A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain).

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Abstract
Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

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