Application of intergenic sequence between ND5 and ND6 as novel target for authentication and quantification of Oviductus Ranae

Abstract

Oviductus Ranae (OR), dried oviducts from Rana dybowskii (RD), is a valuable food and medicine included by Chinese pharmacopeia, mainly produced in Changbai Mountain area of China. Economically motivated adulteration with oviducts from unclear species has become one of the most concerned food safety issues. Existing methods based on DNA sequencing are cost and time consuming, making them unfit for rapid authentication and quantification. Here, we developed a PCR/RT-PCR method targeting a RD specific intergenic sequence region termed IGS56 in mtDNA (Genbank NC_023528.1, 17059–17154 bp). Common primers in RD close species (F1-ND5/R1-ND6), RD specific primers (F2-IGS56/R2-ND6) and universal primers (F0–18 s/R0–18 s) were used for authentication by PCR, while RD specific primers (F3-IGS56-RT/R3-ND6-RT) and reference primers (F0–18 s-RT/R0–18 s-RT) were used for quantification by RT-PCR. For authentication, standard OR sample together with 19 batches OR samples obtained from different sites of Changbai mountain area were all identified to be RD. For RT-PCR based quantification, calibration curve with a good linear correlation (R2 =0.9915), was plotted with Ct ratio (Specificity Ct /Reference Ct) and OR percentage mixed by oviducts of R.nigromaculata. The accuracy of RT-PCR method established was verified to be high, with an average recovery rate of 106.744%.