Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR

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. In China, GM tomato Huafan No 1 with a character of long shelf-life was the first GM plant which was approved for commercialization in 1996. To meet the requirement of the GM tomatoes labeling policy that has been actualized in China since 2001, screening and construct-specific PCR detection methods for detecting the universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti-sense ethylene-forming enzyme (EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No 1 was 68 haploid genome copies in conventional PCR detection, and three copies in TaqMan real-time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No 1 was three copies and was 25 copies for construct-specific quantitative PCR. Two samples with known Huafan No 1 tomato content were detected using the established conventional and real-time PCR systems, and these results also indicated that the established Huafan No 1 screening and construct-specific PCR detection systems were reliable, sensitive and accurate.

Event-Specific Qualitative and Quantitative Polymerase Chain Reaction Analysis for Genetically Modified Canola T45

“…Qualitative and quantitative real-time PCR detection methods were well established for transgenic Huafan No.1 tomato, Bt cottons, MON863 maize, etc. ( − ).…”

Duplex polymerase chain reaction method for detection of unapproved genetically modified tomato (Solanum lycopersicon L.) with cucumber mosaic virus (CMV) satellite RNA gene

“…Recently, it has been reported that tomatoes with cucumber mosaic virus (CMV) resistance, tobacco mosaic virus (TMV) resistance, increased shelf-life, and cold tolerance have been developed or are under development in China (5,8). CMV is classified into the genus Cucumovirus in the family Bromoviridae and is a plant virus found in most countries (9).…”

Development of event‐specific PCR detection methods for genetically modified tomato Huafan No. 1

“…To date, only a few papers have been published describing the development of a PCR detection method for GM tomato events, such as those for B/Da/F and Huafan No. 1 …”

“…1, developed by Huazhong Agriculture University (HZAU) in China, was produced by inserting an expression cassette of anti-sense ethylene-forming enzyme (EFE) gene into the tomato genome with the trait of delayed fruit ripening. 13 To date, only a few papers have been 15,16 In this study, we developed new qualitative and quantitative PCR assays for identification and quantification of GM tomato event Huafan No. 1 based on the cloned 3 flanking sequence of the transgene integration site.…”