantified, it showed that the quantification was very close to the actual value, and the detection sensitivity was as low as 1.47% of a single component, which satisfied most labeling policies. This novel method is realized without fluorescent group labeling, hence the number of targets is not limited by factors inherent in method or equipment, and is proven to be a reliable tool for the quantitative detection.