PRRSV经典株和变异株RT-nPCR鉴别诊断方法的建立

发布时间：2018-09-05 07:31

【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649bp条带,从SXXY/2013变异株扩增出562bp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。
[Abstract]:[objective] to establish a method for the diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) (PRRSV) classic strain and variant strain by RT-nPCR, which can detect the target gene directly from the diseased tissue, serum or semen. [methods] according to the gene sequence of porcine reproductive and respiratory syndrome virus published on GenBank, two pairs of primers were designed and synthesized. To establish a RT-nPCR diagnostic method for classical and variant strains of PRRSV, and to detect the tissues and serum or semen samples of PRRSV by sensitivity test, specificity test. [results] A RT-nPCR diagnostic method for classical and variant strains of PRRSV was established. The limit of cDNA content detected by this method was 1.3 脳 10 ~ (-7) pg/ 渭 L, and only the target bands could be amplified from PRRSV strains and 649bp bands could be amplified from CH-1R strains. The 562bp bands were amplified from the SXXY/2013 mutant strains, but the target bands could not be amplified from other reference strains. 31 positive results could be directly detected from 48 samples of suspected samples and sera. [conclusion] A RT-nPCR method with high sensitivity and good specificity was established for the rapid identification of classical and variant strains of PRRSV from diseased tissue and serum.
【作者单位】：咸阳职业技术学院畜牧兽医研究所动物疫病分子生物学诊断实验室;
【基金】：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)
【分类号】：S852.651

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