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Infectious Bronchitis Virus Detection in Allantoic Fluid using the Polymerase Chain Reaction and a DNA Probe

Mark W. Jackwood, Hyuk Moo Kwon and Deborah A. Hilt
Avian Diseases
Vol. 36, No. 2 (Apr. - Jun., 1992), pp. 403-409
DOI: 10.2307/1591520
Stable URL: http://www.jstor.org/stable/1591520
Page Count: 7
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Infectious Bronchitis Virus Detection in Allantoic Fluid using the Polymerase Chain Reaction and a DNA Probe
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Abstract

A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embyronating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses. /// Se desarrolló un procedimiento de extracción rápida para purificar el ARN del virus de la bronquitis infecciosa, a partir del líquido alantoideo de embriones previamente inoculados. Se usaron la transcripción inversa y la reacción de cadena por la polimerasa para amplificar el genoma viral de 8 cepas del virus de bronquitis infecciosa pertenecientes a 5 serotipos diferentes. En una prueba de hibridización puntual se usó una sonda de ADN con biotina, preparada contra una secuencia dentro del producto amplificado por la reacción de cadena por la polimerasa, de la cepa Beaudette de bronquitis infecciosa. Se detectaron los productos de la amplificación de todas las cepas estudiadas del virus de bronquitis infecciosa. La transcripción inversa y la amplificación por la reacción de cadena por la polimerasa se consideraron específicas para el virus de bronquitis infecciosa, debido a que los productos de amplificación no fueron detectados por electroforesis en geles de agarosa ó por la prueba de hibridización puntual, cuando el patrón usado en la reacción de cadena por la polimerasa fue extraído del líquido alantoideo y de la membrana corioalantoidea de embriones inoculados con otros virus que infectan el tracto respiratorio superior de las aves.

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