Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing

Abstract

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

“…Additionally, these methods can serve as a basis to develop other GMO detection tools using new technologies such as xMAP [52][53][54]. Furthermore, the detection of a certain screening marker in a sample can be exploited for the application of technologies such as NGS [55] and DNA/chromosome walking [36,[55][56][57], that are especially useful for the characterisation of events for which little or no information (i.e. classes 2, 3 and 4) [9] is available (e.g.…”

Statistical framework for detection of genetically modified organisms based on Next Generation Sequencing

“…Other approaches have been developed to exploit the potential of NGS for GMO detection and analysis when a reference genome of the GM crop is available, but only partial or no prior knowledge of the sequence of the transgene insert is available (Wahler, Schauser, Bendiek, & Grohmann, 2013;Yang et al, 2013). Liang et al (2014) have dealt with GMOs by developing a targeted strategy combining a chromosome walking method, based on SiteFinding-PCR, and NGS technology. In this study, a part of the cassette is known and targeted (partial a priori knowledge).…”

Antimicrobial Resistance Is a Global Health Emergency

“…For example, rates of gram-negative resistance reach over 25% in parts of southern Europe, and all 6 regions of the World Health Organization (WHO) include countries that are reporting more than a 50% incidence of resistance of Klebsiella to third-generation cephalosporins. 3,4 There are no reliable estimates of the number of cases globally, largely because surveillance is incomplete; however, fragmentary evidence suggests that the problem is much worse in many developing countries. 1 These infections occur in nearly all medical settings.…”

“…For example, all WHO regions report countries with rates of resistance to penicillin exceeding 50% in community-acquired pneumococcal infections, and half of the WHO regions have countries wherein the rate of gonorrhea resistance to third-generation cephalosporins exceeds 50%. 4 …”