【作者】 陈文炳； 李寿崧； 邵碧英； 郑腾； 江树勋； 黄晓蓉； 蔡开珍； 张志灯；

【Author】 Chen Wenbing Li Shousong Shao Biying ZhengTeng Jiang Shuxun Huang Xiaorong Cai Kaizhen Zhang Zhideng (Fujian Entry-exit Inspection and Quarantine Bureau,Fuzhou 350003)

【机构】 福建出入境检验检疫局技术中心；

【摘要】 以分子生物学方法——聚合酶链式反应(PCR)技术为基础,初步探讨了食品中 SARS 病毒靶基因片段的多重 PCR 检测技术。根据 GenBank 公开发表的 SARS 病毒基因组 cDNA 序列,人工合成克隆特异性靶基因 DNA 片段,以此片段作为阳性对照,并将其添加到罐装蘑菇、牛肉干、大豆、鱼千等食品的 cDNA 中作为阳性样品,再根据世界卫生组织推荐的引物序列合成引物,进行单 PCR 与多重 PCR 检测分析。结果表明：以单 PCR 法获得了121bp、182bp 及302bp3条靶基因片段；以二重 PCR 法获得了121 bp+182bp、121bp+302 bp 与182 bp+302 bp 的靶基因片段组合；以三重 PCR 法获得了121 bp+182 bp+302bp 的靶基因片段组合。检测灵敏度的实验表明：182 bp 片段的模板 DNA 量在0．003 ng 以上时,单 PCR 都能扩出清晰的条带,而121 bp 的靶基因片段只有模板 DNA 量在0．03 ng 以上时,单 PCR 才能扩出清晰的条带。它们的二重 PCR 分析的灵敏度与相应的单 PCR 分析灵敏度相同。更多还原

【Abstract】 The detection of target genes of SARS virus in foods by multiplex PCR was studied primarily basing on PCR technique.The target cDNA fragments of SARS virus synthesized artificially according to the genomic sequence of SARS submitted by Chinese University of Hong Kong to GenBank were used as positive controls.The cDNA fragments were reverse-transcripted from RNA extracted from canned mushroom,dried beef,soybean and dried fish.Mixture of the positive target cDNA fragments and the cDNA of food were used as positive samples.5 primers recommended by WHO were used to amplify by single PCR and multiplex PCR.In the positive controls and positive samples,3 target DNA fragments(121 bp,182 bp and 302 bp)were amplified by single PCR,as well as 3 associations of 2 target fragments including 121 bp & 182 bp,121 bp & 302 bp and 182 bp & 302 bp,and the association of 3 target DNA fragments(121 bp,182 bp and 302bp)were amplified by multiplex PCR.The band of target DNA fragment can be visible when the template content of 182 bp DNA was over 0.003 ng,but that of 121 bp DNA should be over 0.03 ng.These results were same to the Duplex PCR patterns of the association of 182bp & 121 bp.更多还原