Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons

Abstract

Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicate that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

“…The labeling of GM foods is not compulsory in the United States and Canada (Matsuoka, 2001); In China, 17 different foods derived from 5 different plants should be labeled, such as tomato seeds, ketchup, soybean milk, soybean oil, maize oil, rapeseed and cottonseeds etc. (Yang et al, 2005a).…”

“…And the second T-DNA insert contains 242 bp of a portion of the 7S 3¢ polyadenylation sequence from the terminus of the cry1Ac gene (Monsanto Company, 2002b). To suffice to the requirement of GM cotton labeling regulation in China, the qualitative and quantitative PCR systems for event-specific detection of Mon1445 and Mon531 cotton were established according to the reported upstream or downstream integration junctions' sequences of Mon1445 and Mon531 in this study using cotton Sad1 gene as an endogenous reference gene (Yang et al, 2005a).…”

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

“…For total cotton genome quantification, a Sad I real-time PCR assay employing primers (Sad I-1F/2R) and probe Sad I-p was used according to a previous paper. 17 For development of the MON15985 event-specific real-time PCR assay, the quantitative standard curve, repeatability and reproducibility, and limits of detection and quantitation (LOD and LOQ) were performed using a series of MON15985 cotton genomic DNA dilutions as templates in the real-time PCR assay.…”

“…For cotton species detection, the previous reported endogenous reference gene, Sad I, and its qualitative and quantitative PCR primers (Sad I-1F/2R) and probe (Sad I-p), were used in this study. 17 All of the primers and probes were synthesized by Invitrogen Co., Ltd (Shanghai, China). …”

SYBR®Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products

“…Within the commercial applications for GM Food & Feed, several reference genes for the respective crops/ ingredients have already been proposed: the lectin gene for soybean, the adh, hmg, SSIIb and invertase genes for maize, the acc, cruA and fat A genes for oilseed rape, the pld gene for rice, the sad1 and sah7 gene for cotton, and the GS gene for sugar beet [17][18][19][20][21][22][23][24][25][26][27][28]. Several of these and other newly developed markers have been tested independently for each of these crops both in qualitative screening and/or in GM quantification [29].…”