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To establish a specific polymerase chain reaction(PCR) method based on SNP loci for molecular identification of Eupolyphaga Steleophaga formula granules and its counterfeit.
By analyzing the cytochrome oxidase Ⅰ (COⅠ) sequences of Eupolyphaga Steleophaga and its common forgeries，the SNP mutation sites of Eupolyphaga Steleophaga were searched and specifi cprimers were designed，which were TBC-F(5’-TTCTTGTTGGCAAGCAGTATAAT-3’) and TBC-R(5’-AACTACTGCTCAAACAAATAATGGA-3’). Three-step method was used to amplify specific polymerase chain reaction (PCR) and the optimal PCR reaction system was determined by optimizing the PCR reaction procedure. At the same time，in order to ensure the accuracy of the test results，the PCR amplified products were conducted by generation sequencing.
The PCR method with the annealing temperature of 55-57 ℃ and 37 cycles produced a single band at 237 bp for 21 batches of Eupolyphaga Steleophaga，20 batches of standard decoction and 19 batches of its dispensing granules，while it produced no band for the adulterant or the negative control. The experimental result was consistent with the result of Sanger sequencing，which was Eupolyphaga sinensis.
The established specific PCR method can accurately identify the medicinal materials，and standard decoction freeze-dried powder of Eupolyphaga Steleophaga，as well as final products of dispensing granules. It provides a reference for research on the quality standards of Eupolyphaga Steleophaga dispensing granules.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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