流行性乙型脑炎和西尼罗病毒双重微滴数字PCR检测方法的建立

doi:10.11853/j.issn.1003.8280.2023.03.001

中国媒介生物学及控制杂志 ›› 2023, Vol. 34 ›› Issue (3): 285-290.DOI: 10.11853/j.issn.1003.8280.2023.03.001

• 实验研究 • 下一篇

流行性乙型脑炎和西尼罗病毒双重微滴数字PCR检测方法的建立

张俊锋^1,2, 张雅丽^2,3, 王瑞晨², 禄阳², 张天姿², 付士红², 殷启凯², 李樊², 何英², 聂凯², 马超锋^3,4, 梁国栋², 扈瑞平¹, 许松涛², 王环宇²

1. 内蒙古医科大学基础医学院生物化学与分子生物学教研室, 内蒙古呼和浩特 010110;
2. 中国疾病预防控制中心病毒病预防控制所虫媒病毒室, 北京 102206;
3. 陕西中医药大学公共卫生学院, 陕西咸阳 712360;
4. 西安市疾病预防控制中心, 陕西西安 710000

收稿日期:2023-01-18 出版日期:2023-06-20 发布日期:2023-06-16
通讯作者: 扈瑞平,E-mail:783674348@qq.com;许松涛,E-mail:xust@ivdc.chinacdc.cn
作者简介:张俊锋,女,在读硕士,主要从事病原检测方法建立,E-mail:2384999538@qq.com
基金资助:
美国NIH项目U01（AI151810）

Establishment of a duplex droplet digital PCR assay for Japanese encephalitis and West Nile viruses

ZHANG Jun-feng^1,2, ZHANG Ya-li^2,3, WANG Rui-chen², LU Yang², ZHANG Tian-zi², FU Shi-hong², YIN Qi-kai², LI Fan², HE Ying², NIE Kai², MA Chao-feng^3,4, LIANG Guo-dong², HU Rui-ping¹, XU Song-tao², WANG Huan-yu²

1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia 010110, China;
2. Department of Arbovirus, Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
3. School of Public Health, Shaanxi University of Traditional Chinese Medicine, Xianyang, Shaanxi 712360, China;
4. Xi'an Center for Disease Control and Prevention, Xi'an, Shaanxi 710000, China

Received:2023-01-18 Online:2023-06-20 Published:2023-06-16
Supported by:
US NIH Project U01 (No. AI151810)

摘要/Abstract

摘要： 目的建立流行性乙型脑炎病毒（JEV）和西尼罗病毒（WNV）的双重微滴式数字PCR（ddPCR）检测方法。方法基于已设计的JEV和WNV引物探针,建立JEV和WNV双重ddPCR检测反应体系,摸索检测的敏感性、特异性和可重复性,灵敏度与双重荧光定量PCR（qPCR）的每个反应管内荧光信号达到设定的阈值所经历的循环数（Ct）值做对比。结果双重ddPCR检测反应体系对JEV和WNV的检测灵敏度均可达到10²拷贝/μl,该方法的可重复性、特异性良好,未发现与登革病毒、基孔肯雅病毒、寨卡病毒、蜱传脑炎病毒以及人类基因组有交叉反应。结论建立的双重ddPCR方法能敏感、特异检测JEV和WNV,为不同场景下这2种病毒的检测提供了解决方案。

关键词: 流行性乙型脑炎病毒, 西尼罗病毒, 微滴式数字聚合酶链式反应

Abstract: Objective To establish a duplex droplet digital polymerase chain reaction (ddPCR) detection method for Japanese encephalitis virus (JEV) and West Nile virus (WNV).Methods Based on the designed primers and probes of JEV and WNV, a duplex ddPCR detection system for JEV and WNV was established. Its sensitivity, specificity, and repeatability were explored. The sensitivity was compared with the number of cycles required for the fluorescent signal to cross the threshold in each reaction tube of dual quantitative PCR.Results The detection sensitivity of the duplex ddPCR detection system could reach 10² copies/μl for both JEV and WNV, with good specificity and repeatability. No cross-reactivity was observed with the Dengue virus, Chikungunya virus, Zika virus, Tick-borne encephalitis virus, and human genome.Conclusion The established duplex ddPCR method shows high sensitivity and specificity for JEV and WNV detection, which provides a solution for detection for the two viruses in different scenarios.

Key words: Japanese encephalitis virus, West Nile virus, Droplet digital polymerase chain reaction

中图分类号:

R373

张俊锋, 张雅丽, 王瑞晨, 禄阳, 张天姿, 付士红, 殷启凯, 李樊, 何英, 聂凯, 马超锋, 梁国栋, 扈瑞平, 许松涛, 王环宇. 流行性乙型脑炎和西尼罗病毒双重微滴数字PCR检测方法的建立[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 285-290.

ZHANG Jun-feng, ZHANG Ya-li, WANG Rui-chen, LU Yang, ZHANG Tian-zi, FU Shi-hong, YIN Qi-kai, LI Fan, HE Ying, NIE Kai, MA Chao-feng, LIANG Guo-dong, HU Rui-ping, XU Song-tao, WANG Huan-yu. Establishment of a duplex droplet digital PCR assay for Japanese encephalitis and West Nile viruses[J]. Chinese Journal of Vector Biology and Control, 2023, 34(3): 285-290.

参考文献

[1] Sumiyoshi H,Mori C,Fuke I,et al. Complete nucleotide sequence of the Japanese encephalitis virus genome RNA[J]. Virology,1987,161(2):497-510. DOI:10.1016/0042-6822(87)90144-9.
[2] Solomon T,Ni HL,Beasley DWC,et al. Origin and evolution of Japanese encephalitis virus in southeast Asia[J]. J Virol,2003,77(5):3091-3098. DOI:10.1128/jvi.77.5.3091-3098.2003.
[3] Turtle L,Solomon T. Japanese encephalitis:The prospects for new treatments[J]. Nat Rev Neurol,2018,14(5):298-313. DOI:10.1038/nrneurol.2018.30.
[4] Han N,Adams J,Chen P,et al. Comparison of genotypes I and III in Japanese encephalitis virus reveals distinct differences in their genetic and host diversity[J]. J Virol,2014,88(19):11469-11479. DOI:10.1128/jvi.02050-14.
[5] Fischer M,Casey C,Chen RT. Promise of new Japanese encephalitis vaccines[J]. Lancet,2007,370(9602):1806-1808. DOI:10.1016/S0140-6736(07)61753-X.
[6] Caldwell M,Boruah AP,Thakur KT. Acute neurologic emerging flaviviruses[J]. Ther Adv Infect Dis,2022,9:1-19. DOI:10. 1177/20499361221102664.
[7] Ronca SE,Ruff JC,Murray KO. A 20-year historical review of West Nile virus since its initial emergence in North America:Has West Nile virus become a neglected tropical disease?[J]. PLoS Negl Trop Dis,2021,15(5):e0009190. DOI:10.1371/journal.pntd.0009190.
[8] Lu Z,Fu SH,Cao L,et al. Human infection with West Nile virus,Xinjiang,China,2011[J]. Emerg Infect Dis,2014,20(8):1421-1423. DOI:10.3201/eid2008.131433.
[9] Ahlers LRH,Goodman AG. The immune responses of the animal hosts of West Nile virus:A comparison of insects,birds,and mammals[J]. Front Cell Infect Microbiol,2018,8:96. DOI:10.3389/fcimb.2018.00096.
[10] Shao N,Li F,Nie K,et al. TaqMan real-time RT-PCR assay for detecting and differentiating Japanese encephalitis virus[J]. Biomed Environ Sci,2018,31(3):208-214. DOI:10.3967/bes2018.026.
[11] Lustig Y,Sofer D,Bucris ED,et al. Surveillance and diagnosis of West Nile virus in the face of Flavivirus cross-reactivity[J]. Front Microbiol,2018,9:2421. DOI:10.3389/fmicb.2018.02421.
[12] Roberts A,Gandhi S. Japanese encephalitis virus:A review on emerging diagnostic techniques[J]. Front Biosci (Landmark Ed),2020,25(10):1875-1893. DOI:10.2741/4882.
[13] Lei SW,Chen S,Zhong QP. Digital PCR for accurate quantification of pathogens:Principles,applications,challenges and future prospects[J]. Int J Biol Macromol,2021,184:750-759. DOI:10.1016/j.ijbiomac.2021.06.132.
[14] Brandolini M,Taddei F,Marino MM,et al. Correlating qRT-PCR,dPCR and viral titration for the identification and quantification of SARS-CoV-2:A new approach for infection management[J]. Viruses,2021,13(6):1022. DOI:10.3390/v13061022.
[15] Berden P,Wiederkehr RS,Lagae L,et al. Amplification efficiency and template accessibility as distinct causes of rain in digital PCR:Monte Carlo modeling and experimental validation[J]. Anal Chem,2022,94(45):15781-15789. DOI:10.1021/acs.analchem.2c03534.
[16] Pomari E,Piubelli C,Perandin F,et al. Digital PCR:A new technology for diagnosis of parasitic infections[J]. Clin Microbiol Infect,2019,25(12):1510-1516. DOI:10.1016/j.cmi.2019. 06.009.
[17] Gaňová M,Zhang HQ,Zhu HL,et al. Multiplexed digital polymerase chain reaction as a powerful diagnostic tool[J]. Biosens Bioelectron,2021,181:113155. DOI:10.1016/j.bios.2021.113155.
[18] de Almeida PR,Demoliner M,Eisen AKA,et al. SARS-CoV2 quantification using RT-dPCR:A faster and safer alternative to assist viral genomic copies assessment using RT-qPCR[J] bioRxiv,2020. DOI:10.1101/2020.05.01.072728.
[19] de Almeida PR,Eisen AKA,Demoliner M,et al. RT-dPCR in mosquito samples for ZIKV detection:Effects of RNA extraction and reverse transcription in target concentration[J]. Viruses,2020,12(8):827. DOI:10.3390/v12080827.
[20] Ahrberg CD,Lee JM,Chung BG. Microwell array-based digital PCR for influenza virus detection[J]. BioChip J,2019,13(3):269-276. DOI:10.1007/s13206-019-3302-8.
[21] Farkas K, Mannion F,Hillary LS,et al. Emerging technologies for the rapid detection of enteric viruses in the aquatic environment[J]. Curr Opin Environ Sci Health,2020,16:1-6. DOI:10.1016/j.coesh.2020.01.007.
[22] 徐蕾蕊,李丹,汪琦,等. 基于RT-ddPCR技术的食品中诺如病毒定量检测[J]. 食品科学,2022,43(20):313-320.Xu LR,Li D,Wang Q,et al. Quantitative detection of norovirus in foods using reverse transcriptase droplet digital polymerase chain reaction[J]. Food Sci,2022,43(20):313-320. (in Chinese)
[23] 薛晓宁,徐翮飞,张娟,等. 乙型脑炎病毒微滴式数字PCR检测方法的建立[J]. 口岸卫生控制,2021,26(5):60-63.Xue XN,Xu HF,Zhang J,et al. Development of a reverse transcriptase droplet digital PCR assay for the detection of Japanese encephalitis virus[J]. Port Health Control,2021,26(5):60-63. (in Chinese)
[24] Rastogi M,Sharma N,Singh SK. Flavivirus NS1:A multifaceted enigmatic viral protein[J]. Virol J,2016,13:131. DOI:10.1186/s12985-016-0590-7.
[25] Zhang LX,Parvin R,Fan QH,et al. Emerging digital PCR technology in precision medicine[J]. Biosens Bioelectron,2022,211:114344. DOI:10.1016/j.bios.2022.114344.

流行性乙型脑炎和西尼罗病毒双重微滴数字PCR检测方法的建立

Establishment of a duplex droplet digital PCR assay for Japanese encephalitis and West Nile viruses

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