核桃过敏原成分PCR方法的建立

Establishment of PCR Method for Allergen Components in Walnut

建立核桃过敏原成分的PCR检测方法,经查阅文献,并根据核桃中主要的过敏原基因Jug r 1设计出3对引物(Jur 1 primer,Jur 2 primer,Jur 3 primer),建立检测核桃过敏原成分的PCR方法并对其进行优化。3对引物可在核桃DNA中扩增出158 bp,145 bp,199 bp的DNA片段,依据引物的Tm值预测引物的最佳退火温度,一般退火温度要比Tm值低5℃,依据预测的退火温度设立温度梯度,进一步确定最佳退火温度;为了检测3对引物是否适用于加工食品,依据各引物的最佳退火温度建立各自的PCR反应条件,配制含不同植物DNA样品的反应体系,分别进行PCR检测;为提高精确度,将核桃DNA梯度稀释,制备出核桃含量为100.00%,10.00%,1.00%,0.10%,0.01%的DNA,分别进行PCR检测。结果表明,3对引物的最佳退火温度分别为50.7,64.0,64.0℃;各引物的特异性良好;3对引物检测的最低含量为0.10%,0.10%,0.01%。建立了一种食品中核桃过敏原的PCR检测方法,该方法特异性良好,精确度高。

To establish a PCR method for detecting allergens in walnut.By consulting the literature and designing three pairs of primers(Jur 1 primer,Jur 2 primer and Jur 3 primer)according to the main allergen walnut gene Jur 1 in walnut,the PCR method for detecting walnut allergen components was established and optimized.Three pairs of primers can amplify DNA fragments of 158 bp,145 bp,199 bp in walnut DNA.According to the Tm value of the primers,the optimal annealing temperature of the primers was predicted.Generally,the annealing temperature was 5℃lower than the Tm value.According to the predicted annealing temperature,the temperature gradient was set to further determine the optimal annealing temperature.In order to detect whether the three pairs of primers were suitable for processed food,the reaction conditions of PCR were established according to the optimal annealing temperature of each primer,and the reaction systems containing different plant DNA samples were prepared for PCR detection.In order to reduce and improve the accuracy,the DNA of walnut content of 100.00%,10.00%,1.00%,0.10%and 0.01%was prepared,and then detected by PCR.The results showed that the optimal annealing temperature of the three pairs of primers were 50.7,64.0 and 64.0℃respectively.The specificity of each primer was good;the lowest content detected by three pairs of primers was 0.10%,0.10%and 0.01%.A PCR method for detecting walnut allergen in food was established,which had good specificity and high accuracy.