Dataset Information

Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

ABSTRACT: Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/?l for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10(-1) to 10(3) 50% tissue culture infective dose (TCID(50))/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs.

SUBMITTER: Buckwalter SP

PROVIDER: S-EPMC3295144 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

Buckwalter Seanne P SP Teo Rose R Espy Mark J MJ Sloan Lynne M LM Smith Thomas F TF Pritt Bobbi S BS

Journal of clinical microbiology 20111214 3

Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diar ...[more]

PMID: 22170906

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Project description:Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 10(2) to 3.7 x 10(2) CFU/ml in bacterial cultures and 6.6 x 10(2) CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.

Project description:BackgroundAvian pox is a viral disease documented in a wide range of bird species. Disease-related detrimental effects can cause dyspnea and dysphagia, and birds with high metabolic requirements, such as hummingbirds, are thus especially vulnerable to the pathogen. Hummingbirds have a strong presence in California, especially in urban environments. However, little is understood regarding the impact of pox virus on hummingbird populations. Currently, diagnosing a pox infection relies on obtaining a tissue biopsy, which poses significant risks to birds and challenges in the field. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. Our aim was to address whether pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds.Methodology and principal findingsPox-like lesion tissue, pectoral muscle, feathers, toenail clippings, blood, and swabs (both pox-like lesion tissue and non pox-like lesion tissue) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types obtained from 27 individuals. These results were compared to those of conventional PCR and comparisons were also made among sample types, utilizing lesion tissue samples as the gold standard.Conclusions and significanceHummingbird avian pox can be diagnosed without relying on tissue biopsies. We identify that feather samples, of which contour feathers yielded the best results, can be used for diagnosing infected birds, thus reducing sampling risk. In sum, the real-time PCR assay detected viral DNA in various integumentary system sample types and will be useful in future studies of hummingbird disease ecology.

Dataset Information

Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

Publications

Real-time qualitative PCR for 57 human adenovirus types from multiple specimen sources.

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