绵羊肺腺瘤病毒巢式RT-PCR检测方法的建立与验证

发布时间：2018-05-07 22:27

本文选题：绵羊肺腺瘤病 + 巢式PCR　；参考：《中国兽医科学》2017年01期

【摘要】：根据Gen Bank上发表的外源性绵羊肺腺瘤病毒(exJSRV)的基因序列,分别设计合成巢式RT-PCR引物及扩增exJSRV-env全基因的引物,建立exJSRV的检测方法。将病肺的巢式外套与内套RT-PCR扩增产物与载体连接,对重组质粒送检测序进行序列分析。结果显示,巢式外套与内套RT-PCR扩增产物的序列与JQ837489.1序列的同源性分别为99.7%和99.4%。通过对3份鼻液以及75份血样的临床检测,将检测阳性的1份鼻液和2份血样送检测序,序列比对结果显示,鼻液、血样的巢式外套和内套RT-PCR扩增产物与JQ837489.1的同源性分别为99.8%、99.7%、99.7%和98.9%、100%、99.4%,且扩增基因序列的氨基酸序列中具有exJSRV特有的致病性YXXM序列。敏感性试验表明,巢式RT-PCR诊断方法的敏感性是普通RT-PCR的10倍,最低能够检测出9.72 fg的标准模板RNA。说明本试验所建立的方法,具有良好的临床应用价值,灵敏性高、特异性好,对exJSRV的临床早期检测提供了一种方便、可靠、经济实用的诊断方法。
[Abstract]:According to the gene sequence of exJSRV published on Gen Bank, nested RT-PCR primers and primers for amplifying the whole exJSRV-env gene were designed and synthesized, and the detection method of exJSRV was established. The RT-PCR amplification products of the nested coat and the inner sleeve of the diseased lung were connected with the vector and the recombinant plasmids were sequenced and sequenced. The results showed that the homology of RT-PCR amplification products between nested coat and inner jacket was 99.7% and 99.4%, respectively, and the homology of JQ837489.1 sequence was 99.7% and 99.4% respectively. Through clinical examination of 3 nasal fluids and 75 blood samples, 1 nasal fluid and 2 blood samples were tested and sequenced. The homology of the nested RT-PCR amplification products of blood samples with JQ837489.1 was 99.8% and 99.7%, respectively, and 98.9% and 99.4%, respectively. The amino acid sequence of the amplified gene sequence had the pathogenicity YXXM sequence of exJSRV. The sensitivity test showed that the diagnostic sensitivity of nested RT-PCR was 10 times as high as that of RT-PCR, and the lowest 9.72 fg standard template could be detected. This method has good clinical application value, high sensitivity, good specificity, and provides a convenient, reliable, economical and practical diagnostic method for early clinical detection of exJSRV.
【作者单位】：内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室;
【基金】：国家自然科学基金项目(31360597) 内蒙古科技厅应用研究项目(201502070) 内蒙古草原英才创新团队项目(20151031)
【分类号】：S852.654

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