In this study, we are reporting for the first time the elucidation of single nucleotide polymorphisms (SNPs) of clinically important alleles from consenting human subjects using a disposable electrochemical printed (DEP) chip in connection with differential pulse voltammetry (DPV) and a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)]. Post-PCR products were analyzed directly without any purification process. The aggregation of the DNA-H33258 complex causes a significant drop in the peak current intensity of H33258 oxidation. The phenomenon of DNA aggregation induced by H33258 in addition to changes in anodic current peak are used to detect SNPs. Since laborious probe immobilization was not required, our biosensor offers several benefits due to its simplicity and rapid response as a promising device for genetic analysis.

A new ion-exchange capture technique is introduced for label-free sample preparation in single nucleotide polymorphism (SNP) genotyping. The DNA sample is hybridized with a new pyrrolidinyl peptide nucleic acid (PNA) probe and treated with a strong anion exchanger. The complementary PNA.DNA hybrid is selectively captured by the anion exchanger in the presence of noncomplementary or unhybridized PNA, allowing direct detection of the hybridization event on the anion exchanger by MALDI-TOF mass spectrometry after simple washing. The high specificity of the pyrrolidinyl PNA allows simultaneous multiplex SNP typing to be carried out at room temperature without the need for enzyme treatment or heating. Exemplary applications of this technique, in the identification of meat species in feedstuffs and in multiplex SNP typing of the human IL-10 gene promoter region are demonstrated, clearly suggesting the potential for much broader applications.

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

No abstract

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.