DNA based identification of medicinal materials in Chinese patent medicines

Abstract

Chinese patent medicines (CPM) are highly processed and easy to use Traditional Chinese Medicine (TCM). The market for CPM in China alone is tens of billions US dollars annually and some of the CPM are also used as dietary supplements for health augmentation in the western countries. But concerns continue to be raised about the legality, safety and efficacy of many popular CPM. Here we report a pioneer work of applying molecular biotechnology to the identification of CPM, particularly well refined oral liquids and injections. What's more, this PCR based method can also be developed to an easy to use and cost-effective visual chip by taking advantage of G-quadruplex based Hybridization Chain Reaction. This study demonstrates that DNA identification of specific Medicinal materials is an efficient and cost-effective way to audit highly processed CPM and will assist in monitoring their quality and legality.

Figure 1

(a) Agarose gel electrophoresis of PCR results from ginseng samples. Lane 1–6: negative control (water was used as sample), tablet, pill, oral liquid, injection, positive control (DNA extracted from reference medical material is used as sample). Tablet and pill samples are subjected to one-step simple PCR while oral liquid and injection samples are subjected to nested PCR. (b) Research on the specificity of PCR amplification. In the left four lanes ginseng primers are used. Lane 1–4: negative control (water was used as sample), ginseng oral liquid, codonopsis oral liquid, mixture of ginseng and codonopsis oral liquid. In the right four lanes codonopsis primers are used. Lane 5–8: negative control (water was used as sample), ginseng oral liquid, codonopsis oral liquid, mixture of ginseng and codonopsis oral liquid.

Figure 2. Agarose gel electrophoresis of PCR results from sample a and b.

Lane 1–3: isolation negative control (water is used as sample when extracted DNA), sample, positive control (DNA extracted from P. ternata is used as template), 4–6: isolation negative control (water is used as sample when extracted DNA), sample, positive control (DNA extracted from P. pedatisecta is used as template), 7–9: isolation negative control (water is used as sample when extracted DNA), sample, positive control (DNA extracted from T. flagelliforme is used as template).

Figure 3. The principle of the chip-basd detection strategy.

Step 1: a 5′-phosphorylated primer was used in PCR to get duplex product with all antisense sequences containing phosphate groups at 5′ ends. Step 2: the 5′-phosphorylated stand of duplex DNA was selectively digested by Lambda Exonuclease. Step 3: the ssDNA initiates the two probes open and polymerize to DNA polymers bearing a lot of G-quadruplexes. Step 4: the G-quadruplex bind to hemin and catalyze the oxidization of ABTS to a green product in the presence of H₂O₂. Sequences with same color are the same or complementary to each other.

Figure 4

(a) Agarose gel electrophoresis of G-quadruplex integrated hybridization chain reactions (GQ-HCR) which were initiate by the digested PCR product from different kinds of ginseng CPM samples.Lane 1–5: negative control (water is used as sample), tablet, pill, oral liquid, injection. (b) Research on the reproducibility of color reaction: the ginseng DNA samples were subjected to three parallel detections (the samples on the same column are the same kind of CPM). All the circles on the chip were pre-printed with special probes for gensing. From 1–5: digested PCR products from negative control (water is used as sample), tablet, pill, oral liquid, injection samples. (c) Research on the specificity of chip-based detection: the two lines were pre-printed with ginseng probes and Codonopsis probes respectively. From 1–4: digested PCR products from negative control (water is used as sample), ginseng oral liquid, Codonopsis oral liquid, mixture of ginseng and Codonopsis oral liquid were added.