Journal
JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION
Volume 19, Issue 6, Pages 625-634Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/104063870701900603
Keywords
avian influenza virus; real-time reverse transcription polymerase chain reaction (RRT-PCR); subtypes H6; H9; and H11; virus detection
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Real-time reverse transcription polymerase chain reaction (RRT-PCR) is commonly used for the rapid detection, as well as to determine the subtype, of avian influenza viruses (AIVs). There are 16 known serologically distinct hemagglutinin (HA) subtypes of AIV described. Currently, determination of the subtypes of AlVs by RRT-PCR tests has been limited to the H5 and H7 subtypes. In this study, RRT-PCR assays were developed in simplex formats for rapid detection of AIV subtypes H6, H9, and H11. The primers and probes for RRT-PCR were designed from nucleotide sequences of the HA genes, which were either downloaded from GenBank (for H6 and H9) or sequenced for this study. The specificity and sensitivity of the RRT-PCR assays were determined based on the detection of the virus from a proficiency panel consisting of 15 different HA subtypes of AIVs and from serial dilutions of target viral RNA. The subtype-specific RRT-PCR assays were used to detect the virus in cloacal and oropharyngeal swabs of experimental chickens inoculated with H6, H9, and H I I AIVs, and the test results were compared with validated RRT-PCR assays based on the amplification of AI matrix (MA) gene. A high correlation of the matrix test and the specific H6, H9, and HI 1 by the RRT-PCR assays was observed; kappa coefficients for the agreement of test results in cloacal and oropharyngeal swabs combined were 0.927, 0.962, and 0.981, respectively.
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