H7亚型禽流感病毒一步法RT-PCR检测方法的建立

Development of one step RT-PCR technique for detection of H7 subtype avian influenza

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501 bp。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105.5 EID50/mL; 阳性棉拭子最低检出量为103 EID50/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。

Abstract:

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.