Simultaneous identification of Fritillariae cirrhosae bulbus and its common adulterants in one reaction by multiplex ligation-dependent probe amplification and high-resolution melting curve assay

• Published in: Gene Vol. 785; p. 145620
• Main Authors: Zhu, Hailan, Wang, Wenbin, Zhou, Yuxin, Wang, Bo, Nie, Jing
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.06.2021
• Subjects: adulterated products; China; cough; cross reaction; detection limit; DNA; DNA Probes; DNA, Plant; Fritillaria - classification; Fritillaria - genetics; Fritillariae cirrhosae bulbus; genes; internal transcribed spacers; medicine; MLPA-HRM; Multiplex Polymerase Chain Reaction - methods; Nucleic Acid Denaturation; prices; Sensitivity and Specificity; Simultaneous identification method; Traditional Chinese medicine; China; HRM; DHPLC; MLPA-HRM; Traditional Chinese medicine; PCR-RFLP; bp; MLPA; ITS1 regions; LHS; NCBI; RHS; Fritillariae cirrhosae bulbus; Simultaneous identification method
• ISSN: 0378-1119; 1879-0038; 1879-0038
• DOI: 10.1016/j.gene.2021.145620

•F. cirrhosae bulbus and its adulterants can be identified with two pairs of probes.•This assay is highly sensitive and specific, rapid, simple, and cost-effective.•As low as 10% adulteration can be detected in F. cirrhosae bulbus in one reaction.•Our method can be used as a prototype for detecting other species for drug safety. Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.