Su Wenijn, Song Siyang, Long Minnan, Liu Guangming
School of Biotechnology, Jimei University, Shi-Gu Road, 9 Xiamen, Fujian 361021, China.
J Biotechnol. 2003 Nov 6;105(3):227-33. doi: 10.1016/j.jbiotec.2003.07.001.
To improve detection efficiency and result accuracy, four screening primer pairs, four identifying primer pairs, one common primer pair and corresponding probes were designed for the development of multiplex polymerase chain reaction/membrane hybridization assay (MPCR-MHA) for detection of the foreign genes insert in genetically modified organisms (GMOs). After detecting condition and parameter were optimized and determined, MPCR reactions were developed for amplifying several target genes simultaneously in one tube. Primers were labeled with biotin at the 5'-end; biotinylated MPCR products were detected by hybridization to the oligonucleotide probes immobilized on a membrane with subsequent colorimetric detection to confirm hybridization. The testing of screening primers can judge whether the sample contains GMOs, and that of identifying primers can further judge what kinds of trait genes are contained in the sample. We detected nine soybean samples, six maize samples, seven potato samples and two rice samples by the MPCR-MHA method; at the same time we also detected them with single PCR-MHA method. The results between two methods have good consistency.
为提高检测效率和结果准确性，设计了四对筛选引物、四对鉴定引物、一对通用引物及相应探针，用于开发多重聚合酶链反应/膜杂交检测法（MPCR-MHA），以检测转基因生物（GMO）中插入的外源基因。在优化并确定检测条件和参数后，开展MPCR反应，以便在同一管中同时扩增多个靶基因。引物在5'-端用生物素标记；生物素化的MPCR产物通过与固定在膜上的寡核苷酸探针杂交进行检测，随后进行比色检测以确认杂交。筛选引物的检测可判断样品是否含有转基因生物，鉴定引物的检测可进一步判断样品中含有何种性状基因。我们采用MPCR-MHA方法检测了九个大豆样品、六个玉米样品、七个马铃薯样品和两个水稻样品；同时我们也用单重PCR-MHA方法对它们进行了检测。两种方法的结果具有良好的一致性。
