Multiplex polymerase chain reaction/membrane hybridization assay for detection of genetically modified organisms.
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.
School of Biotechnology, Jimei University, Shi-Gu Road, 9 Xiamen, Fujian 361021, China.
Wenijn
W
Su
Siyang
S
Song
Minnan
M
Long
Guangming
G
Liu
D017931:DNA Primers; D015342:DNA Probes; D018744:DNA, Plant / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005504:Food Analysis / Q000379:methods; D030841:Food, Genetically Modified; D012275:Oryza / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000737:chemistry / Q000235:genetics; D013025:Glycine max / Q000737:chemistry / Q000235:genetics; D003313:Zea mays / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D015342:DNA Probes; D018744:DNA, Plant; D012337:RNA, Ribosomal, 18S