Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing

- A novel multiplex qualitative detection method using pyrosequencing is proposed.
- One sequencing primer was used in the multiple pyrosequencing employing UP-M-PCR.
- The multiplex qualitative detection was realized based on the unique base peak during pyrosequencing.
- This method could realize multiplex qualitative and semi-quantitative detection of target genes.

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events.