犬细小病毒实时荧光环介导等温扩增检测方法的建立

发布时间：2018-08-06 09:54

【摘要】：参照GenBank中犬细小病毒(CPV)VP2基因保守区域序列设计合成特殊的内、外引物,在扩增反应体系中加入荧光染料SYBR GreenⅠ,通过恒温荧光检测仪Deaou-308C扩增检测,建立了一种快速检测CPV的实时荧光环介导等温扩增(LAMP)方法。结果显示,这种方法对CPV具有特异性的扩增反应,对照病毒核酸的扩增结果均为阴性。灵敏度试验最低可检出3.72 copies/μL的病毒核酸。重复性试验结果表明,其检测重复性良好。对30份临床宠物犬的粪便样品进行检测,与免疫胶体金检测试纸方法进行比较,符合率为90%。将本方法初步应用于大熊猫粪便中犬细小病毒的检测,结果表明,从大熊猫基地采集的84份粪便样品中有16份为细小病毒阳性,阳性率为19.0%。本方法在63℃下恒温45 min可以完成检测,操作简单、灵敏度高、特异性强,通过恒温实时荧光检测仪可实时检测及自动判读检测结果,对大熊猫的早期诊断非常重要。
[Abstract]:Specific internal and external primers were designed and synthesized according to the conservative region sequence of the canine parvovirus (CPV) VP2 gene in GenBank. The fluorescent dye SYBR Green I was added to the amplification reaction system, and a real-time fluorescence ring mediated isothermal amplification (LAMP) method for rapid detection of CPV was established. The results showed that this method was used for rapid detection of CPV. CPV has a specific amplification reaction, and the amplification result of the virus nucleic acid is negative. The sensitivity test can detect the virus nucleic acid of 3.72 copies/ mu L. The repeatability test results show that the detection repeatability is good. The fecal samples of 30 clinical pet dogs are detected and the colloid gold detection test paper method is carried out. The results showed that the method was applied to the detection of canine parvovirus in the feces of pandas by 90%.. The results showed that 16 of the 84 fecal samples collected from the giant panda base were positive for parvovirus, and the positive rate was 19.0%. at the constant temperature of 45 min at 63 C, which was simple, sensitive and specific. The constant temperature real-time fluorescence detector can detect and interpret the test results in real time. It is very important for the early diagnosis of giant pandas.
【作者单位】：四川农业大学动物医学院;东莞出入境检验检疫局;
【基金】：成都大熊猫繁育研究基金会项目(CFP研2012-12、CFP研2012-9)
【分类号】：S852.655

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