Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus

Author Posting. © Wildlife Disease Association, 2015. This article is posted here by permission of Wildlife Disease Association for personal use, not for redistribution. The definitive version was published in Journal of Wildlife Diseases 51 (2015): 454-465, doi:10.7589/2014-05-142.Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 100 to 109 copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 100 to 109 copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.This project was possible thanks to the John H. Prescott Marine Mammal Rescue Assistance Grant Program (Grant NA10NMF4390260) and with support from the National Oceanographic and Atmospheric Administration/University of Connecticut Oceans and Human Health I-RICH Fellowship.2016-04-0

Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots

Segment distribution along the repeat-rich gene SFI1. Visualization of 500 bp segments (250 bp sliding window) at the SFI1 locus for hDBS, rDBS and oDBS. (PDF 113 kb

Utility of (11)C-methionine and (11)C-donepezil for imaging of Staphylococcus aureus induced osteomyelitis in a juvenile porcine model:comparison to autologous (111)In-labelled leukocytes, (99m) Tc-DPD, and (18)F-FDG

The aim of this study was to compare (11)C-methionine and (11)C-donepezil positron emission tomography (PET) with (111)In-labeled leukocyte and (99m)Tc-DPD (Tc-99m 3,3-diphosphono-1,2-propanedicarboxylic acid) single-photon emission computed tomography (SPECT), and (18)F-fluorodeoxyglucose ((18)F-FDG) PET to improve detection of osteomyelitis. The tracers’ diagnostic utility where tested in a juvenile porcine hematogenously induced osteomyelitis model comparable to osteomyelitis in children. Five 8-9 weeks old female domestic pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus. The sequential scan protocol included Computed Tomography, (11)C-methionine and (11)C-donepezil PET, (99m)Tc-DPD and (111)In-labelled leukocytes scintigraphy, and (18)F-FDG PET. This was followed by necropsy of the pigs and gross pathology, histopathology, and microbial examination. The pigs developed a total of 24 osteomyelitic lesions, 4 lesions characterized as contiguous abscesses and pulmonary abscesses (in two pigs). By comparing the 24 osteomyelitic lesions, (18)F-FDG accumulated in 100%, (111)In-leukocytes in 79%, (11)C-methionine in 79%, (11)C-donepezil in 58%, and (99m)Tc-DPD in none. Overall, (18)F-FDG PET was superior to (111)In-leukocyte SPECT and (11)C-methionine in marking infectious lesions