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It is difficult to differentiate pigs that have been injected attenuated vaccines from those that are infected by a virulent virus. In this study, a duplex reverse transcription polymerase chain reaction (RT-PCR) was developed for simultaneous detection and identification of the pathogenic strain (Shimen-strain) and attenuated vaccine strain (C-strain) of classical swine fever virus (CSFV). A fragment of 438 bp or 265 bp was amplified from the genomic RNA of the Shimen-strain or the C-strain, respectively. Both types of viruses were simultaneously identified from the mixed samples of Shimen-strain and C-strain, while no PCR bands were obtained from the templates of some other porcine original viruses with the designed primers. The lowest limit of detection was 0.0616 pg for the C-strain RNA and 0.0844 pg for the Shimen-strain RNA. The developed RT-PCR was adopted to test CSFV from 65 collected samples. Compared with quantitative real-time PCR, the results indicated that the developed RT-PCR can not only be used to distinguish the virulent and attenuated CSFV, but also possesses high sensitivity for detecting CSFV. This is a convenient method to detect and differentiate pigs infected with virulent CSFV from the pigs immunized with the C-strain. [ABSTRACT FROM AUTHOR]