Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95 % for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.     

Four new SYBR<Superscript>®</Superscript>Green qPCR screening methods for the detection of Roundup Ready<Superscript>®</Superscript>, LibertyLink<Superscript>®</Superscript>, and CryIAb traits in genetically modified products

SYBR^®Green qPCR methods for the detection of the Roundup Ready^® “CP4-EPSPS”, LibertyLink^® “PAT” and “BAR,” and the Bacillus thuringiensis “CryIAb” traits as present in genetically modified organisms (GMO) were developed. Their specificity, sensitivity, and PCR method efficiency were determined. All methods are specific and generate amplicons of 108, 73, 109, and 69 bp, respectively, for “CP4-EPSPS,” “CryIAb,” “PAT,” and “BAR” targets. They perform well at low target levels and can detect down to 5 copies of their respective targets extracted from a sample. The PCR efficiency of the methods ranges between 91 and 109%. Due to their trait-specific nature, these methods allow an efficient screening between the different GMO. In this way, the number of possible GMO candidates present in a sample can be reduced what results in lower global costs due to limiting of further the number of analytical identification steps. The application of these methods in CoSYPS GMO analysis is illustrated using two GEMMA proficiency test samples and a reference material from the GM rapeseed event RF3. This set of SYBR^®Green qPCR trait-specific methods represents a very interesting novel set of GMO analysis methods allowing cost-effective identification of GM materials in products.     

Associations between paratuberculosis ELISA results and test-day records of cows enrolled in the Irish Johne's Disease Control Program

The effect of the Mycobacterium avium ssp. paratuberculosis (MAP) ELISA status on test-day milk performance of cows from Irish herds enrolled in the pilot national voluntary Johne's disease control program during 2013 to 2015 was estimated. A data set comprising 92,854 cows and 592,623 complete test-day records distributed across 1,700 herds was used in this study. The resulting ELISA outcome (negative, inconclusive, and positive) of each cow within each year of the program was used to allocate the cow into different scenarios representing the MAP status. At MAPscenario₁, all cows testing ELISA nonnegative (i.e., inconclusive and positive) were assigned a MAP-positive status; at MAPscenario₂ only cows testing ELISA-positive were assigned a MAP-positive status; at MAPscenario₃ only cows testing ELISA nonnegative (inconclusive or positive) and gathered exclusively from herds where at least 2 further ELISA nonnegative (inconclusive or positive) cows were found were assigned a MAP-positive status; at MAPscenario₄ only cows testing ELISA-positive that were gathered exclusively from herds where at least 2 further ELISA-positive cows were found were assigned a MAP-positive status. Milk outputs based on test-day records were standardized for fat and protein contents (SMY) and the effect of MAP ELISA status on the SMY was estimated by a linear mixed effects model structure. The SMY mean difference recorded at test day between cows with a MAP-positive status and those with a MAP-negative status within MAPscenario₁ was estimated at ?0.182 kg/test day; the mean difference was ?0.297 kg/test day for MAPscenario₂; for MAPscenario₃ mean difference between MAP-positive status and MAP test-negative cows was ?0.209 kg/test day, and for MAPscenario₄, the difference was ?0.326 kg/test day.     

Diversity of the Volatile Organic Compounds Emitted by 55 Species of Tropical Trees: a Survey in French Guiana

Volatile organic compounds (VOCs) are produced by a broad range of organisms, from bacteria to mammals, and they represent a vast chemical diversity. In plants, one of the preeminent roles of VOCs is their repellent or cytotoxic activity, which helps the plant deter its predators. Most studies on VOCs emitted by vegetative parts have been conducted in model plant species, and little is known about patterns of VOC emissions in diverse plant communities. We conducted a survey of the VOCs released immediately after mechanical damage of the bark and the leaves of 195 individual trees belonging to 55 tropical tree species in a lowland rainforest of French Guiana. We discovered a remarkably high chemical diversity, with 264 distinct VOCs and a mean of 37 compounds per species. Two monoterpenes (α-pinene and limonene) and two sesquiterpenes (β-caryophyllene and α-copaene), which are known to have cytotoxic and deterrent effects, were the most frequent compounds in the sampled species. As has been established for floral scents, the blend of VOCs is largely species-specific and could be used to discriminate among 43 of the 55 sampled species. The species with the most diverse blends were found in the Sapindales, Laurales, and Magnoliales, indicating that VOC diversity is not uniformly distributed among tropical species. Interspecific variation in chemical diversity was caused mostly by variation in sesquiterpenes. This study emphasizes three aspects of VOC emission by tropical tree species: the species-specificity of the mixtures, the importance of sesquiterpenes, and the wide-ranging complexity of the mixtures.