Establishing a TaqMan qPCR method for identification and quantification of Fritillaria hupehensis as a common adulterant of Fritillaria thunbergii Miq

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Abstract
Background: Fritillaria hupehensis (HBBM) is frequently found as an adulterant in Fritillaria thunbergii Miq. (ZBM) owing to its similar appearance and lower price, resulting in risk of unstable medicinal effects of ZBM. Methods: A highly specific TaqMan real-time quantitative polymerase chain reaction (qPCR) method was established to identify HBBM from ZBM based on the 47 bp amplicon located on the internal transcribed spacer region of the nuclear ribosomal DNA. Results: The qPCR assay developed herein could specifically distinguish ZBM from HBBM. The sensitivity study showed that the detectable DNA template concentration of HBBM for this assay was 0.001 ng/μL. The efficiency of the optimized qPCR assay was evaluated to be 90.2% (R 2 = 0.9990, slope = −3.581). A standard-curve-based quantification method was established to determine the proportion of HBBM adulteration in a binary mixture with ZBM, and the equation of the standard curve was determined as y = −3.192x + 18.316 with R 2 = 0.9949. This quantification approach was evaluated using the ΔCt parameter (difference in Ct values between sample and reference standard); furthermore, the test accuracy was calculated using the recovery rate that ranged from 81.60% to 113.85% for mixed powder samples containing different proportions of HBBM (1%, 5%, 25%, 50%, and 100%). The short amplicon of length 47 bp ensured efficient detection and accurate quantification of dried decoction pieces. Conclusion: The TaqMan real-time qPCR assay developed in this study has high efficiency, sensitivity, and specificity; this method is applicable for not only authentication of ZBM and HBBM but also quantitative detection of a binary mixture or adulterated botanical drugs. Furthermore, it provides a reference for qualitative control of other traditional Chinese medicines.

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