| 孙俊颖,李吉初,伍绮文,等.流行性乙型脑炎病毒、西尼罗病毒、黄热病毒和寨卡病毒四重Luminex xTAG检测方法的建立与应用[J].广东农业科学,2024,(11-12):- |
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流行性乙型脑炎病毒、西尼罗病毒、黄热病毒和寨卡病毒四重Luminex xTAG检测方法的建立与应用 |
| Development and application of Quadruple Luminex xTAG method for simultaneous detection of Japanese encephalitis virus, West Nile virus，Yellow fever virus and Zika virus |
| 投稿时间：2024-05-29 修订日期：2024-11-20 |
| DOI： |
| 中文关键词: 虫媒病毒 流行性乙型脑炎病毒 西尼罗病毒 黄热病毒 寨卡病毒 液相芯片 Luminex xTAG技术 |
| 英文关键词: arbovirus Japanese encephalitis virus West Nile virus Yellow fever virus Zika virus liquid chip Luminex xTAG |
| 基金项目:广州市开发区国际科技合作项目（2019GH08）；十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目（2022SDZG02）；海南省重点研发计划项目（ZDYF2022SHFZ085）；国家自然科学基金项目（82060378） |
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| 中文摘要: |
| 【目的】黄病毒科虫媒病毒引起的人畜共患传染病对我国畜牧业及公共卫生造成严重影响。虫媒病毒种类多且症状相似，临床监测工作繁重，建立危害严重的4种黄病毒科虫媒病毒快速、高通量检测技术为虫媒病毒诊断和流行病学监测提供技术支撑。【方法】利用Luminex xTAG技术，针对流行性乙型脑炎病毒（JEV）5' UTR基因、西尼罗病毒（WNV）5' UTR基因及部分C基因、黄热病毒（YFV）5' UTR基因和寨卡病毒（ZIKV）NS5基因保守区，设计4对特异性引物并对引物进行TAG和Biotin修饰，以标准毒株为模板，进行多重PCR扩增；将扩增产物与带有反向TAG序列的不同编号荧光MagTAG磁珠、链霉亲和素-藻红蛋白进行核酸杂交反应，利用Luminex 200系统检测磁珠荧光信号和藻红蛋白荧光信号，对病原进行分类和定量检测。【结果】利用Luminex xTAG方法检测JEV、WNV 、 YFV 、ZIKV 4种病毒时，其最优引物工作比例：JEV : WNV : YFV : ZIKV为0.5 μmol/L: 0.5 μmol/L : 0.75 μmol/L : 0.5 μmol/L；建立杂交工作体系为：磁珠工作液20 μL、PCR扩增产物5 μL、SAPE报告缓冲液75 μL；杂交温度为37 ℃，杂交时间为30 min，缓冲体系pH值为8.0；其能特异性检测JEV、WNV、YFV和ZIKV，不与登革病毒等其他虫媒病毒发生交叉反应。重复性实验结果表明，Luminex xTAG方法的批内变异系数为2.50%~5.63%，批间变异系数为3.61%~12.50%。四重Luminex xTAG方法对JEV和ZIKV的检测限为1×104 copies/μL，对WNV和YFV的检测限为1×103 copies/μL，其检测WNV、YFV和ZIKV的灵敏度较常规PCR高10-100倍。用Luminex xTAG方法和RT-qPCR方法检测209份临床样品和模拟样品，JEV、WNV、YFV、ZIKV符合率分别为100%、100%、100%和98.7%， Luminex xTAG方法检出率高于RT-qPCR方法。【结论】建立的四重Luminex xTAG方法具有高通量、高特异性和灵敏度、成本效益高的优点，可为虫媒病毒临床诊断和流行病学监测提供一种高通量技术手段。 |
| 英文摘要: |
| Abstract: 【Objective】The zoonotic infectious diseases caused by arbovirus of family Flavivirus have a severe impact on China’s animal husbandry and public health. This study intends to establish a fast and high-throughput detection technologiesy for four serious Flavivirus arbovirus to provide technical support for clinical diagnosis and epidemiological monitoring of arbovirus. 【Method】Based on Luminex xTAG technology, four pairs of specific primers were designed for 5' UTR gene of Japanese encephalitis virus (JEV), 5' UTR gene and part of C gene of West Nile virus (WNV), 5' UTR gene of Yellow fever virus (YFV), NS5 gene of Zika virus (ZIKV), and were modified with TAG sequence and Spacer 18 at the 5' end of upstream primer and biotin at the 5' end of downstream primer, multiplex PCR amplification was carried out; Then PCR products were hybridized with streptavidin-phycoerythrin (SAPE) and microspheres with complementary TAG sequences, and the hybridization results were detected by Luminex 200 instrument to indicate the qualification and quantification of the arbovirus of the samples. 【Result】While the Luminex xTAG method was applied to detect JEV、WNV、YFV and ZIKV，The results showed that the optimized primer working ratio: JEV : WNV : YFV : ZIKV is 0.5 μMμmol/L : 0.5 μMμmol/L : 0.75 μMμmol/L : 0.5 μMμmol/L ; the established hybridization system and reaction conditions: 20 μL of magnetic bead working solution, 5 μL of PCR amplification product, and 75 μL of SAPE working buffer; hybridization temperature is 37 ℃, hybridization time is 30 min, and buffer pH is 8.0. The quadruple Luminex xTAG assay can detect JEV, WNV, YFV and ZIKV simultaneously, and there was no cross reaction with dengue virus (DENV). The coefficient of variation of the intra-assay for quadruple Luminex xTAG assay was 2.50% - 5.63% and inter-assay was 3.61% - 12.50%. The detection limits of JEV and ZIKV were 1×104 copies/μL, and that of WNV and YFV were 1×103 copies/μL respectively. The sensitivity for WNV, YFV and ZIKV is 10 to 100 times that of conventional PCR. A total of 209 clinical samples and simulated samples were detected by Luminex xTAG assay and RT-qPCR method, the coincidence rate of JEV, MNV, YFV and ZIKV was 100%, 100%, 100% and 98.7%, accordingly. The positive detection rate of Luminex xTAG assay was higher than that of RT-qPCR method. 【Conclusion】The established quadruple Luminex xTAG assay has high throughput, high specificity, and sensitivity,with and high cost-effectiveness, providing a high-throughput technology platformmethod for clinical diagnosis and epidemiological monitoring of arboviruses. |
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