Establishing a TaqMan qPCR method for the identification and quantification of Fritillaria Hupehensis, the common adulterants of Fritillaria Thunbergii Miq

Abstract

Background: The Fritillaria Hupehensis(HBBM) is frequently found as the adulterant of Fritillaria thunbergii Miq.(ZBM) due to the similar appearance and the lower price, resulting in the certain risk of unstable medicinal effect of ZBM. Methods: A highly specific TaqMan real-time quantitative polymerase chain reaction (qPCR) method was firstly established to identify HBBM from ZBM with the amplicon of 47bp located on the nrDNA internal transcribed spacer (ITS) region. Results: The constructed qPCR assay could specifically discriminate ZMB from HBBM. The sensitivity study showed that the detectable DNA template concentration of HBBM for this qPCR assay was 0.001ng/μl.The efficiency of the optimized qPCR assay was evaluated as 90.2%(R2=0.9990, slope=-3.581). A standard curve quantification method were established to determining the proportion of HBBM in binary mixture or adulterated botanical drugs, and the standard curve equation was determined to be y = -3.192x + 18.316, R²=0.9949. The quantification is evaluated by the parameter ΔCt (the Ct value difference between samples and reference standards), and the accuracy was evaluated by recovery rate which ranged from 81.60% to 113.85% with mixed powder samples containing HBBM at different proportion (1%,5%,25%,50%,100%) for testing. The short enough amplicon (47bp) assured the efficient detection and the accurate quantification of the dried decoction pieces. Conclusions: The TaqMan real-time qPCR assay we developed was efficient, sensitive and specificity, which could not only be applied to supervise the authenticity of the ZBM and HBBM but also provide a quantitative detection method of binary mixture or adulterated botanical drugs. It also provided a reference to the qualitative quality control of other TCMs.