Rift valley fever(RVF) and peste des petits ruminants(PPR) are both highly contagious diseases of ruminants caused by RVF virus (RVFV) and PPR virus (PPRV), which are both the notifiable multiple species diseases in the OIE list. In order to establish a rapid method for detecting of RVF and PPR, two pairs of primers were designed according to the nucleotide sequence information of RVFV and PPRV published in GenBank. The duplex RT-PCR which contained two pairs of the primers and two templates in one PCR system was established and then the reaction conditions were optimized including temperature and primers concentration. The results showed that the target sequence-products of RVFV and PPRV were 318bp and 589bp, and the duplex RT-PCR assay could be used to detect the RVFV and PPRV with good sensitivity and the detection limit were approximately 53 copies of PPRV and 68 copies of RVFV. Besides, the specificity test proved that there were no cross-reaction with Foot and mouth disease virus, sheep pox virus, E.coli , Salmonella and Pasteurella multocida isolated from ruminants and blood sample of healthy sheep.
To develop the rapid method for detecting of Spring viremia of carp virus (SVCV) and Cyprinid herpesvirus 2(CyHV2), 2 pairs of specific primers for duplex (reverse transcriptase) polymerase chain reaction (RT-PCR) were designed according to the nucleotide sequence information of CyHV2 and SVCV published in GenBank, based on the construction of recombinant plasmids pMD19-T-SVCVG and pMD19-T-CyHV2, a duplex RT-PCR method were established via a serial of tests, including reaction temperature optimization test, sensitivity and specificity tests, and primary application tests. The results showed that the detection limits of the duplex RT-PCR for CyHV2 and SVCV detection were both approximately 80 copies of the cloned viral genomic fragments, as well as resulted in no cross-reaction for GCRV, Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus isolated from fish, and the DNA(cDNA) of the normal Carassius auratus gibelio samples, and the application test results showed all of the 3 CyHV2 samples proved to be CyHV2 positive and there were not any amplification for the 15 clinical samples detection by the this method. The duplex RT-PCR established here is a rapid method with high specificity and sensitivity which are suitable for CyHV2 and SVCV detection.
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