A reverse transcriptase aid-enzymatic recombinase isothermal amplification-based method for detection of West Nile virus

doi:10.12140/j.issn.1000-7423.2022.03.010

Abstract

Abstract:

Objective To establish a reverse transcription-enzymatic recombinase isothermal amplification (RT-ERA) assay for detection of specific gene segment of West Nile virus. Methods The highly conserved region of NS5 gene (120 bp) was selected as the target gene fragment to be detected. The primers and fluorescence probes were designed and synthesized based on the isothermal amplification principle to establish a fluorescence RT-ERA assay system. The fluorescence RT-ERA assay was performed to detect serial diluted recombinant plasmids (10⁴, 10³, 10², 10, 1 copy/μl) containing target gene fragments and West Nile virus RNA at different concentrations (10, 1, 10^-1, 10^-2, 10^-3 ng/μl) to determine the sensitivity. Further, this assay was applied to detect the genomic RNA of Tick-Borne Encephalitis virus, Chikungunya virus, Yellow fever virus, Dengue virus type I, Japanese Encephalitis virus and Influenza A virus (H2N1) to evaluate the specificity. Results A fluorescence RT-ERA assay was successfully established, which was effective in amplifying the specific gene fragments of West Nile virus within 20 min at 39 ℃. The minimum detectable limit of the fluorescence RT-ERA assay was 1 copy/μl using recombinant plasmids as templates and 10^-3 ng/μl using West Nile virus RNA samples as templates. The results of the fluorescent RT-ERA assays were all negative for detecting the genomic RNA from tick-borne encephalitis virus, chikungunya virus, yellow fever virus, dengue virus type I, Japanese encephalitis virus, and Influenza A virus (H2N1). Conclusion A fluorescence RT-ERA assay for detection of West Nile virus RNA is successfully established, which is easy to use, sensitive and specific.

Key words: West Nile virus, Gene detection, Reverse transcription-enzymatic recombinase, Isothermal amplification of nucleic acid, Fluorescent probe

CLC Number:

R392

ZHANG Yi-long, YE Run, LE Bin, CHEN Wen-zhu, PAN Wei-qing, ZHANG Dong-mei. A reverse transcriptase aid-enzymatic recombinase isothermal amplification-based method for detection of West Nile virus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(3): 344-348.

Figures/Tables 6

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 19

[1]	Fan XX,, Wang SJ,, Zhang YQ, et al. Current situation and Countermeasures for prevention and control of West Nile fever in China in the future[J]. Chin J Vet Med, 2018, 54(1): 117-120. (in Chinese)
	( 樊晓旭,, 王淑娟,, 张永强, 等. 西尼罗河热现状及未来我国防控应对思考[J]. 中国兽医杂志, 2018, 54(1): 117-120.)
[2]	Chancey C,, Grinev A,, Volkova E, et al. The global ecology and epidemiology of west Nile virus[J]. Biomed Res Int, 2015, 2015: 376230.
[3]	Lv Z,, Yu YZ,, Hu GX, et al. Advancement of West Nile virus disease research[J]. J Econ Animal, 2012, 16(1): 55-59. (in Chinese)
	( 吕喆,, 于亚洲,, 胡桂学, 等. 西尼罗河病毒病的研究进展[J]. 经济动物学报, 2012, 16(1): 55-59.)
[4]	Napp S,, Petrić D,, Busquets N. West Nile virus and other mosquito-borne viruses present in Eastern Europe[J]. Pathog Glob Health, 2018, 112(5): 233-248. doi: 10.1080/20477724.2018.1483567
[5]	Wang SJ,, Song XH,, Chi TY, et al. The epidemic history and current situation of West Nile fever and its prevention and control[J]. Chin Animal Health Insp, 2016, 33(12): 4-6, 21. (in Chinese)
	( 王淑娟,, 宋晓晖,, 迟田英, 等. 西尼罗河热的流行历史、现状及防控[J]. 中国动物检疫, 2016, 33(12): 4-6, 21.)
[6]	Zheng XL,, Zhu LH,, Wang Q, et al. Progress on diagnostic techniques of West Nile virus disease[J]. Prog Vet Med, 2014, 35(3): 92-97. (in Chinese)
	( 郑小龙,, 朱来华,, 王群, 等. 西尼罗病毒病诊断技术研究进展[J]. 动物医学进展, 2014, 35(3): 92-97.)
[7]	Cao ZG,, Wang HL,, Li L, et al. Development, verification and application of an indirect ELISA for West Nile virus antibody[J]. Chin J Biol, 2016, 29(1): 70-74. (in Chinese)
	( 曹增国,, 王化磊,, 李岭, 等. 西尼罗病毒抗体间接ELISA检测方法的建立、验证及其应用[J]. 中国生物制品学杂志, 2016, 29(1): 70-74.)
[8]	Lv B,, Cheng HR,, Yan QF, et al. The development and recent improvements of in vitro nucleic acid amplification technology[J]. China Biotechnol, 2011, 31(3): 91-96. (in Chinese)
	( 吕蓓,, 程海荣,, 严庆丰, 等. 体外核酸快速扩增技术的发展和不断创新[J]. 中国生物工程杂志, 2011, 31(3): 91-96.)
[9]	Song J,, Tang TS,, Tian LL, et al. Nest RT-PCR to detect the West Nile virus[J]. Chin J Vet Sci, 2008, 28(12): 1411-1414, 1418. (in Chinese)
	( 宋捷,, 唐泰山,, 田玲玲, 等. 西尼罗病毒套式RT-PCR检测方法的建立[J]. 中国兽医学报, 2008, 28(12): 1411-1414, 1418.)
[10]	Linke S,, Ellerbrok H,, Niedrig M, et al. Detection of West Nile virus lineages 1 and 2 by real-time PCR[J]. J Virol Methods, 2007, 146(1/2): 355-358. doi: 10.1016/j.jviromet.2007.05.021
[11]	Qiu L,, Li XL,, Jiang J, et al. Detection of West Nile virus by real-time RT-PCR[J]. Animal Husb Vet Med, 2012, 44(9): 4-7. (in Chinese)
	( 邱璐,, 李小林,, 蒋静, 等. 实时荧光PCR检测西尼罗病毒方法的建立[J]. 畜牧与兽医, 2012, 44(9): 4-7.)
[12]	Li T,, Yang K. Application of isothermal amplification technology for pathogen detection in parasitic and other diseases[J]. Chin J Schisto Control, 2018, 30(2): 232-236. (in Chinese)
	( 李婷,, 杨坤. 等温扩增技术在寄生虫及其他病原体检测中的应用[J]. 中国血吸虫病防治杂志, 2018, 30(2): 232-236.)
[13]	Li SH,, Lu WY,, Cao GW. Loop-mediated isothermal amplification in detection of West Nile virus genome[J]. Acad J Second Mil Med Univ, 2010, 31(6): 590-594. (in Chinese)
	( 李淑华,, 鹿文英,, 曹广文. 利用环介导等温扩增技术检测西尼罗病毒[J]. 第二军医大学学报, 2010, 31(6): 590-594.)
[14]	Nyan DC,, Swinson KL. A novel multiplex isothermal amplification method for rapid detection and identification of viruses[J]. Sci Rep, 2015, 5: 17925. doi: 10.1038/srep17925
[15]	Kumar JS,, Saxena D,, Parida M, et al. Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients[J]. Indian J Med Res, 2018, 147(3): 293-298. doi: 10.4103/0971-5916.234607
[16]	Wu J,, Lin YX,, Jia P, et al. Establishment and applications of the RT-RPA method for detection of West Nile virus[J]. Chin J Vet Med, 2021, 57(2): 35-37, 41, 126. (in Chinese)
	( 吴江,, 林彦星,, 贾鹏, 等. 西尼罗河病毒RT-RPA检测方法的建立及初步应用[J]. 中国兽医杂志, 2021, 57(2): 35-37, 41, 126.)
[17]	Lv B,, Cheng HR,, Yan QF, et al. Recombinase-aid amplification: a novel technology of in vitro rapid nucleic acid amplification[J]. Sci Sin Vitae, 2010, 40(10): 983-988. (in Chinese) doi: 10.1360/zc2010-40-10-983
	( 吕蓓,, 程海荣,, 严庆丰, 等. 用重组酶介导扩增技术快速扩增核酸[J]. 中国科学: 生命科学, 2010, 40(10): 983-988.)
[18]	Lv QF,, Liao J,, Luo P, et al. Development of a reverse transcription recombinase aided amplification assay for detec-tion of West Nile virus[J]. Microbiol Chin, 2020, 47(2): 659-664. (in Chinese)
	( 吕沁风,, 廖静,, 罗鹏, 等. 西尼罗病毒的逆转录重组酶介导扩增检测方法[J]. 微生物学通报, 2020, 47(2): 659-664.)
[19]	Su S. Construction of recombinase-aid nucleic acid amplification system and its using in SNP genotyping[D]. Nanchang: Jiangxi Agricultural University, 2013: 8-10. (in Chinese)
	( 苏帅. 重组酶介导的核酸恒温扩增体系构建及其在SNP分型中的应用[D]. 南昌: 江西农业大学, 2013: 8-10.)

引物/探针Primers/probes	序列（5′→3′） Sequence （5′→3′）
WNV-F	CGTCCGCGACACTGCTTGTCTGGCTAAGTCTT
WNV-R	CAATTCACAGGGACAGCGGAGCAAATGGCGTT
WNV-EXO	TTGTCTGGCTAAGTCTTATGCCCAGATG(FAM-dT)G(THF)C(BHQ1-dT)GCTTCTGTACTTC-C3Spacer