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乳糜泻是患者对小麦、大麦、黑麦和燕麦中的谷蛋白产生的一种不适当免疫反应。患者只能通过终身无谷蛋白食疗来避免严重并发症。本文建立了一种常规PCR方法来同时检测食品中大麦和黑麦致敏原。引物BR108bp是针对大麦醇溶谷蛋白3编码序列设计的,在GenBank上经Blast,发现理论上特异性很好。然而通过试验发现,该引物除了在大麦处出现相应扩增外在黑麦处也扩增出大小约为108bp的条带。经测序和比对,发现以大麦和黑麦为模板的扩增产物测序结果一致,并且只与模板序列相差一个碱基,这说明黑麦也含有这段序列,只是在核酸数据库中没公布,因此可以说是发现了黑麦的部分基因序列。通过多次试验,结果表明:该引物只在大麦和黑麦处出现相应扩增,具有良好特异性,针对大麦和黑麦的理论检出限均为1.0ng/μL,实际检出限均为1.0%。而且能检测热加工食品中的大麦和黑麦DNA,故可用于日常检测。
Celiac disease is an inappropriate immune response that patients develop against gluten in wheat, barley, rye and oats. Patients can only avoid serious complications through life-long gluten-free diet. This article established a conventional PCR method for simultaneous detection of barley and rye allergens in food. Primer BR108bp was designed for the barley prolamin 3 coding sequence and was found to be theoretically specific in GenBank by Blast. However, it was found through experiments that this primer also amplified a band of about 108 bp in size at rye except for the corresponding amplification at barley. Sequencing and alignment showed that the sequencing results of the amplified product from barley and rye were identical and only one base difference from the template sequence. This indicated that rye also contained this sequence, but it was not published in the nucleic acid database, So it can be said that part of the rye gene sequence was found. Through several experiments, the results showed that the primer only showed the corresponding amplification in barley and rye, with good specificity. The theoretical detection limits for both barley and rye were 1.0 ng / μL, the actual detection limits were 1.0%. But also to detect barley and rye DNA in hot processed foods, it can be used for routine testing.