Dataset Information

Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

ABSTRACT: Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds.Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawai'i 'amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawai'i but were observed in two individuals in this study.We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.

SUBMITTER: Farias ME

PROVIDER: S-EPMC2877708 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

Farias Margaret E M ME LaPointe Dennis A DA Atkinson Carter T CT Czerwonka Christopher C Shrestha Rajesh R Jarvi Susan I SI

PloS one 20100527 5

<h4>Background</h4>Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawai'i, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds.<h4>Methodology/principal findings</h4>Here, ...[more]

PMID: 20523726

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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.

Project description:BackgroundDNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations.MethodsMinor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors.ResultsCOLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors.ConclusionsThe major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Dataset Information

Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

Publications

Taqman real-time PCR detects Avipoxvirus DNA in blood of Hawai'i 'amakihi (Hemignathus virens).

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