Abstract
Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.
Highlights
And traditionally, earthworms are widely used as medicine for treatment of various diseases with its many medicinal effects in Asia [1,2,3,4,5,6,7]
Traditional methods of morphology, microscopy, and chemistry are reliable in the identification of fresh medicinal materials, but medicinal herbal products are usually sold in heavily processed forms
loop-mediated isothermal amplification (LAMP) method has changed our view on the complexity of identification procedures of herbal medicines
Summary
And traditionally, earthworms are widely used as medicine for treatment of various diseases with its many medicinal effects in Asia [1,2,3,4,5,6,7]. Guang-dilong (Pheretima aspergillum), commonly known as the best quality, is one of species of earthworms that is recorded by the Chinese Pharmacopoeia. These species (Pheretima aspergillum, Eisenia foetida, Amynthas obscuritoporus, Pheretima guillelmi, and Metaphire magna) have similar appearance (Figure 1), they do not have the same medicinal value as Guang-dilong, which is the main problem effecting the quality of herbal medicine. With the price of commercial Guang-dilong growing rapidly, many different species and a wide range of sources sprang up in herbal markets, it increases the difficulty for consumers to quickly differentiate amongst them. DNA level identification is needed to differentiate between Guang-dilong and other species
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