摘要
【目的】建立一种快速、敏感的ENTV-2检测方法,用于ENT的早期诊断与流行病学调查。【方法】通过生物信息学的方法将ENTV-2与ENTV-1、ERVs、JSRV进行比对,寻找ENTV-2的保守序列并设计1对特异性引物,建立SYBR-GreenⅠ实时荧光定量PCR检测方法,对所建立的PCR反应条件进行优化,将PCR扩增产物连接T载体构建的阳性质粒作为标准品,对SYBR-Green Ⅰ实时荧光定量PCR检测方法的特异性、敏感性和重复性进行验证。【结果】建立的检测ENTV-2 qPCR方法标准曲线呈现良好的线性关系(R^(2)=0.992);该方法的特异性良好,对ENTV-2可以产生特异性扩增曲线,与ENTV-2高度同源的ERVs没有交叉反应,也无法扩增羊口疮病毒(ORFV)、绵羊肺炎支原体(Mo)、丝状支原体山羊亚种(Mmc)等常见病原;敏感性良好,最低检测限度为7.5×10^(2) copies·μL^(-1),敏感性可达常规PCR检测方法的100倍;批内、批间的变异系数CV<1%,重复性良好;对81份临床样品的阳性检出率为17.3%。【结论】建立的SYBR-Green Ⅰ实时荧光定量PCR方法特异性良好,敏感性较高,重复性良好,为山羊地方性鼻内肿瘤的早期、快速检测提供了技术支持。
【Objective】A rapid,sensitive detection method for early diagnosis and epidemiological survey on enzootic nasal tumor(ENT)in goats was established.【Method】Bioinformatics methods were employed for sequence alignment of ENTV-2 with ENTV-1,ERVs,and JSRV in search for the conserved sequence of the virus.Primers for qPCR were designed to establish a SYBR-Green Ⅰ RT-qPCR methodology for its detection.Reaction conditions were optimized,and a standard positive plasmid used to determine the specificity,sensitivity,and reproducibility of the newly developed assay.【Result】A linear standard curve was found for the detection with a R^(2)=0.992.The method specifically detected only ENTV-2,not the highly homologous ERVs,nor amplified ORFV,MO,or Mmc.It showed a detection sensitivity of up to 7.51×10^(2) copies·μL^(-1),which was 100 times greater than the conventional PCR can deliver,a repeatability with a coefficient variations(CV)of less than 1% on intraand inter-batch tests,and a positive detection rate of 17.3% on 81 clinical samples.【Conclusion】The newly established sensitive,repeatable,and considered adequate for early and rapid detection of ENTV-2 in goats.
作者
张靖鹏
江锦秀
林裕胜
游伟
刘道泉
毛坤明
江斌
胡奇林
ZHANG Jingpeng;JIANG Jinxiu;LIN Yu Sheng;YOU Wei;LIU Daoquan;MAO Kunming;JIANG Bin;Hu Qilin(Institute of Animal Husbandry and Veterinary Medicine,Fuzhou,Fujian 350013,China;Fuzhou Animal Disease Prevention and Control Center,Fuzhou,Fujian 350002,China;Fuqing Technical Service Center of Animal Husbandry and Veterinary Medicine,Fuzhou,Fujian 350300,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2021年第7期779-784,共6页
Fujian Journal of Agricultural Sciences
基金
福建省科技重大专项(2019NZ09007)
福建省科技计划公益类专项(2018R1023-1)。
