Molecular detection of human adenovirus in sediment using a direct detection method compared to the classical polyethylene glycol precipitation

2015 ◽  
Vol 213 ◽  
pp. 65-67 ◽  
Author(s):  
Rodrigo Staggemeier ◽  
Marina Bortoluzzi ◽  
Tatiana Moraes da Silva Heck ◽  
Tiago da Silva ◽  
Fernando Rosado Spilki ◽  
...  
2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Yong Wang ◽  
Bin-Quan Luan ◽  
Zhiyu Yang ◽  
Xinyue Zhang ◽  
Brandon Ritzo ◽  
...  

Abstract Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.


1994 ◽  
Vol 42 (6) ◽  
pp. 815-822 ◽  
Author(s):  
D Egger ◽  
M Troxler ◽  
K Bienz

We performed light and electron microscopic in situ hybridization, according to the same protocol and without pretreatment of sections, on Lowicryl- and LR Gold-embedded cells. Digoxigenin (DIG)- or biotin-labeled riboprobes were visualized by direct or indirect immunodetection using commercially available gold-antibody conjugates with 0.8-10-nm gold grains. At the ultrastructural level, the main findings were that DIG-labeled probes gave a slightly higher labeling intensity (grains per signal) than biotin. The direct detection method produced a more compact signal, which led to better resolution at medium and high magnifications. Labeling intensities of all gold grain sizes were essentially equal. Grain sizes of 5 nm and larger were highly preferable because available enhancement methods are unsatisfactory for ultrasmall grains. The optimized immunodetection protocols are suitable for double hybridization with two different probes and for combined hybridization and immunocytochemistry.


2014 ◽  
Vol 77 (1) ◽  
pp. 6-14 ◽  
Author(s):  
MELISSA L. PARTYKA ◽  
RONALD F. BOND ◽  
JEFF FARRAR ◽  
ANDY FALCO ◽  
BARBARA CASSENS ◽  
...  

Consumption of raw produce commodities has been associated with foodborne outbreaks in the United States. In a recent Centers for Disease Control and Prevention report outlining the incidence of food-related outbreaks from 1998 to 2008, produce of all kinds were implicated in 46% of illnesses and 23% of deaths. Methods that quickly identify fecal contamination of foods, including produce, will allow prioritization of samples for testing during investigations and perhaps decrease the time required to identify specific brands or lots. We conducted a series of trials to characterize the sensitivity and specificity of scent detection dogs to accurately identify fecal contamination on raw agricultural commodities (romaine lettuce, spinach, cilantro, and roma tomatoes). Both indirect and direct methods of detection were evaluated. For the indirect detection method, two dogs were trained to detect contamination on gauze pads previously exposed to produce contaminated with feces. For the direct detection method, two dogs were trained to identify fecal contamination on fresh produce. The indirect method did not result in acceptable levels of sensitivity except for the highest levels of fecal contamination (25 g of feces). Each dog had more difficulty detecting fecal contamination on cilantro and spinach than on roma tomatoes. For the direct detection method, the dogs exhibited >75% sensitivity for detecting ≥0.25 g of feces on leafy greens (cilantro, romaine lettuce, and spinach) and roma tomatoes, with sensitivity declining as the amount of feces dropped below 0.025 g. We determined that use of a scent detection dog to screen samples for testing can increase the probability of detecting ≥0.025 g of fecal contamination by 500 to 3,000% when samples with fecal contamination are rare (≤1%).


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