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Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness.

ABSTRACT: BACKGROUND: Influenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak. METHODS: We developed a multiplexed PCR (MT-PCR) assay for the simultaneous diagnosis of respiratory viruses causing influenza-like illness, including the specific recognition of influenza A haemagglutinin subtypes H1, H3, and H5. We tested several hundred clinical specimens in two diagnostic reference laboratories and compared the results with standard techniques. RESULTS: The sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens. The MT-PCR assays provided differential diagnoses as well as potentially useful quantitation of virus in clinical samples. CONCLUSIONS: MT-PCR is a potentially powerful tool for the differential diagnosis of influenza-like illness in the clinical diagnostic laboratory.

SUBMITTER: Szewczuk E

PROVIDER: S-EPMC2881921 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness.

Szewczuk Elektra E Thapa Kiran K Anninos Terry T McPhie Kenneth K Higgins Geoff G Dwyer Dominic E DE Stanley Keith K KK Iredell Jonathan R JR

BMC infectious diseases 20100511

<h4>Background</h4>Influenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak.<h4>Methods</h4>We developed a multiplexed PCR (MT-PCR) assay for the simultaneous diagnosis of respiratory viruses causing influenza ...[more]

PMID: 20459845

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Project description:BackgroundThe diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.MethodsTwo multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC.ResultsMultiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.ConclusionsOur multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

Dataset Information

Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness.

Publications

Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness.

Similar Datasets

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