Identification of Fritillaria pallidiflora Using Diagnostic PCR and PCR-RFLP Based on Nuclear Ribosomal DNA Internal Transcribed Spacer Sequences

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Fritillaria pallidiflora Schrenk (Liliaceae) is a commonly used antitussive herb. There are 9 species of Fritillaria recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as F. pallidiflora, and thus, the therapeutic effects of F. pallidiflora are not achieved. Methods to distinguish F. pallidiflora from the 8 other species of Fritillaria are limited by the current morphological and chemical methods. In this study, we report two molecular authentication methods based on the sequences of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) regions. For diagnostic PCR, we designed a pair of species-specific primers to authenticate F. pallidiflora. The PCR program consisted of only two steps for every repeated cycle. For PCR-RFLP, we identified a distinctive site which can be recognized by the restriction endonuclease Eco81I in the nrDNA ITS1 region of F. pallidiflora. PCR-RFLP analysis was established to differentiate F. pallidiflora from the other species of Fritillaria. These methods provide effective and accurate identification of F. pallidiflora.

Fritillaria pallidiflora Schrenk (Liliaceae) is an antitussive herb widely used in China. F. pallidiflora is one of nine species that come from the genus Fritillaria L. and can be used as Bulbus fritillariae (BF) (Chinese Pharmacopoeia, 2000). Data show that F. pallidiflora has a stronger antitussive effect compared to the other species of BF [1] due to its higher alkaloid concentration [2]. Moreover, safety of herbal medications is an important issue [3]. It has been shown that F. pallidiflora has the least toxic alkaloids compared to all other Fritillaria species [1]. We have previously conducted some investigations on F. pallidiflora with different morphological and chemical methods [4]. These methods, however, lacked sensitivity because of the similarities between different species of BF. In this study, we have developed two identification methods based on the sequences of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region: fast polymerase chain reaction (PCR) amplification with species-specific primers and a PCR-restriction fragment-length polymorphism (PCR-RFLP) procedure.

The sequences of nrDNA in the ITS regions of nine species from the genus Fritillaria L. were determined using an ABI 3700 Sequencer (ABI, Italy). The fragment length of the ITS region of these nine species amounts to between 608 - 632 bases. F. pallidiflora had a 628 base sequence, and the percentage of G + C was 65.9 %. The sequences were aligned with Clustal X1.5. and analyzed with Mega 2.0. The genetic distance in F. pallidiflora compared with other species from BF was 2.9 - 3.4 %. The species-specific primers for the identification of F. pallidiflora are shown in Fig. [1]. The 3′ end of the sense primer FpdP₃ (5′-acc gtg ttc acg att gcc tca gg-3′) is in a gap similar to that of the seven other species and has three different bases compared with F. ussuriensis. The anti-sense primer FpdP2 (5′-tcc ggg tct ctt gag ccc ctt g-3) has just one special base in the 3′ end. With these species-specific primers, using a template of F. pallidiflora, the length of the PCR products was 495 bp.

Before diagnostic PCR amplification, the quality of total plant DNA must be tested [5]. The universal outer primers (ITS-P1, ITS-P2) amplified the 3′ end of 18S nrDNA and the 5′ end of 26S nrDNA, including the ITS1, 5.8S and ITS2 regions from the nine species of the BF genus. Each total DNA template from BF got a single amplification fragment with the basic PCR program (Fig. [2] A upper part).

During the process of increasing the annealing temperature, F. pallidiflora showed a bright amplification fragment even at an annealing temperature of 72 °C. We developed a quick PCR program (for diagnostic PCR) so that every repeated cycle included only two steps: 30 sec of denaturing at 95 °C and 30 sec of extension at 72 °C for identification of F. pallidiflora (Fig. [2] A lower part). Although the species-specific primer PCR authentication method has been used for Dendrobium candidum [6], and Fritillaria cirrhosa [7], in our present study, the diagnostic PCR required only 43 min while the normal PCR process takes 1 - 2 h. The quick PCR program was used during the process of identification.

For the analysis of restriction maps [8] in the nrDNA ITS regions among the nine species of BF, the nrDNA ITS sequence from F. pallidiflora had one restricted site (CC*TNAGG) of endonucleases Eco81I. The other eight species did not have this restricted site. The PCR products (with primer ITS-P1 and ITS-P2) were digested with Eco81I and only F. pallidiflora could be cleaved into two fragments of 146 bp and 573 bp (Fig. [2] B upper part). Because the length of the longer digested fragments (573 bp) was near that of the PCR product of F. pallidiflora (719 bp), the two DNA bends were so close that the DNA fingerprinting of RFLP was not clear. Since the restricted site of F. pallidiflora was in the ITS1 region, we amplified the nrDNA ITS1 regions with the primers ITS-P1 and ITS-P3, and digested PCR products of ITS1 regions from BF with endonucleases Eco81I. The RFLP fingerprinting showed that the amplified ITS1 fragment of F. pallidiflora was digested to 146 bp and 179 bp while the PCR product was a 325 bp band. PCR products from the other eight species could not be digested by endonucleases Eco81I. Identification was easy with the RFLP fingerprinting and this modified protocol (Fig. [2] B lower part). The fragments of 719 bp and 325 bp existed with two other digested fragments due to partial digestion of the PCR products of the ITS and ITS1 regions from F. pallidiflora (Fig. [2] B). We have tested several reaction conditions such as increased quantities of enzymes and prolonged reaction time, incomplete digestion is most likely a result of hybridization between related species. However, partial digestion did not influence the identification of F. pallidiflora because the PCR products from other species cannot be digested at all.

We tested four commercially prepared crude drugs purchased in the market. Three samples were recognized as F. pallidiflora, and one sample was not F. pallidiflora. The two identification methods produced the same results. It confirmed that both the methods can solve the problem of the authentication of F. pallidiflora. Each method has distinct aspects. Diagnostic PCR is faster and less expensive. PCR-RFLP requires only one distinctive site. Diagnostic PCR needs at least two species-specific sites in target sequences. The PCR-RFLP method can be used to verify commercial drugs that have been stored for a long time because our PCR fragment was only 325 bp long while the length of the PCR product is 719 bp with the first step of diagnostic PCR. This aspect makes the positive PCR amplification easier for the more hydrolyzed total DNA.

Materials and Methods

F. pallidiflora Schrenk (pa, No. F2001038), F. ussuriensis Maxim. (us, No. F2001051), F. cirrhosa D. Don (ci, No. F2001041), F. unibracteata Hsiao et K. C. Hsia. (un, No. F2001031), F. przewalskii Maxim. ex Batal (pr, No. F2001048), F. delavayi Franch. (de, No. F2001029), F. thunbergii Miq. (th, No. F2001042), F. hupehensis Hsiao et K. C. Xia (hu, No. F2001043), F. puqiensis G. D. Yu et G. Y. Chen (pu, No. F2001045), F. walujewii Regel (wa, No. F2001040) were collected in China. The plant specimens in this study were identified by the authors, and voucher specimens were deposited in the University Herbarium at the China Pharmaceutical University, Nanjing, China.

Total DNA was extracted from 100 - 200 mg of dried bulbs or leaves using cetyltrimethylammonium bromide (CTAB) as previously described [9]. The total DNA was dissolved in TE buffer and was stored at 4 °C for PCR.

For the PCR the reaction mixture, 30 μL, was composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1 % Triton X-100, 2 mM MgCl₂, 200 μM of each dNTP, 15 pmol of each primer, 1 U of Taq DNA polymerase (Promega), and 10 to 50 ng of template DNA. Amplification was carried out in an Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany). For the whole ITS region amplification, the primers of ITS-P1: (5′-CGT AAC AAG GTT TCC GTA GGT GAA-3′) and ITS-P2: (5′-TTA TTG ATA TGC TTA AAC TCA GCG GG-3′) were used, which are based on rice rDNA sequences [10]. The basic PCR program included predenaturing of 3 min at 95 °C, 30 cycles of 30 sec at 95 °C, 30 sec at 53 °C, and 30 sec at 72 °C, followed by one cycle of 2 min at 72 °C. Sequences were determined by using PCR products with the two primers (ITS-P1, ITS-P2). Automated DNA sequencing was run on an ABI 3700 Sequencer (ABI, Italy) by United Gene Holdings, Ltd. (Shanghai, China).

The species-specific primer was designed based on the nrDNA ITS region of BF with the software Primer-Premier 5.0 (Palo Alto, Canada). The DNA templates which got positive amplification with primers ITS-P1 and ITS-P2 recognized quality DNA for the later identification process. For the diagnostic studies of the PCR condition, one factor in the basic program of PCR, the annealing temperature, was increased step by step. After settling, nine species of BF were identified with this method.

Restriction maps of the ITS sequences were done using the software Primer-Premier 5.0. From the restriction maps, Eco81I was selected as a suitable candidate for discrimination of F. pallidiflora and other species of BF. Digestions with Eco81I recommended by the manufacturer; incubation was at 37 °C for 4 hours. The DNAs were fractionated by a 2 % agarose gel electrophoresis and visualized by ethidium bromide (EB) staining under ultraviolet light. Method modification was carried out with the PCR products amplified using primers of ITS-P1 (described earlier) and ITS-P3 (5′-GCT ACG TTC TTC ATC GAT-3′, which was designed according to the nrDNA 5.8S homologous sequences of sibling species). Nine species of BF were analyzed with the PCR-RFLP method.

Acknowledgements

This work was supported in part by grants from the National Science Foundation of China (30 070 886), Trans-Century Talents Training Program Foundation of the Ministry of Education, P.R.C. (No. ETF[2002]48), and Excellent Youth Teacher Foundation of the Ministry of Education, P.R.C (No. EPD[2001]39).

References

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Prof. Ping Li, Ph. D.

Department of Pharmacognosy

School of Traditional Chinese Medicine

China Pharmaceutical University

1 Shennong Road

Nanjing

Jiangsu Province 210038

People’s Republic of China

Phone: +86-25-539-1244

Fax: +86-25-324-2299

Email: lipingli@ public1.ptt.js.cn

References

• 1 Chan S W, Kwan Y W, Lin G, Ho Y P, Li P. The effects of Fritillaria alkaloids on rat isolated trachea and bronchi.  Pharm Sci. 1998;  1 365-9
  PubMedSearch in Google Scholar
• 2 Wang C Z, Sun J, Li P. Research on determination of alkaloid and gluco-alkaloid in Bulbus fritillariae.  Chinese Pharm J. 2003;  51 415-8
  PubMedSearch in Google Scholar
• 3 Mehendale S R, Bauer B A, Yuan C S. Ephedra-containing dietary supplements in the US versus Ephedra as a Chinese medicine.  Am J Chin Med. 2004;  32 1-10
  CrossrefPubMedSearch in Google Scholar
• 4 Li P, Xu G J. Studies on resources of Chinese drugs Beimu.  J Plant Resour Environ. 1993;  2 12-7
  PubMedSearch in Google Scholar
• 5 Matsuki T, Watanabe K, Tanaka R. Genus- and species-specific PCR primers for the detection and identification of bifidobacteria.  Curr Issues Intest Microbiol. 2003;  4 61-9
  PubMedSearch in Google Scholar
• 6 Zhang M, Huang H R, Liao S M, Gao J Y. Cluster analysis of Dendrobium by RAPD and design of specific primer for Dendrobium candidum .  China Journal of Chinese Materia Medica. 2001;  26 442-7
  PubMedSearch in Google Scholar
• 7 Li Y F, Li Y X, Lin J, Xu Y, Yan F, Tang L, Chen F. Identification of bulb from Fritillaria cirrhosa by PCR with specific primers.  Planta Med. 2003;  69 186-8
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Espinosa J C, Fernandez-Gonzalez M, Ubeda J, Briones A. Identification of wine yeasts by PCR-RFLP without previous isolation on plate.  Food Technol Biotechnol. 2002;  42 157-60
  PubMedSearch in Google Scholar
• 9 Pirttilä A M, Hirsikorpi M, Kämäräinen T, Jaakola L, Hohtola A. DNA isolation methods for medicinal and aromatic plants.  Plant Mol Biol Rep. 2001;  19 273a-f
  PubMedSearch in Google Scholar
• 10 Takaiwa F, Oono K, Sugiura M. Nucleotide sequence of the 17S-25S spacer region from rice rDNA.  Plant Mol Biol. 1985;  4 355-64
  PubMedSearch in Google Scholar

Prof. Ping Li, Ph. D.

Department of Pharmacognosy

School of Traditional Chinese Medicine

China Pharmaceutical University

1 Shennong Road

Nanjing

Jiangsu Province 210038

People’s Republic of China

Phone: +86-25-539-1244

Fax: +86-25-324-2299

Email: lipingli@ public1.ptt.js.cn