Differential diagnosis for pythiosis using thermophilic helicase DNA amplification and restriction fragment length polymorphism (tHDA-RFLP).

Pythiosis is caused by Pythium insidiosum, a fungus-like microbe belonging to the kingdom Stramenopila. Its diagnosis is challenging due to clinical and histopathological similarities with the fungal microbes that cause mucormycosis and entomophthoramycosis. In addition, the proper identification of P. insidiosum in the clinical laboratory is difficult. We have developed a rapid and accurate, species-specific identification method using a thermophilic helicase DNA amplification (tHDA) technique, to differentiate this pathogen from closely related pathogenic fungi. Sixty-seven fungal isolates, including 39 of P. insidiosum, were evaluated. A 91 base-pair (bp) DNA fragment was consistently amplified using a COX2 primer. The limiting concentrations of the one- and two-step tHDA protocols were 100 picograms (1.74 × 102 copies) and 100 femtograms (1.74 × 10-1 copies), respectively. The CviKI-1 enzyme in restriction fragment length polymorphism (RFLP) with the 91 bp amplicons accurately separated P. insidiosum from other fungal species. The data suggest that this tHDA-RFLP assay is a rapid and accurate test for the identification of P. insidiosum. The potential use of the assay directly in clinical samples is also discussed.

Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

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.

Multiplex mass spectrometric genotyping of single nucleotide polymorphisms employing pyrrolidinyl peptide nucleic acid in combination with ion-exchange capture.

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.

Simple Screening of Listeria monocytogenes Based on a Fluorescence Assay via a Laminated Lab-On-Paper Chip.

Monitoring food safety is essential for protecting the health and safety of consumers. Conventional methods used are time consuming and laborious, requiring anywhere from three to seven days to obtain results. Thus, better monitoring methods are required. In this study, a laminated lab-on-paper chip was developed, and its use for the screening of ready-to-eat seafood was demonstrated. The assay on a chip was based on loop-mediated isothermal DNA amplification (LAMP) of the hly gene of Listeria monocytogenes and fluorescence signal detection via SYBR GoldTM. Overall assay processes were completed in 4.5 h., (including 3.5 h. incubation for the bacteria enrichment, direct DNA amplification with no DNA extraction, and signal detection), without relying on standard laboratory facilities. Only positive samples induced fluorescence signals on chip upon illumination with UV light (λ = 460). The method has a limit of detection of 100 copies of L. monocytogenes DNA per 50 g of sample. No cross-reactivity was observed in samples contaminated with other bacteria. On-site monitoring of the seafood products using this chip revealed that one of 30 products from low sanitation vendors (3.33%) were contaminated, and these agreed with the results of PCR. The results demonstrated a benefit of this chip assay for practical on-site monitoring.

Rapid detection of aflatoxigenic Aspergillus sp. in herbal specimens by a simple, bendable, paper-based lab-on-a-chip.

Postharvest herbal product contamination with mycotoxins and mycotoxin-producing fungi represents a potentially carcinogenic hazard. Aspergillus flavus is a major cause of this issue. Available mold detection methods are PCR-based and rely heavily on laboratories; thus, they are unsuitable for on-site monitoring. In this study, a bendable, paper-based lab-on-a-chip platform was developed to rapidly detect toxigenic Aspergillus spp. DNA. The 3.0-4.0 cm(2) chip is fabricated using Whatman™ filter paper, fishing line and a simple plastic lamination process and has nucleic acid amplification and signal detection components. The Aspergillus assay specifically amplifies the aflatoxin biosynthesis gene, aflR, using loop-mediated isothermal amplification (LAMP); hybridization between target DNA and probes on blue silvernanoplates (AgNPls) yields colorimetric results. Positive results are indicated by the detection pad appearing blue due to dispersed blue AgNPls; negative results are indicated by the detection pad appearing colorless or pale yellow due to probe/target DNA hybridization and AgNPls aggregation. Assay completion requires less than 40 min, has a limit of detection (LOD) of 100 aflR copies, and has high specificity (94.47%)and sensitivity (100%). Contamination was identified in 14 of 32 herbal samples tested (43.75%). This work demonstrates the fabrication of a simple, low-cost, paper-based lab-on-a-chip platform suitable for rapid-detection applications.

Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 µL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

Electrochemical DNA biosensor using a disposable electrochemical printed (DEP) chip for the detection of SNPs from unpurified PCR amplicons.

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.