Rapid and simultaneous identification of Lonicera Japonicae Flos and Lonicerae Flos using MLPA-HRM and rhPCR-HRM

Abstract

Background

Lonicera Japonicae Flos (LJF) and Lonicerae Flos (LF), derived from different species of Lonicera, are two different types of traditional Chinese medicine (TCM) according to their differences in composition and effectiveness. Because of the high price, good effectiveness of the medicinal, and functional values, LJF is often adulterated by LF in the market. Therefore, two single nucleotide polymorphism-based genotyping methods, multiplex ligation-dependent probe amplification high-resolution melting (MLPA-HRM) and RNase H2-dependant PCR (rhPCR)-HRM, were established for the identification of LF and LJF.

Results

Highly specific MLPA probes and rhPCR-primers were designed based on the trnL-trnF regions. The melting temperature (T_m) of amplicons was derived from the peak of the HRM curve, and the differences in T_m values were used to identify LJF and LF. The two methods have both shown good specificity for identifying LJF and LF without cross-reaction, and have high sensitivity with the detection limits of 0.1 ng DNA template. For mixed samples, MLPA-HRM could detect LJF mixed with 10% LF while rhPCR-HRM could detect 5% LF in mixed samples. The MLPA-HRM and rhPCR-HRM were performed on DNA extracted from 54 samples of LJF and LF randomly selected from the medicinal material market, all samples were successfully identified and further verified with DNA barcoding.

Conclusion

MLPA-HRM and rhPCR-HRM methods developed in our study not only provided fast, simple, sensitive, and practical identification of LJF and LF, but also provided technical references for the adulteration identification of other herb-medicines, they are of great significance to the quality control and safe use of plant medicinal materials.