| Peste des petits ruminants is an acute virus disease characterized as febricity, a great deal of secretion from eyes and nose, anabrosis, necrotic stomatitis, gastricism, diarrhea and pneumonia. In July 2007, PPR broke out in Ritu county, Ali area, Tibet of China. It was the first PPR outbreak in China and a number of 272 goats and sheep died. After that, PPR also broke out in Naqu area. As an important transnational animal epidemic disease, PPR does a serious threat to the safety of animal heath and the development of stock raising in China. Therefore, enforcement actions of prevention, control and eradication must be adopted. In this study, a rapid and specific TaqMan-based, one-step real-time qRT-PCR for the detection of PPRV was established, and the molecular characteristic of F gene was analyzed.According to the sequences of F gene of PPRV reported in India, Turkey, Nigeria and other countries in Genebank, primers and probe were designed based on the fusion protein gene sequences. The PPRV detection system was optimized in the use of primers and probe.Most RNA positive control currently used for monitoring the qRT-PCR assays have some disadvantages, such as instability and inaccuracy for quantification. To avoid these disadvantages, a simple method to prepare stable RNA positive standards was demonstrated with qRT-PCR assay for the detection of PPRV. T7 promoter was added to the 5′of the forward primer designed above. RNA extracted from the PPRV vaccine strain Nigeria 75/1 cell culture medium was amplified by forward primer added T7 promoter and reverse primer, and the PCR products were reclaimed as the template of transcription in vitro. After transcription in vitro, OD value was detected. The results indicated the template has good linearity and specificity. After storing at -80℃for six month, the template was also stable as before. And the assay has a linear range from 4.9×103 to 4.9×108 copies/μl as well as excellent intra-assay and inter-assay reproducibilities. The coefficient of variation value for intra-assay and inter-assay reproducibilities were ranged from 0.13% to 0.5% and from 0.44% to 1.71% respectively. The correlation coefficient of the standard curve is 0.9997. The specificity of the assay was assessed by detecting four lines of PPRV, RPV, CDV and healthy sheep. The analytical sensitivity of the real-time qRT-PCR assay was achieved through the construction of in-house PPRV cRNA standard for the generation of a standard curve. The sensitivity of the assay was found to be 490 RNA copies per reaction. And the method of one-step real-time RT-PCR is 100 times more sensitive than the method of conventional RT-PCR.Samples including blood, nasopharyngeal swabs and organs from goats were collected in Tibet for PPRV detection using qRT-PCR established in this study and another qRT-PCR detecting N gene, conventional RT-PCR detecting N gene, conventional RT-PCR detecting F gene. The results indicated that the positive detection rates of the two qRT-PCR assays are matched and their positive detection rates were higher than that of the two conventional assays.The F gene nucleotide sequence of Chinese PPRV (China/Tib/Gej/07-30) was firstly determined. It is 2411 nucleotides long, encoding a protein of 546 amino acids. The nucleotide and deduced amino acid sequences were compared with the homologous regions of other PPRV isolates. The nucleotide sequence of F gene of the"China/Tib/Gej/07-30"was 85.5-96.1% identical to other PPRV isolates, respectively. While a homology of 94.3-98.2% could be observed in the amino acids level. Several sequence motifs in the F genes has been identified on the basis of conservation in the PPRVs and the morbilliviruses. The 5' untranslated region of F gene is 634 nucleotides long with 76.2-91.7% identical to other PPRV isolates. |
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