| 摘要: |
| 【目的】建立一种能够区分猪繁殖与呼吸综合征病毒（PRRSV）经典株和变异株的RT-nPCR诊断方法，能够从病料组织、血清或精液中直接进行目的基因检测，达到快速诊断的目的。【方法】根据GenBank上发表的猪繁殖与呼吸综合征病毒基因序列，设计并合成了2对引物，通过改变不同的反应体系和反应条件，建立PRRSV经典株和变异株的RT-nPCR诊断方法，并通过灵敏性试验、特异性试验、病料组织和血清或精液样品检测，验证建立RT-nPCR方法的适用性。【结果】建立了PRRSV经典株和变异株的RT-nPCR诊断方法，该方法检测的cDNA含量极限为1.3×10-7 pg/μL；且仅能从PRRSV毒株中扩增到目的条带，从CH-1R经典株扩增出649 bp条带，从SXXY/2013变异株扩增出562 bp条带，电泳后能清晰区别，而从其他参考毒株中均不能扩增出目的条带。从48份疑似病料组织和血清中能直接检测出31份阳性结果。【结论】建立了一种灵敏度高、特异性好、可直接从病料组织和血清中快速鉴别PRRSV经典株和变异株的RT-nPCR方法。 |
| 关键词: 猪繁殖与呼吸综合征 经典株 变异株 诊断方法 |
| DOI： |
| 分类号: |
| 基金项目:咸阳市科学技术研究发展计划项目（2014K02-21）；咸阳职业技术学院重点科研项目（2013KYA01） |
|
| Establishment of a RT-nPCR diagnostic method for PRRSV classical and variant strains |
|
ZHU Xiao-fu,WU Xu-jin
|
| Abstract: |
| 【Objective】This study established a RT-nPCR diagnostic method to distinguish classical and variants strains of porcine reproductive and respiratory virus (PRRSV) quickly from tissue and serum.【Method】Based on PRRSV gene sequences in GenBank,two pairs of primers were designed and synthesized and a RT-nPCR diagnostic method was established to distinguish classical and variants strains of PRRSV through sensitive test,specificity test,and detection of tissue and serum samples.【Result】The established method had the detection limit of 1.3×10-7 pg/μL and it only amplified target band from PRRSV.A total of 649 bp bands were amplified from classic strain CH-1R and 562 bp bands were amplified from variant strain SXXY/2013.These bands were clearly distinguished after electrophoresis,while no bands were amplified from other reference strains.31 out of the 48 suspected tissues and serum were detected positive.【Conclusion】A highly sensitive and specific RT-nPCR method was established and it can directly differentiate PRRSV classic strains and variant strains from diseased tissues and serum. |
| Key words: porcine reproductive and respiratory syndrome classical strains variant strain diagnostic methods |
