Objective: To establish a fast and accurate identification method for adulteration of wheat powder in Xiaohuoluo Wan by real-time PCR combined with high-resolution melting curve (HRM) analysis technique. Methods: The genome of the sample of Xiaohuoluo Wan was extracted by optimizing the pretreatment method. The specific primers of wheat flour were obtained by literature retrieval and bioinformatic analysis. The samples (10 batches of wheat, 16 batches of Gramineae plants and 7 batches of high-starch food) were amplified by real-time fluorescence PCR-HRM technique, and the results were further confirmed by clone sequencing. The limit of detection (LOD), precision and repetitiveness were investigated so as to validate the real-time fluorescence PCR-HRM method. In addition, the commercially available samples of Xiaohuoluo Wan were also tested. Results: Specific primers suitable for this study were screened, and a real-time fluorescencePCR-HRM detection method for adulteration of wheat powder in Xiaohuoluo Wan was established. The LOD of adulterated wheat flour in Xiaohuoluo Wan was 0.01g·g-1. The precisions (RSD, %) were 2.15% for response curve (Ct value, 26.63) and 0.05% for high-resolution melting curve (Tm value, 89.53℃), respectively. And the repetitiveness values (RSD, %) were 3.20% for Ct value (26.65) and 0.12% for Tm value (89.53℃), respectively. A total of 81 samples of 9 batches of Xiaohuoluo Wan from 4 manufacturers on the market were analyzed in the test, and it was found that 3 batches of samples of Xiaohuoluo Wan from Manufacturer A were adulterated with wheat flour. The PCR amplification products were verified by cloning and sequencing assay, and the adulterated goods were wheat (Triticum Aestivum L.). Conclusion: A real-time fluorescence PCR-HRM detection method was estalbished to quickly identify the adulterated wheat flour in Xiaohuoluo Wan.
Key words
Xiaohuoluo Wan； wheat powder； real-time PCR； high-resolution melting curve； Gramineae plants
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