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Harshman D.K.,University of Arizona | Reyes R.,University of Arizona | Park T.S.,University of Arizona | You D.J.,University of Arizona | And 2 more authors.
Biosensors and Bioelectronics | Year: 2014

There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67s/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1ng/μL or 105genomes/μL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity. © 2013 Elsevier B.V. Source

Lee Y.-N.,Georgia State University | Lee Y.-T.,Georgia State University | Kim M.-C.,Georgia State University | Kim M.-C.,Animal and Plant Quarantine Agency | And 4 more authors.
Immunology | Year: 2014

Summary: The ectodomain of matrix protein 2 (M2e) of influenza virus is considered a rational target for a universal influenza A vaccine. To better understand M2e immune-mediated protection, Fc receptor common γ chain deficient (FcRγ-/-) and wild-type mice were immunized with a tandem repeat of M2e presented on virus-like particles (M2e5x VLP). Levels of M2e-specific antibodies that were induced in FcRγ-/- mice after immunization with M2e5x VLP were similar to those in wild-type mice. In addition, M2e antibodies induced in FcRγ-/- mice were found to be equally protective as those induced in wild-type mice. However, M2e5x VLP-immunized FcRγ-/- mice were not well protected, as shown by severe weight loss, higher lung viral titres and interleukin-6 inflammatory cytokine production upon influenza virus challenge compared with M2e5x VLP-immunized wild-type mice. Importantly, FcRγ-/- mice that were immunized with inactivated influenza virus induced haemagglutination inhibition activity and were well protected without a significant weight loss. Interestingly, interferon-γ-producing CD4 T and CD8 T cells were found to be prevalent in lungs from M2e5x VLP-immunized FcRγ-/- mice, which appeared to be correlated with a faster recovery after infection. These results indicate that Fc receptors play a primary role in conferring M2e-specific antibody-mediated protection whereas T cells may contribute to the recovery at later stages of infection. © 2014 John Wiley & Sons Ltd. Source

Shin Y.-G.,Animal and Plant Quarantine Agency | Rho J.-Y.,Dankook University
Plant Pathology Journal | Year: 2014

Iris yellow spot virus (IYSV) is a plant pathogenic virus which has been reported to continuously occur in onion bulbs, allium field crops, seed crops, lisianthus, and irises. In South Korea, IYSV is a “controlled” virus that has not been reported, and inspection is performed when crops of the genus Iris are imported into South Korea. In this study, reverse-transcription polymerase chain reaction (RT-PCR) and nested PCR inspection methods, which can detect IYSV, from imported crops of the genus Iris at quarantine sites, were developed. In addition, a modified positive plasmid, which can be used as a positive control during inspection, was developed. This modified plasmid can facilitate a more accurate inspection by enabling the examination of a laboratory contamination in an inspection system. The inspection methods that were developed in this study are expected to contribute, through the prompt and accurate inspection of IYSV at quarantine sites to the plant quarantine in South Korea. ©The Korean Society of Plant Pathology. Source

Sundaram J.,U.S. Department of Agriculture | Park B.,U.S. Department of Agriculture | Kwon Y.,Animal and Plant Quarantine Agency | Lawrence K.C.,U.S. Department of Agriculture
International Journal of Food Microbiology | Year: 2013

A biopolymer encapsulated with silver nanoparticles was prepared using silver nitrate, polyvinyl alcohol (PVA) solution, and trisodium citrate. It was deposited on a mica sheet to use as SERS substrate. Fresh cultures of Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus and Listeria innocua were washed from chicken rinse and suspended in 10ml of sterile deionized water. Approximately 5μl of the bacterial suspensions was placed on the substrate individually and exposed to 785nm HeNe laser excitation. SERS spectral data were recorded over the Raman shift between 400 and 1800cm-1 from 15 different spots on the substrate for each sample; and three replicates were done on each bacteria type. Principal component analysis (PCA) model was developed to classify foodborne bacteria types. PC1 identified 96% of the variation among the given bacteria specimen, and PC2 identified 3%, resulted in a total of 99% classification accuracy. Soft Independent Modeling of Class Analogies (SIMCA) of validation set gave an overall correct classification of 97%. Comparison of the SERS spectra of different types of gram-negative and gram-positive bacteria indicated that all of them have similar cell walls and cell membrane structures. Conversely, major differences were noted around the nucleic acid and amino acid structure information between 1200cm-1 and 1700cm-1 and at the finger print region between 400cm-1 and 700cm-1. Silver biopolymer nanoparticle substrate could be a promising SERS tool for pathogen detection. Also this study indicates that SERS technology could be used for reliable and rapid detection and classification of food borne pathogens. © 2013. Source

Zhu S.,University of Kentucky | Jeong R.-D.,University of Kentucky | Lim G.-H.,University of Kentucky | Yu K.,University of Kentucky | And 8 more authors.
Cell Reports | Year: 2013

Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R) proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT), recognizes the turnip crinkle virus (TCV) coat protein (CP). HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4) even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling Source

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