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The article presents information on a study which evaluated a multiplex real-time reverse transcript polymerase chain reaction assay for detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus strains that occur in Mexico and North and Central America. It explores the sequence of primers and probes used in real-time reverse transcription polymerase chain reaction. It reveals the results of diagnostic sensitivity, specificity, and accuracy of Vesicular stomatitis virus real-time reverse transcription polymerase chain reaction tests on samples using two alternative cutoff threshold cycle values.