Successful detection of foreign inserts in transgenic rice TT51-1 (BT63) by RNA-sequencing combined with PCR.

Abstract

As event-specific sequence information for most unauthorised genetically modified organisms (GMOs) is currently still unavailable, detecting unauthorised GMOs remains challenging. Here, we used insect-resistant rice TT51-1 as an example to develop a novel approach via detecting GMOs by RNA-seq (sequencing) and PCR. RNA-seq of TT51-1 generated 4.8 million (M) 21-nt cDNA tags. Alignment to the Oryza sativa subsp. japonica reference genome revealed 24 098 unmapped tags. Foreign tags from the nopaline synthetic enzyme gene (NOS) terminator and insect-resistant genes were then identified by searching against the NCBI VecScreen and NT databases. To further isolate foreign DNA sequences, putative NOS terminator and insect-resistant gene tags were combined and used directly as primer pairs for long-range PCR, producing a 5016-bp fragment. The inserted DNA sequence of TT51-1 has been submitted to a database, and thus, similarity analysis using the database could identify a test sample. The novel approach has a great potential for application to the detection and identification of unauthorised GMOs in food and feed products. © 2016 Society of Chemical Industry.