Authentication of Bulbus Fritillariae Cirrhosae by RAPD-derived DNA markers

Abstract

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

Figure 1

RAPD profiles of Fritillaria species amplified with primer S158. The specific primers revealed species-specific patterns for Fritillaria species of Bulbus Fritillariae Cirrhosae. M: 1 kb DNA ladder; B: Blank (nuclease-free distilled water) Lane 1–11: species of Bulbus Fritillariae Cirrhosae: Fritillaria cirrhosa (1–2), F. unibracteata (3–4), F. przewalskii (5–6), F. delavayi (7–8), F. taipaiensis (9–10), and F. unibracteata Hsia et K. C. Hsia var. wabuensis (11) Lane 12–17: Other Fritillaria species: F. hupehensis (12), F. ussuriensis (13), F. thunbergii (14), F. pallidiflora (15), F. walujewii (16), and F. puqiensis (17).

Figure 2

Nucleotide sequence of the RAPD amplicon CBM08; the RAPD primers (a pair of S158) and SCAR primers (CBM-S and CBM-AS) were indicated.

Figure 3

(A) Schematic diagram of RAPD and SCAR amplifying region. (B) PCR amplification of Fritillariae species using SCAR (CBM-S and CBM-AS) primers. The specific band (231 bp) was only detected in species of Bulbus Fritillariae Cirrhosae. M: 1kb DNA ladder; B: Blank (nuclease-free distilled water). Lane 1-11: species of Bulbus Fritillariae Cirrhosae: Fritillaria cirrhosa (1–2), F. unibracteata (3–4), F. przewalskii (5–6), F. delavayi (7–8), F. taipaiensis (9–10), and F. unibracteata Hsia et K. C. Hsia var. wabuensis (11). Lanes 12–17: Other Fritillaria species: F. hupehensis (12), F. ussuriensis (13), F. thunbergii (14), F. pallidiflora (15), F. walujewii (16), and F. Puqiensis (17).

Figure 4

(A) PCR amplification of nine commercially available crude drugs of Bulbus Fritillariae Cirrhosae using SCAR marker amplification (CBM-S and CBM-AS); A specific band (231 bp) for Bulbus Fritillariae Cirrhosae was detected in samples 1–5. (B) PCR-RFLP analysis of Bulbus Fritillariae Cirrhosae validated results obtained using the SCAR marker. The detail protocol was displayed in part 3.8. The five samples generated two fragments between 100–200 bp after being digested by SmaI, confirming that they were Bulbus Fritillariae Cirrhosae (B).