Identification and Monitoring of Amomi Fructus and its Adulterants Based on DNA Barcoding Analysis and Designed DNA Markers

Abstract

Amomi Fructus is one of the traditional medicines derived from the ripe fruits of the Zingiberaceae family of plants, which include Amomum villosum, A. villosum var. xanthioides, and A. longiligulare. Owing to their highly similar morphological traits, several kinds of adulterants of Amomi Fructus have been reported. Therefore, accurate and reliable methods of identification are necessary in order to ensure drug safety and quality. We performed DNA barcoding using five regions (ITS, matK, rbcL, rpoB, and trnL-F intergenic spacer) of 23 Amomi Fructus samples and 22 adulterants. We designed specific DNA markers for Amomi Fructus based on the single nucleotide polymorphisms (SNPs) in the ITS. Amomi Fructus was well separated from the adulterants and was classified with the species of origin based on the detected SNPs from the DNA barcoding results. The AVF1/ISR DNA marker for A. villosum produced a 270 bases amplified product, while the ALF1/ISF DNA marker produced a 350 bases product specific for A. longiligulare. Using these DNA markers, the monitoring of commercially distributed Amomi Fructus was performed, and the monitoring results were confirmed by ITS analysis. This method identified samples that were from incorrect origins, and a new species of adulterant was also identified. These results confirmed the accuracy and efficiency of the designed DNA markers; this method may be used as an efficient tool for the identification and verification of Amomi Fructus.

Keywords: A. longiligulare; Amomi Fructus; Amomum villosum; Amomum villosum var. xanthioide; DNA barcode; DNA marker; adulterants; multiplex PCR.

Figure 1

Multiple alignments of the 32 single nucleotide polymorphisms (SNPs) from five regions (ITS, matK, rbcL, rpoB, trnL-F intergenic spacer) in Amomi Fructus. Numbers above the bases indicate the position of single-nucleotide polymorphisms in each region. The dots indicate the consensus nucleotide; Sample code shown in Table 1. Heterozygous sites were defined according to IUPAC. AX01 (A. villosum var. xanthioides): KJ151892 and MH161417; AX02 (A. villosum var. xanthioides): KJ151893 and MN067432.

Figure 2

Phylogenetic analysis of Amomi Fructus and its adulterants based on the nucleotide sequences of internal transcribed spacer (ITS), including 5.8S rDNA region using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. As outgroups, ITS nucleotide sequences of Brassica rapa (MK424344.1) and Oryza sativa (MH744632.1) were used.

Figure 3

Multiplex PCR products of the primer set AVF1/ISR/ISF/ALF1 from randomly chosen samples in Table 1 for distinguishing Amomi Fructus from its adulterants. Lane numbers above: The sample number listed in Table 1. M: 100 bases ladder size marker.

Figure 4

The monitoring results of commercial Amomi Fructus by using multiplex PCR with the designed AVF1/ISR/ISF/ALF1 primer set in this study. Lane numbers above: The sample number as listed in Table 2. M: 100 bases ladder; Box shows different amplified results than expected.

Figure 5

Phylogenetic analysis of Amomi Fructus and its adulterants based on the concatenate nucleotide sequences of four plastid regions (matK, rbcL, rpoB and trnL-F intergenic spacer) using the MrBayes.