Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs
ABSTRACT Identifying contaminating bovine constituents in feed has been a major means to help prevent the spread of bovine spongiform encephalopathy (BSE). The phenomenon of DNA aggregation induced by Hoechst 33258 in conjunction with the change in anodic current measurement was, for the first time, applied for bovine DNA detection in feedstuffs. By using the PCR amplification system specific to bovine parathyroid and common 12S rRNA genes, anodic current peaks measurements of these PCR products on linear sweep voltammetry could be determined. Anodic peaks measurement of bovine parathyroid gene among ruminant meat and pet foods containing bovine constituents were at 1.18–1.52 μA while anodic peaks among non bovine samples were greater than 1.78 μA. In the study, anodic current peaks greater than 1.75 μA could be used to distinguish non-bovine from other samples in a qualitative analysis. For quantitative analysis, bovine content was measured using the comparative ratio between copy number of bovine parathyroid and 12S rRNA genes. This ratio reflected the proportion of target bovine cells to total cell numbers. In the experiment, contents of bovine constituents in four kinds of tested pet foods were 10.88, 8.76, 6.39 and 2.69%. Compared with the first two samples on which defined content had been addressed, the estimated content with 90.66 and 87.60% accuracy could be obtained, respectively. Although, this quantitative detection was not a real-time determination, the method had several merits on its rapidity and simplicity in performing the test and on its cost effectiveness since no sophisticated devices and expensive reagents were needed.