6种虫媒病毒微孔膜芯片检测方法的研制与应用

目的研制能同时检测6种口岸重要虫媒病毒的微孔膜芯片。方法针对包括1—4型登革病毒、乙型脑炎病毒、西尼罗病毒、黄热病毒、基孔肯雅病毒和裂谷热病毒等6种虫媒病毒，选择合适的保守基因，设计特异性的PCR引物（5’端标记生物素）和检测探针，通过参数优化建立单管多重RT—PCR扩增体系；然后按每个阵列5X5的格式，并确保点样区域为96孔板的微孔大小，将探针喷点到处理后的尼龙膜上，通过条件优化建立稳定的PCR产物与固化探针的杂交体系；采用碱性磷酸酯酶标记链亲和素和化学显色底物NBT／BCIP来检测特异性的PCR杂交产物。选取2012年1—6月份从口岸输入的疑似登革热发热病例的临床血清标本，提取RNA后，直接采用本研究建立的微孔膜芯片进行未知虫媒病毒的快速检测。结果用1～4型登革病毒、乙型脑炎病毒、西尼罗病毒和基孔肯雅病毒等7种毒株、1种黄热病毒疫苗株和1种裂谷热病毒核酸体外转录的RNA模板验证已建立的微孔膜芯片，获得比较特异和稳定的实验结果。应用该研究建立的方法，从3份疑似登革热发热病例的临床血清标本中检出了1例登革1型病毒和2例登革2型病毒，与实时荧光PCR检测结果相符。结论该研究建立的6种虫媒病毒微孔膜芯片检测方法，具有快速、准确、自动化和高通量等特点，为快速应对口岸输入性发热病例提供了非常有价值的检测手段，也为进一步开发更多指标的病原体检测方法提供良好的示范作用。

Development and application of microwell membrane array for detection of six species of arboviruses

Objective To develop a microwell membrane array for simultaneous detection of six species of important arboviruses at frontier port. Methods Specific PCR primers labeled with biotin and ol- igonucleotide probes were designed based on the sequence of conservative genes of six arboviruses specises, including four serotypes of dengue virus, Japanese encephalitis virus, West Nile virus, Yellow Fever virus, Chikungunya virus and Rift Valley Fever virus. A one-tube multiplex RT-PCR system for simultaneous am- plification of these viruses was established. DNA probes were sprayed and immobilized on the membrane in 5 x 5 array format. Also, we confirmed the array size of the spray areas were suitable for the 96-microwell. Stable hybridization system between PCR products and oligonucleotide probes was established. Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products. Clin- ical serum samples from suspected cases of dengue fever imported at frontier port from January to June 2012 were selected, then a rapid detection of unknown arboviruses was performed directly using the microwell membrane array developed in this study after RNA was extracted. Results The microwell membrane array was tested by using six strains of viruses including four serotypes of dengue virus, Japanese encephalitis vi- rus, West Nile virus, Chikungunya virus, one vaccine strain of Yellow Fever virus, and one in-vitro-tran-scribed RNA template of Rift Valley Fever virus. Highly specific and stable experiment results were ob- tained. When applying this assay, one case of dengue virus type 1 and two cases of dengue virus type 2 were detected in three clinical serum samples from suspected cases of dengue fever. These results were con- sistent with the real-time PCR data. Conclusion The novel microwell membrane array allows rapid, accu- rate, automated and high-throughput arboviruses detection. It is especially valuable for quick response to fever cases imported at frontier port. Also, it becomes a very good model for development of pathogen de- tection method with additional parameters.