Dataset Information

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay.

ABSTRACT: H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was recently discovered and proposed to cause runting-stunting syndrome and urate deposition in geese. At present, however, there is a lack of epidemiological information pertaining to the dynamics and distribution of H146-like GCV. The development of novel molecular diagnostic approaches capable of rapidly and accurately detecting this virus would support the strengthening, the prevention, and control of H146-like GCV infection. In the present study, we therefore used a TaqMan probe and primers specific for the viral nonstructural (NS) gene to develop a highly sensitive and specific PCR assay capable of detecting this H146-like GCV. The assay reproducibly detected 5.07 × 10² copies of a recombinant DNA plasmid containing the NS gene, with a dynamic range of 8 orders of magnitude (10²-10⁹ copies). Importantly, no cross-reactivity was observed with common viruses that affected waterfowl, and when we used this assay to evaluate clinical samples, we found it to be more sensitive and faster than traditional PCR. In summary, herein, we developed a novel TaqMan-based real-time PCR approach that could reliably detect and diagnose H146-like GCV. This tool will allow for the real-time diagnosis of H146-like GCV infections, enabling researchers to better understand the epidemiology and clinical presentation of this disease.

SUBMITTER: Zheng M

PROVIDER: S-EPMC7858078 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay.

Zheng Min M Lin Su S Zhang Shizhong S Chen Xiuqin X Jiang Dandan D Chen Shaoying S Wang Shao S Chen Shilong S

Poultry science 20201119 2

H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was recently discovered and proposed to cause runting-stunting syndrome and urate deposition in geese. At present, however, there is a lack of epidemiological information pertaining to the dynamics and distribution of H146-like GCV. The development of novel molecular diagnostic approaches capable of rapidly and accurately detecting this virus would support the strengthening, the ...[more]

PMID: 33518100

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Project description:BackgroundClassic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV.ResultsAfter genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings.ConclusionsWe established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Project description:BACKGROUND:The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples. METHODS:A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples. RESULTS:Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/?L and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/?L of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA. CONCLUSIONS:This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

Dataset Information

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay.

Publications

Rapid detection of H146-like goose calicivirus using a TaqMan-based real-time PCR assay.

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