Development Of Real-time RT-PCR Assay For Detection Of The Highly Pathogenic Porcine Reproductive And Respiratory And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV) was the causative agent of porcine reproductive and respiratory syndrome(PRRS),a newly recognized Pig disease of economic importance,characterized by reproductive failures(abortions, stillbirths,mummied fetuses)and respiratory problems in any age.One of the most variated in the genome of PRRSV was the Nsp2 gene.May of 2006,serious outbreaks of a highly contagious disease marked by high fever and high morbidity and mortality have occurred in China.Etiology studies confirmed that the disease was caused by a highly pathogenic PRRSV characterized by two non-continued deletions in the Nsp2 region of viral genome.1 In this study,the obtained partial SC-JX01 Nsp2 gene was cloned into pMD-T vector.Got the pMD-Nsp2 plasmid and the gene was got by sequencing.The result showed that there was high homology between the SC-JX01 Nsp2 gene and other Highly pathogenic PRRSV Nsp2 gene sequences published from the GenBank,which was an identity of 98.1%-99.1%,and the indentity of deduced amino acids was 96.3%-98.3%.It also showed that the obtained PRRSV strain in SC-JX01 belonged to Highly pathogenic PRRSV in genotype;Meanwhile,30 amino acids deletion were found by sequence analysis and comparison with the published sequences of other strains by software DNASTAR. Results showed that the Nsp2 gene of SC-JX01 strain had the same deletion compared with that of EF11245,EF112446,EF112447,EU106888,EU109502,EU109503 that isolated from other districts in 2006.The Hydrophobicity,antigenic index and surface probability analysis of SC-JX01 Nsp2 protein were the same as the feature of the SC-JX01 Nsp2 protein.2 A pair of primers was designed and synthesized according to the deletion information of Nsp2 gene sequences of the highly pathogenic PRRSV,and a real-time RT-PCR assay based on Power SYBR Green was developed for detection of the highly pathogenic PRRSV Nsp2 mutant strain.The standard curve was constructed by using the pMD-Nsp2 plasmid.Subsequently,specificity,sensitivity as well as repeatability of the assay were evaluated.In addition,the sensitivity between real-time RT-PCR and a conventional RT-PCR was compared.The assay exhibited high specificity as positive amplification could only be obtained from the highly pathogenic PRRSV Nsp2 mutant strain,and the PCR product was confirmed to be the specific fragment of the highly pathogenic PRRSV Nsp2 gene sequence.The real-time RT-PCR had a broad linear range (3.15×10~9 copies -3.15×10~0copies,R~2=0.998 355).The detection limit of the assay was 3.15 virus copies.The intra-assay and inter-assay coefficient of variations of the assay were in the range of 0.38～2.22%and 1.74%～3.57%,respectively,showing a good repeatability.3 Based on the Principle of TaqMan Probe quantitative assay,the TaqMan probe and primers were designed and synthesized with Primer Express 3.0 software according to the Nsp2 gene sequence of the highly pathogenic PRRSV available in GenBank.Then,a Real-time TaqMan RT-PCR for differential diagnosis was established.The plasmid pMD-Nsp2 which contained the whole gene of the highly pathogenic PRRSV Nsp2 was used as the control template,and then reaction parameters were optimized to develop Real-time TaqMan RT-PCR assay.At the same time the sensitivity,specifity,reproducibility and quantitation range of this method were also determined.A known concentrion of one 10×series of dilutions of pMD- Nsp2 DNA were detected by using the established quantitative RT-PCR assay,and the results was compared with that of routine RT-PCR.The developed quantitative RT-PCR assay could detect 3.15 copies with an excellent linear relation of plasmid DNA and its sensitivity was 100 times higher than that of the routine RT-PCR.Three plasmid standards were examined using the Real-time RT-PCR repeatedly and the results indicated that the Nsp2-TaqMan RT-PCR was reproducible.The intra-assay and inter-assay coefficient of variations of the assay were in the range of 0.47%～1.61%and 1.64%～3.89%,respectively.Furthermore,the serum and other tissue samples from pigs on farms were detected by using the Nsp2-TaqMan RT-PCR. We found the sensitivity of Nsp2-TaqMan RT-PCR was prior to that of conventional RT-PCR by detecting the positive field samples,while the results of the quantitative RT-PCR were the same as that of the virus isolation.The Nsp2-TaqMan RT-PCR had a good accuracy and fewer samples was enoug and could be used for the diagnosis of the highly pathogenic PRRSV Nsp2 mutant strain infection.4 The experiments revealed that the developed two techniques(Power SYBR Green RT-PCR and TaqMan RT-PCR) offer the high specifity,sensitivity,rapid,reproducibility and high throughput for quantitative assay with the advantage of savings in both time and material,the closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2h or less.The assay provided a new way for rapid and early diagnosis and molecular epidemiological investigation of the highly pathogenic PRRSV Nsp2 mutant strain,and has a strong clinical relevance.