应用多重PCR检测食品中SARS病毒靶基因的初步研究

Preliminary Study on the Detection of Target Genes of SARS Virus in Foods by Multiplex PCR

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、鱼干等食品的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121bp+182bp、121bp+302bp与182bp+302bp的靶基因片段组合;以三重PCR法获得了121bp+182bp+302bp的靶基因片段组合。检测灵敏度的实验表明:182bp片段的模板DNA量在0.003ng以上时,单PCR都能扩出清晰的条带,而121bp的靶基因片段只有模板DNA量在0.03ng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。

The detection of target genes of SARS virus in foods by muhiplex 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.