Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

Engineering Properties of Edible Transglutaminase Cross-Linked Caseinate-Based Films

In this work, the effect of the weight ratio (Y _E/C) between transglutaminase (TGase) and sodium caseinate on the main engineering properties and microstructure of maltodextrin/caseinate/glycerol-based films, as well as cross-linking kinetics of the corresponding film-forming mixtures, was assessed. In the absence of TGase, the film forming solution did not jelly at all even after a 20-h gel cure experiment at 30 °C. As Y _E/C was increased from 0.5 to 8.3 mg/g, the gel point time exhibited quite a hyperbolic dependence on Y _E/C, whereas the pseudo-first order reversible kinetic constant rate of casein reticulation linearly increased with the enzyme concentration. Despite a certain data scattering, the initial and equilibrium complex shear moduli appeared to be practically constant and independent of Y _E/C. This finding was also extended to the mechanical and water barrier properties of the films under study. This was also indirectly confirmed by scanning electron microscopy, the film structure being quite compact, dense, and about free of air bubbles. In particular, their complex tensile modulus (E*) resulted to be not only independent of the cross-linking agent dosage but also interconvertible with the elastic modulus (E) determined in the quasi-static mode at an initial strain rate equal to the angular frequency. The water vapor permeability (WVP) for all the films examined was found to be extremely dependent on the water, desiccant, or Permatran-W standard methods used as well as on the difference in water activity (a _W) at the inside (a _Wi = 1.0 or 0) and outside (a _Woi = 0 or ∼0.5) of the test cup. By maintaining a _Wi = 0 and a _Woi = 0.5, the Permatran-W method yielded practically constant WVP values (30 ± 4 pg m⁻¹ s⁻¹ Pa⁻¹) whatever the enzyme/caseinate ratio used. Nevertheless, TGase appeared to decrease the water vapor permeability of not cross-linked maltodextrin–caseinate films.