Publications (3)2.43 Total impact
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Article: A multiplex real-time reverse transcription polymerase chain reaction assay for detection and differentiation of Bluetongue virus and Epizootic hemorrhagic disease virus serogroups.
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ABSTRACT: Bluetongue virus (BTV) causes disease in domestic and wild ruminants and results in significant economic loss. The closely related Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Therefore, rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the early detection of indigenous and exotic BTV and EHDV is described. This sequence data foundation focused on 2 conserved target genes: one that is highly expressed in infected mammalian cells, and the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for both a virus group-comprehensive and virus group-specific gene amplification diagnostic test. This information has been used as the basis for the development of a rapid multiplex BTV-EHDV real-time RT-PCR that detects all known serotypes of both viruses and distinguishes between BTV and EHDV serogroups. The sensitivity of this rapid, single-tube, real-time RT-PCR assay is sufficient for diagnostic application, without the contamination problems associated with standard gel-based RT-PCR, especially nested RT-PCR tests.Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 11/2009; 21(6):760-70. · 1.21 Impact Factor -
Article: Detection of all eight serotypes of Epizootic hemorrhagic disease virus by real-time reverse transcription polymerase chain reaction.
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ABSTRACT: Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection. Differentiation of Bluetongue virus (BTV) and EHDV is necessary because diagnosis of infection caused by these viruses is often confused. The previously developed nested reverse transcription polymerase chain reaction (nRT-PCR) test for indigenous EHDV disease is sensitive and specific, but it is prone to contamination problems. Additionally, the EHDV nRT-PCR only detects 7 of the 8 serotypes. To develop an improved diagnostic test, sequence analysis was performed on 2 conserved target genes; one is highly expressed in infected mammalian cells, whereas the other is highly expressed in infected insect cells. This information was used to develop a rapid EHDV real-time PCR that detects all 8 EHDV serotypes. The EHDV assay did not cross-react with BTV strains and performed similarly to the nRT-PCR tests with archived clinical samples. In addition, it is superior to the nRT-PCR, not only because it is a closed system with fewer cross-contamination problems, but also because it detects all 8 serotypes and is less labor and time intensive.Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 04/2009; 21(2):220-5. · 1.21 Impact Factor -
Article: Detection of all eight serotype of Epizootic hemorrhagic disease virus by real-time reverse transcription polymerase chain reaction.
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ABSTRACT: Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection. Differentiation of Bluetongue virus (BTV) and EHDV is necessary because diagnosis of infection caused by these viruses is often confused. The previously developed nested reverse transcription polymerase chain reaction (nRT-PCR) test for indigenous EHDV disease is sensitive and specific, but it is prone to contamination problems. Additionally, the EHDV nRT-PCR only detects 7 of the 8 serotypes. To develop an improved diagnostic test, sequence analysis was performed on 2 conserved target genes; one is highly expressed in infected mammalian cells, whereas the other is highly expressed in infected insect cells. This information was used to develop a rapid EHDV real-time PCR that detects all 8 EHDV serotypes. The EHDV assay did not cross-react with BTV strains and performed similarly to the nRT-PCR tests with archived clinical samples. In addition, it is superior to the nRT-PCR, not only because it is a closed system with fewer cross-contamination problems, but also because it detects all 8 serotypes and is less labor and time intensive.
