减蛋综合征病毒套式PCR检测技术的研究

Detection of egg drop syndrome viruses by nested polymerase chain reaction

从GeneBank的 1段含六邻体蛋白基因、蛋白酶基因、DNA结合蛋白基因的序列中 ,应用Primer软件设计了 2对引物。用这 2对引物在分别对反应体系成分和聚合酶链反应 (PCR)试验条件进行优化后 ,成功地建立了减蛋综合征病毒(EDSV)套式PCR检测技术。对鹌鹑源EDSVQAVC 94株、AV xb西北分离株、AV 1 2 7国际标准株扩增结果 ,第一轮PCR得到约2 90bp的特异性片段 ,第二轮PCR得到约 90bp的片段 ,与预期的 2 89bp、89bp的设计相符合。而传染性腔上囊病病毒(IBDV)疫苗株B87、鸡新城疫病毒 (NDV)Ⅳ系疫苗株 (LaSota)和正常尿囊液等对照的扩增结果均未显示任何扩增条带 ,有很好的特异性。所建立的套式PCR检测方法具有特异、灵敏、快速的优点 ,其灵敏度比常规PCR灵敏 10 0倍 ,可检测出 0 .0 1fg的DNA模板。

With a sequence from the Genebank that includes part of hexon gene, DNA binding protein gene and the whole protease gene, two pairs of oligonucleotide primer sets were designed by using PRIMER program and synthesized. Through the PCRoptimization of the ingredients of the reaction system and the PCR reaction conditions. The nested PCR technology for detecting EDSV was successfully established. With this Nested PCR, a 290 bp DNA fragment was obtained from QAV C 94 , AV 127 standard strain and Xibei strain (northwest strain) of EDSV after first PCR, and a 90 bp DNA amplified product was got after second PCR (Nested PCR). The sizes of fragments were just as expected. No fragments could be amplified from IBDV B87, NDV Lasota strain and normal allantoic fluid from duck. This Nested PCR could detect EDSV in 0.01 fg with its sensitivity 100 times higher than routine PCR assay. The experimental allantoic fluid samples boiled for 10 min were used as templates for amplifcation to replace the complcated method of DNA extraction, so that the accurate results can be gotten within 24 hours. Therefore, our newly developed nested PCR is a more sensitive, accurate, specific and rapid detective method, which can be applied in clinical practices.