关键词: 环介导等温扩增, 牛近缘种, 检测方法

Abstract: In recent years, adulteration of meat products frequently occurs,  it is urgent to establish a rapid and reliable detection method for animal-derived components in meat products to ensure the safety of meat products. Compared with conventional PCR and other detection methods, loop-mediated isothermal amplification (LAMP) method has the advantages of high specificity, high sensitivity, quick reaction and easy operation. Based on this, the whole genome sequences of common cattle and their related species (yak, buffalo, bison) were screened by bioinformatics algorithm to obtain the similarity sequences among species, and then the specific LAMP method for detecting the components of common cattle and their related species was established and optimized. The final optimized LAMP reaction system was as follows: 0.25 μL of 10 μmol·L-1 outer primers F3 and B3, 2 μL of 10 μmol·L-1 inner primers FIP and BIP, 2.5 μL of 2 mmol·L-1 dNTP, 4 μL of 25 mmol·L-1 MgSO4, 3.5 μL of 5 mol·L-1 betaine, 1 μL of 8.0 U·μL-1 Bst DNA polymerase, 2.5 μL of 10×ThermoPol Buffer, 2 μL of DNA template, and adding double distilled water to 25 μL. The reaction conditions of LAMP were: amplification at 65 ℃ for 1 h and then amplification at 80 ℃ for 10 min. After adding SYBR GreenⅠfluorescent dye into the amplification product, the detection results could be directly observed by naked eyes. The optimized LAMP method could specifically detect the components of common cattle, yak, buffalo and bison within 1 h, with a detection sensitivity of 0.020 ng·μL-1, and could specifically detect the samples containing beef components in meat products on the market. The LAMP method established in this study had the characteristics of high specificity, high sensitivity, quick response, simple operation and no need of precision instruments, which could be applied to the actual detection of common cattle and their related species in meat products, and provide a strong guarantee for the safety of meat products in China.

Key words: loop-mediated isothermal amplification, bovine-derived materials, detection method