摘要
为建立一种可同时检测食品中弓形虫和旋毛虫的液相基因芯片方法,本研究以弓形虫B1基因和旋毛虫18S rDNA基因为靶序列,设计并合成特异性探针和引物,通过PCR方法扩增目的片段并测序,建立一种基于双重PCR的液相基因芯片检测方法。结果显示:在约180bp和90bp处分别扩增出目的条带;测序结果与GenBank登录的弓形虫和旋毛虫的相关基因一致性分别为99.47%和100%;液相基因芯片对单重PCR产物和双重PCR产物的检测结果一致,特异性和重复性良好。应用该方法检测弓形虫和旋毛虫变异系数(CV%)均在7%以内;灵敏度分别为65.6ng/mL和39.06ng/mL,比琼脂糖凝胶电泳灵敏高约8倍;模拟污染试验准确率达98%以上。本研究建立的液相基因芯片检测食品中弓形虫和旋毛虫的方法具有高效、灵敏和特异等优点,为食源性寄生虫的检测和监控提供了新方法。
A duplex PCR based on liquid gene chip technique for detecting Toxoplasma gondii and Trichinella spiralis in foods was developed using primers designed according to the T.gondii B1 and T.spiralis 18S rDNA gene sequences,respectively.The amplified B1 and 18S rDNA PCR fragments were 188 bp and 92 bp in length,had 99.47% and 100% identities to the corresponding sequences in GenBank.The liquid chip was able to detect both single and duplex PCR products from the targets with high specificity and reproducibility.The detection limit of the assay was 65.6 ng/mL for T.gondii and 39.06 ng/mL for T.spiralis,which was 8-fold more sensitive than that of agarose gel method.The simulation of pollution test showed that the accuracy rate of blind test was more than 98%.The results showed that liquid gene chip technique was a rapid,sensitive and specific method for detecting T.gondii and T.spiralis in food.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第10期777-780,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
"十一五"国家科技支撑计划项目(2006BAD06A09)
国家质检总局科技计划项目
黑龙江省教育厅科学技术研究重点项目(1153Lz04)
关键词
液相基因芯片
弓形虫
旋毛虫
liquid gene chip
Toxoplasma gondii
Trichinella spiralis
