A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays.
Rapid diagnostic technologies for bovine mastitis caused by Staphylococcus aureus (S. aureus) are urgently needed. In the current study, we generated an anti-ribosomal protein-L7/L12 antibody to detect S. aureus and an anti-ribosomal protein-L7/L12 antibody-coated immune-chromatographic strip (ICS) test. Moreover, we determined the ability of the ICS test to detect S. aureus from milk samples collected from cows with clinical mastitis. The developed ICS reacted to S. aureus in a bacteria load-dependent manner with a detection limit of ∼10 4 CFU/mL. In the evaluation of possible cross-reactivity of the ICS test, six strains of coagulase-negative Staphylococci showed slightly positive reactions, although at a lower level; however, other bacteria were completely negative. Next, we investigated the sensitivity and specificity of the ICS test compared with the bacteriological culture method using milk samples from clinical bovine mastitis. The results of the experiments demonstrated that the ICS test had high sensitivity [100%, 95% confidence interval (CI): 91.3-100%] and specificity (91.9%, CI: 90.5-91.9%) compared with culture tests. In addition, the kappa statistic demonstrated that ICS tests showed substantial agreement (k = 0.77, CI: 0.66-0.87) with culture tests. Positive correlations were observed for the statistical analysis between S. aureus (nuc gene) copy numbers and ICS test scores in mastitic milk infected by S. aureus. Therefore, we assume that this new detection method using ICS may be useful as a highly sensitive S. aureus-screening method for the diagnosis of bovine mastitis. Our findings support the ongoing effort to develop an ICS method for bovine S. aureus-induced mastitis, which can contribute to the rapid diagnosis of this disease.
We evaluated the relationship between the severity of coliform mastitis and bacterial load in 106 quarter milk samples. We found no significant relationship between somatic cell count and coliform bacterial load in milk in bovine clinical coliform mastitis. Results of the Cochran-Armitage test for trend in milk bacterial load proportions indicated a significant decreasing low group (P<0.001), increasing medium group (P<0.002) and increasing high group (P<0.02) with increasing clinical grade. The present study indicates that the coliform bacterial load in milk is significantly associated with clinical severity states in cases of bovine coliform mastitis, and can be a useful indicator for optimal management of this disease.
Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. In this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly (dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C (MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen-and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time.-3 -
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