| 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):- |
| 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：2024-12-19 |
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| Abstract: |
| 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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