南美对虾主要过敏原检测样品的快速简便预处理方法

A Rapid and Simple Method Pretreating Samples of Penaeus vannamei for Detecting Major Allergens

为了开发一种适合于试纸条法快速检测南美白对虾主要过敏原原肌球蛋白(Tropomyosin,Tm)的简单快速的样品前处理方法。本实验设计了8种预处理方法,并通过BCA蛋白定量、十二烷基硫酸钠聚丙烯酰胺凝胶电泳、免疫印迹和层析试纸条检测比较分析了8种方法的可行性。结果发现,采用方法8制备的样品提取液蛋白质浓度适中(2.5mg/mL),杂蛋白较少且对磁性纳米探针标记的免疫层析检测干扰少,较适合于现场快速检测。本研究优化的虾类过敏原Tm的样品前处理方法为:用丙酮抽提虾肉至无色,加入20mMTris-HCl(pH9.2)匀浆30s,超声处理10min,加热5min,冷却后用迷你离心机离心10min,所得上清液即为样品。

We designed eight pretreatment methods to develop the sample pretreatment method suitable for rapid detection of primary allergen tropomyosin of Penaeus vannamei major allergen tropomyosin (Tm) of P. vannamei by the test strip method, the application feasibility of eight pretreatment methods was compared and analyzed by BCA Protein Assay Kit, SDS-PAGE, Western Blot, and magnetic immunoblotting chromatographic test. As a result, it was found that the sample prepared by the eighth method had a moderate protein concentration (2.5 mg/mL), less miscrine protein and less immunochromatography detection interference of the magnetic nano-probe mark,this is suitable for field rapid detection. The optimized sample pretreatment method of shrimp allergen Tm was as follows: the shrimp was extracted with acetone until colorless, 20 mM Tris-HCl (pH9.2) homogenate was added for 30s, ultrasonic treatment for 10min and heated for 5 min. After cooling, it was centrifuged by mini centrifuge for 10 min, the supernatant was the sample.