Detection of genetically modified tomato using PCR coupled with muParaflo microfluidics microarrays.

ABSTRACT

Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR SAVR was permitted for planting in 1994. To meet the requirement of the GM tomatoes labeling policy, in this study we firstly set up the conventional PCR and multiplex PCR detection system for screening the universal elements transformed into tomato, such as cauliflower mosaic virus 35s (CaMV 35s) promoter, nopaline synthase (nos) terminator of Agrobacterium tumefaciens, neomycinphosphotransferase (nptII) gene, and the specifically inserted heterologous DNA sequence between CaMV 35s promoter and anti-sense ethylene-forming enzyme (anti-EFE) gene in GM tomato "Huafan No. 1." Tomato lat52, mcpi, fru and apx genes were used as endogenous reference genes. Besides these, a muParaflo microfluidic microarray was also developed to screen the exogenous or endogenous genes of GM tomatoes. A total of 957 probes were designed, which can be classified into two categories according to their purpose: the first for screening GM plants from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes, and the second for specific gene confirmation based on target sequences such as anti-EFE or aminocyclopropane cyclase synthase (acc) gene. To ensure the reliability of this method, different kinds of positive and negative controls (such as the probes complementary to cp gene of CaMV) were included in microarray detection system. Four tomato species were identified by means of these methods, and the results indicated that microarray is a high-throughput and more efficient screening method, which could complement PCR-based screening procedures by providing direct conclusive evidence and also may be useful to resolve masking of unknown events by known events.