摘要
通过分析流感数据库45个H7亚型禽流感病毒的HA序列,在保守区内设计并合成引物,建立了一步法RT-PCR检测方法,扩增片段大小为501bp。通过对H7亚型禽流感病毒尿囊液和棉拭子浸出液不同滴度检测,证实病毒尿囊液最低检出量为105.5EID50/mL;阳性棉拭子最低检出量为103EID50/mL。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原进行检测,仅有H7亚型AIV有特异性目的条带,与其他均无交叉反应。从脏器及咽喉、泄殖腔棉拭子样品的病毒分离和RT-PCR方法比较,表明在10-1的样品浓度下,两者可以达到相同的检出量。表明该一步法RT-PCR方法具有特异性强、敏感性高和准确率高的特点。
According to 45 hemagglutinin（HA） gene sequences of H7 subtype of avian influenza virus（AIV）,a pair of specific oligonucleotide primers was designed.We developed one step RT-PCR for detecting AIV subtype H7.Sensitivity to detection of allantoic fluid by one step RT-PCR reached 105.5 EID50/mL and detection of swab samples reached 103 EID50/mL.We simultaneity detected the tissue and swab samples infected with H7 subtypes AIV by one step RT-PCR and virus isolation method.The results showed that the sensitivity of the assay gave an excellent correlation with the conventional virus isolation method.H1-H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR.The results showed the assays were high specific,without cross-reaction with other subtypes or other avian diseases.Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of AIV subtype H7.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第11期1658-1663,共6页
Chinese Journal of Biotechnology
基金
防禽流感高效生物制剂试生产开发项目(No.2007DFR30360)资助~~
