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Pi L.,Shanghai JiaoTong University | Li X.,Technical Center for Animal | Cao Y.,Shanghai JiaoTong University | Wang C.,Shanghai JiaoTong University | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

Abstract Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90 %, and the squared regression coefficients (R2) were more than 0.999. The quantification bias varied from 0.21 % to 19.29 %, and the relative standard deviations were from 1.08 % to 9.84 % in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events. [Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg.

Yang J.,Technical Center for Animal | He Y.,Technical Center for Animal | Jiang J.,Technical Center for Animal | Chen W.,Bright Dairy and Food Co. | And 3 more authors.
Food Control | Year: 2016

Cronobacter sp., an opportunistic foodborne pathogen, can cause high mortality in neonatal and premature infants because this pathogen is associated with powdered infant formula (PIF) contamination. Biofilm formation is recognized as the most effective means to help Cronobacter survive throughout the long shelf-life of PIF, thus promoting resistance to cleaning agents and disinfectants. Crystal violet (CV) staining and fluorescence microscopy analyses were used to compare the biofilm formation ability among different Cronobacter strains, and then the weak biofilm former Cronobacter sakazakii ATCC 29544 and the strong biofilm former Cronobacter dubliniensis DSM 18707 were selected for further proteomic analysis. Two-dimensional liquid chromatography-tandem mass spectrometry, which was coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling, was employed to quantitatively identify the proteins that were differentially expressed in C. sakazakii ATCC 29544 compared to C. dubliniensis DSM 18707. In total, 1190 proteins were detected. Gene Ontology (GO) analysis indicated that these differentially expressed proteins are related to biological binding, cell structure, signal transduction, cell adhesion, and cellular interaction. Among these differential proteins, the expression levels of 177 non-redundant proteins were altered significantly by more than 5-fold, with 96 up-regulated proteins and 81 down-regulated. This study provides important information regarding the cellular requirements for the underlying mechanism of Cronobacter biofilm formation and its survival in harsh environments. © 2015 Elsevier Ltd.

Cai Y.,Technical Center for Animal | Li X.,Technical Center for Animal | Lv R.,Technical Center for Animal | Yang J.,Technical Center for Animal | And 3 more authors.
BioMed Research International | Year: 2014

In this project, a highly precise quantitative method based on the digital polymerase chain reaction (dPCR) technique was developed to determine the weight of pork and chicken in meat products. Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of species-specific DNAs in meat products. However, it is limited in amplification efficiency and relies on standard curves based Ct values, detecting and quantifying low copy number target DNA, as in some complex mixture meat products. By using the dPCR method, we find the relationships between the raw meat weight and DNA weight and between the DNA weight and DNA copy number were both close to linear. This enabled us to establish formulae to calculate the raw meat weight based on the DNA copy number. The accuracy and applicability of this method were tested and verified using samples of pork and chicken powder mixed in known proportions. Quantitative analysis indicated that dPCR is highly precise in quantifying pork and chicken in meat products and therefore has the potential to be used in routine analysis by government regulators and quality control departments of commercial food and feed enterprises. © 2014 Yicun Cai et al.

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