Methodology Case Study (II)

2.6 Case Study (II)

A new molecular identification method: anchored primer amplification polymorphism DNA

Since the inception of PCR technology, researches on the molecularidentification of Panax ginseng and P. quinquefolius have attracted particular concern.

In 1994, AP-PCR was used for the identification of P. ginseng and P. quinquefolius for the first time [ 48 ] , and there have been 24 related reports presently, including RAPD [ 49 – 53 ] , DNA sequence analysis [ 54 – 56 ] , PCR-RFLP [ 50 , 57 ], AFLP [ 58 ], SCAR [ 59 ], MARMS [ 60 ], repetitive sequence, DALP, minisatellite, and so on [ 56, 61 ]. Constant innovation of these methods lies in the gradual understanding of genomic information of P. ginseng and P. quinquefolius in which, MARMS is highly specific, fast, and accurate, but the primer design must be built on the basis of a large number of known sequences. RAPD is the most widely used method because there is no need to predict genome sequence, and the operation is simple and quick, but RAPD has defects including poor reproducibility, vulnerable to origin, and storage time of medicinal materials, thus restricting its application in the field of molecular identifcation. Therefore, it is important and difficult to explore molecular marker methods which are simple and easy to operate as well as have a good stability and strong operability in the molecular identification of Chinese materia medica. A new method reported in this chapter is based on RAPD method. And innovations were conducted on its two main factors including primers and annealing temperature. The method was named as anchored primer amplification polymorphism DNA (APAPD). First, APAPD method was established taking P. ginseng and P. quinquefolius as examples. Then, a wide range of review was conducted on the stability of its reaction system, and the stability of amplification results of different material. Meanwhile, validation and comparison were conducted combining with MARMS method reported in the literature. On this basis, APAPD method was applied to the identification of Tianhua-fen (Trichosanthes Radix) and Bai-zhi (Angelica Radix), achieving desired results. This indicates that APAPD method is a very promising new method for molecular identi fi cation of Chinese materia medica.

2.6.1 Materials and Methods

Thirty-four samples of P. ginseng , P. quinquefolius , and their adulterants; 28 samples of Tian-hua-fen; and 8 samples of Bai-zhi were collected from different areas of China. All samples were identi fied by the researcher Huang Luqi et al. in the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and they were placed in the herbarium of the institute.

Total DNA of Chinese materia medica was extracted using the modified CTAB method [ 62 ]. Primers were designed according to the existing ITS sequences of P. ginseng, P. quinquefolius (GenBank accession number: AJ786235, AY548192, U41680, U41689, U41688, U41687), Tianhua-fen [ 63 ], and Bai-zhi [ 64 ]. Primers were about 20 bp in length. Following the general principles of primer design, areas which had large differences with adulterant sequences to be identified should be selected. MARMS primers used for the identification of P. ginseng by Shu Zhu et al. were applied for the verification of P. ginseng and P. quinquefolius [ 60 ]. Primers were synthesized by Sangon Biological (Shanghai) Co., Ltd.

The PCR reaction system (25 m L) was as follows: 10 mmol · L −1 Tris–HCl (pH 9.0), 50 mmol · L −1 KCl, Mg 2+ 1.5 mmol · L −1 , dNTP 0.15 mmol · L −1 , Taq E 1U (Invitrogen, Promega, etc.), primer 0.15 m mol · L −1 , and template DNA 50–200 ng. PCR amplification was conducted on AB I9700 ampli fi cation instrument. Primer screening and optimization of PCR conditions were conducted on all APAPD primers first using typical materia medica (usually four samples) to be identified.

Preliminary screening was conducted following RAPD general procedures: predenaturation at 94 °C for 5 min, followed by 40 cycles: denaturation at 94 °C for 45 s, annealing at 37 °C for 1 min, and extension at 72 °C for 1 min 30 s, with a final extension at 72 °C for 5 min after 40 cycles. Annealing temperature was gradually increased in primers which could produce polymorphic bands in quality products and adulterants to eliminate nonspecific bands to determine the optimal reaction PCR parameters. Amplification products were electrophoresed on a 2.0% agarose gel containing EB in 1 × TAE buffer. And observation and photographing were conducted under SYNGENE gel imaging system.

2.6.2 Results

2.6.2.1 Establishment of APAPD Method of P. ginseng and P. quinquefolius

When PCR annealing temperature was 37 °C, the primer Pg-q36F showed good effects on the amplification of P. ginseng and P. quinquefolius, presenting clear bands and significant polymorphic bands. Gradual increase in annealing temperature displayed that primers could amplify at 37–60 °C, but when at 40–50 °C, PCR amplification results were stable, single 849-bp band was amplified from P. ginseng, and 864-bp and 792-bp bands were amplified from P. quinquefolius; the bands gradually blurred at 55–60 °C. To ensure that all sources of materia medica and adulterants could be effectively amplified, PCR conditions were determined as follows: predenaturation at 94 °C for 5 min, followed by 40 cycles: 94 °C 45 s, 40 °C 1 min, and 72 °C 1.5 min, with a final extension at 72 °C for 5 min after 40 cycles.

2.6.2.2 Study on Accuracy of APAPD Method

To test the accuracy of primer Pg-q36F identifying P. ginseng and P. quinquefolius, 11 kinds of adulterants which have been presented on the market were amplified using primer Pg-q36F, respectively. Meanwhile, all P. ginseng and P. quinquefolius samples were verified using MARMS primers PgjqtK1966R, PqtK896F, PgS481F, and P-S712R. Results showed that in primer Pg-q36F, there were only 849-bp band amplified from P. ginseng , 864-bp and 792-bp bands amplified from P. quinquefolius, while no corresponding band presented in all adulterants. In MARMS primers, all P. ginseng presented 649-bp and 249-bp bands, and all P. quinquefolius presented 649-bp band. It indicated that the identification result of primer Pg-q36F was consistent with that of primers in the literature, and quality products could be distinguished from all kinds of adulterants, indicating that primer Pg-q36F could be used as identification primer of P. ginseng and P. quinquefolius .

2.6.2.3 Study on Stability of APAPD Method

In the PCR reaction system, the quality of Taq enzyme was the main factor to affect identifcation results. In the MARMS identi fi cation of P. ginseng and P. quinquefolius, 249-bp band was amplified from both P. ginseng and P. quinquefolius using ordinary Taq polymerase, so they could not be identi fi ed, and correct results could be obtained only using the high-fi delity Taq polymerase. Using primer Pg-q36F, ordinary Taq polymerase of Invitrogen, Promega, and fi ve domestic companies were selected for amplification respectively, and the results obtained from all Taq polymerase were consistent. It indicated that the primer is undemanding in PCR reaction system and ordinary Taq polymerase could meet the requirements, being easy to be reproduced in laboratories.

In the long-term cultivation process of P. ginseng, different farm species such as Da-maya, Er-maya, Huangguo, and changbo were presented [ 63 ]. P. quinquefolius is native to the USA and Canada. Since the successful introduction into China, largescale cultivation has been started in many areas. The prices of P. quinquefolius showed great differences according to its different qualities, for example, 3.80 yuan/g, 1.80 yuan/g, and 0.98 yuan/g of P. quinquefolius were sold in Tong Ren Tang Pharmacy. In addition, a lot of P. quinquefolius were processed into decoction pieces, thereby increasing the difficulty in the identification of P. ginseng and P. quinquefolius .

Therefore, the correct identification of different sources of herbs, such as different areas, different prices, different processing methods, and different storage time, is the first step to ensure the safety of clinical pharmacy. Therefore, in this chapter, four farm species of P. ginseng, medicinal materials, samples and powder of P. ginseng sold in different pharmacies as well as medicinal materials, decoction pieces, and samples of different areas and different prices of P. quinquefolius were selected as experimental materials, with broad representation. PCR amplification was conducted on all samples using primer Pg-q36F. The results showed P. ginseng of different sources steadily amplified 849-bp band, and P. quinquefolius of different sources steadily ampli fi ed 864-bp and 792-bp bands. It indicated that P. ginseng and P. quinquefolius could be identified steadily using the primer.

2.6.2.4 Study on Applicability of APAPD Method in Chinese Material Medica of Tian-hua-fen and Bai-zhi

Among Tian-hua-fen primers TkS1-64 F, TkS2-112 F, and TkS2-130R, TkS1-64 F showed the best amplification effect, manifesting that the polymorphism of quality products and adulterants was obvious, so quality products and adulterants could be accurately identified. PCR cycles were identi fi ed as follows: predenaturation at 94 °C for 5 min, followed by 40 cycles: 94 °C 30 s, 50 °C 45 s, and 72 °C 1 min, with a final extension at 72 °C for 5 min after 40 cycles. By detecting 19 batches of Chinese material medica of Tian-hua-fen of different sources, the 560-bp and 960-bp bands were determined as characteristic identification bands of Tian-hua-fen, while other bands such as 1,930-bp, 1,400-bp, 839-bp, and 715-bp bands could be used as secondary identification bands because they could not steadily reproduce among different PCR reaction systems or material medica from different areas.

Characteristic identification bands of each adulterant were the following:

Trichosanthes hupehensis 686 bp, 800 bp, 938 bp, 1,260 bp; Trichosanthes laueribractea Hayata 686 bp, 800 bp, 938 bp, 1,260 bp; Guizhou Trichosanthes 760 bp, 1,259 bp; Trichosanthes pedata Merr. et Chun 900 bp; Trichosanthes truncata C. B. Clarke 760 bp; Momordica cochinchinensis 770 bp, 1,373 bp; Melothria heterophylla (Lour.) Cogn. 673 bp, 786 bp, 919bp, 1,189 bp; Trichosanthes cucumeroides Maxim 865 bp, 1,296, 2,118 bp, 2,669 bp; and Trichosanthes lepiniana (Naud.) Cogn had no amplification bands.

In Bai-zhi primers AfS1-100 F and AfS1-120R, AfS1-100 F showed obvious amplification polymorphism, Bai-zhi could be clearly distinguished from Angelica porphyrocaulis Nakai et Kitagawa, Angelica dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. dt. Sav, and Angelica amurensis Schischk. PCR cycles were determined as: predenaturation at 94 °C for 5 min, followed by 40 cycles: 94 °C 30 s, 40 °C 45 s, and 72 °C 1 min, with a final extension at 72 °C for 5 min after 40 cycles. By detecting 17 batches of Chinese material medica of Bai-zhi of different sources, 740-bp band was determined as characteristic identification bands of Bai-zhi; 740-bp, 917-bp, and 1 032-bp bands as those of Angelica porphyrocaulis Nakai et Kitagawa; 740-bp and 1 032-bp bands as those of Angelica dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. et. Sav; and 500-bp and 1 032-bp bands as those of Angelica amurensis Schischk. Being different from polymorphic bands of P. ginseng, P. quinquefolius, and Tian-hua-fen, characteristic bands of quality products of Bai-zhi also presented in Angelica porphyrocaulis Nakai et Kitagawa and A. dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. dt. Sav, but A. porphyrocaulis Nakai et Kitagawa increased 740-bp and 917-bp bands compared with Bai-zhi; A. dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. dt. Sav increased 740-bp bands compared with Bai-zhi; the combination of their bands formed characteristic fingerprints that could be used for accurate identification.

2.6.3 Discussion

Tian-hua-fen is an important class of Chinese materia medica. Trichosanthin which can terminate pregnancy and has anti-HIV activity is extracted from the root of Trichosanthes kirilowii Maxim. Studies on Trichosanthes serving as a composite species are known as “the most intractable taxonomic problem in eastern Asia Cucurbitaceae center.” There are up to 28 kinds of commercial herbs of Tian-hua-fen, including 19 species of congeneric plants; some of them are highly toxic [ 63 ]. This study group has identified Tian-hua-fen, Trichosanthes hupehensis, and Momordica cochinchinensis using RAPD technique and protein immunoassay technique [ 65 ], but the results using RAPD method are affected by storage time. Certain difficulties exist in the identification of Tian-hua-fen by applying the method [ 66 ], while primer TkS1-64F can amplify samples from different sources, and DNA fingerprint can be obtained from quality products and eight kinds of adulterants of Tian-hua-fen only using one primer, providing a guarantee for the application of materia medica of Tian-hua-fen.

Existing commercial herbs of Bai-zhi are cultivated and divided into A. anomala (Radix angelicae dahuricae) and A. dahurica (Radix angelicae dahuricae). Because the original source of wild plants has not been really figured out, the specific name of traditional Chinese medicine Bai-zhi has been changed for many times in identification, and there has been no unifiedfinal conclusion. This study group conducted exhaustive research on germplasm resources of Bai-zhi from morphology, chemical composition, ITS sequence analysis, and RAPD to prove that the source of wild germplasm of the traditional Chinese medicine Bai-zhi (including Angelica anomala, Radix angelicae dahuricae, Angelica dahurica, and Radix angelicae dahuricae) is Angelica formosana H. Boiss. only distributed in the southeast region of China (Taiwan Province based) currently. While Angelica formosana H. Boiss., A. dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. et. Sav, and A. porphyrocaulis show a close genetic relationship. Angelica amurensis Schischk is an outgroup in the study. Results of the polymorphism of primer AfS1-100F used in this chapter also support the above conclusion; characteristic identification bands of Angelica formosana de Bioss are consistent with those of 4 kinds of commercial Bai-zhi, both A. dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. et. Sav and A. porphyrocaulis contain identification bands of Bai-zhi but increase 1–2 bands compared with Bai-zhi, while Angelica am urensis Schischk has no characteristic bands which Bai-zhi, A. dahurica (Fisch. ex. Hoffm.) Benth. ex. Franch. dt. Sav and A. porphyrocaulis commonly have, suggesting APAPD method can also be used for the study of genetic relationship among plants.

The identi fi cation of P. ginseng , P. quinquefolius , Tian-hua-fen, and Bai-zhi indicates that APAPD method has the following advantages:

* Simple and easy to operate. Although the primer design is more difficult, ideal identification primers can be obtained by designing 2 or 3 primers, thus avoiding the trouble of screening a large number of random primers

* Good stability and reproducibility. Due to the increased primer length and specificity, there are only 1–5 ampli fi cation bands generally; the origin and storage time of medicinal materials have no effect on PCR results; moreover, APAPD primers are undemanding in PCR reaction conditions, so they are easy to be promoted and reproduced in laboratories

* Large amount of information provided. Both quality products and most of adulterants can be amplified using the method; therefore, standard identification electrophoretogram of quality products and adulterants can be established, respectively, to achieve an accurate identification of quality products and adulterants.

The increasing of APAPD primers will provide standard DNA identification fingerprint for more materia medica and provide a powerful tool for the quality control of Chinese materia medica.