Direct quadruplex real-time VPCR for simultaneous rapid detection of Pinelliae Rhizoma, Rhizoma Pinelliae Pedatisectae and Rhizoma Typhonii Flagelliformis

Known for its health benefits, Pinelliae Rhizoma is frequently utilized in many botanical dietary supplements and herbal products. However, the substantial demand has led to the frequent adulteration with morphologically similar species, such as Rhizoma Pinelliae Pedatisectae and Rhizoma Typhonii Flagelliformis . Addressing this challenge, a direct quadruplex real-time VPCR approach has been developed in this study. Firstly, a direct lysis protocol has been implemented to facilitate the rapid release of genomic DNA within only 2 min. Secondly, specific primers and probes were designed for Pinellia ternata (Thunb.) Breit., Pinellia pedatisecta Schott and Typhonium flagelliforme (Lodd.) Blume, respectively. A conserved sequence on the ITS was also included as an internal control to mitigate the potential false negatives arising from DNA extraction or DNA amplification errors. Furthermore, the amplification process has been refined through a VPCR heating model, curtailing the detection to a scant 30 min. After a series of optimizations, the developed direct quadruplex VPCR allows for the simultaneous detection of these three plant materials, with each material having a detection sensitivity as low as 1 pg/µL. At last, the method's applicability was validated by using several commercial samples previously identified by Sanger sequencing. • Developed a quadruplex real-time VPCR for Pinelliae Rhizoma and its two adulterants. • A conserved sequence is chosen as an internal control to avoid false negatives. • The use of VPCR technology allows the assay to be completed in less than 30 min. • Method achieves high repeatability and sensitivity, down to 1 pg/µL. • Validated using 48 different samples to confirm accuracy and reliability.