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 mA while anodic peaks among non bovine samples were greater than 1.78 mA. In the study, anodic current peaks greater than 1.75 mA 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. q

Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene–streptavidin–magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

“…This allows a cost-effective and rapid determination complying with available enforcing regulations. Determination based on this system was very satisfactory for feedstuff analysis [7].…”

Methodology Semi-quantitative detection of gene expression using bisbenzimide dye

“…There was a previous report of using an electrochemical biosensor for detecting gene expression in plant tissues, but the assay principle was based on enzyme-linked DNA hybridization assay (Horaková-Brazdilova et al, 2008). The electrochemical biosensor using Hoechst 33258 has been applied to detect bovine constituents in feedstuff (Chaumpluk et al, 2006), but that particular investigation did not provide any related information with regard to gene expression. In this study, electrochemical biosensor aggregation with Hoechst 33258 was applied to detect gene expression of the highly expressed gene, b-actin, and compared with the conventional agarose gel electrophoresis method.…”

“…This particular dye has commonly been used because the effect of DNA aggre-gation by this molecule is better than the others (Kobayashi et al, 2004). The electrochemical biosensor using Hoechst 33258 has been applied for analysis of DNA in several areas including detection of bovine constituents in feedstuff (Chaumpluk et al, 2006), detection of single nucleotide polymorphisms (SNPs) of clinically important alleles using a disposable electrochemical printed (DEP) chip (Ahmed et al, 2007) and identification of meat species using DEP chips with a loop mediated isothermal amplification technique (Ahmed et al, 2010). The electrochemical biosensor aggregation with Hoechst 33258 is easy to use, inexpensive and less time-consuming because it eliminates the immobilization step and reduces expense for specific probes.…”

Electrochemical detection of the neomycin phosphotransferase gene (NPT-II) in transgenic plants with a novel DNA biosensor

“…PCR, in turn, must be terminated before saturation and a threshold must be defined to reflect the difference of the original template number. Chaumpluk et al found that the amplification of target genes (Pth and 12S rRNA genes) at 30 cycles was effective in solving the problem of similar signals caused by PCR saturation, thus successfully demonstrating the relationship between signals and gene content with the biosensor [29]. A proper cycle number of PCR in the exponential phase of amplification is a critical factor to determine copy number of transgenes in future experiments.…”