适于检测非洲猪瘟病毒的点亮Spinach-p54 RNA适配体的设计及应用

doi:10.16288/j.yczz.21-335

遗传 ›› 2021, Vol. 43 ›› Issue (12): 1170-1178.doi: 10.16288/j.yczz.21-335

适于检测非洲猪瘟病毒的点亮Spinach-p54 RNA适配体的设计及应用

韩程程^1,²(), 夏凯^1,³, 龚茹莹¹, 吴栩涵¹, 张蕾^1,²(), 梁新乐^1,²()

1. 浙江工商大学食品与生物工程学院,杭州 310018
2. 浙江工商大学食品生物工程研究所,杭州 310018
3. 浙江科技学院生物与化学工程学院,杭州 310023

收稿日期:2021-09-21 修回日期:2021-11-30 出版日期:2021-12-20 发布日期:2021-12-10
作者简介:韩程程,博士研究生,研究方向：食品生物技术。E-mail: 707884766@qq.com

Design and application of lightening Spinach-p54 RNA apatmer to detect African swine fever virus

Chengcheng Han^1,²(), Kai Xia^1,³, Ruying Gong¹, Xuhan Wu¹, Lei Zhang^1,²(), Xinle Liang^1,²()

1. School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China
2. Institute of Food Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China
3. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China

Received:2021-09-21 Revised:2021-11-30 Published:2021-12-20 Online:2021-12-10

摘要/Abstract

摘要：

非洲猪瘟病毒(African swine fever virus, ASFV)是近年来流行的一种主要致病病毒,对我国的国民经济生活造成了严重影响。本研究以非洲猪瘟病毒的编码保守蛋白p54基因序列片段为检测目标,结合点亮 Spinach RNA适配体的开关功能,设计了Spinach-p54的嵌合式RNA适配体。其特异地结合目标序列并产生荧光,实现了在RNA水平上对非洲猪瘟病毒的快速检测。研究结果表明,核酸适配体浓度200 nmol/L,反应温度为37 ℃,该方法检出限为200 nmol/L,线性范围为200~400 nmol/L。该方法特异性强,操作简单,灵敏度高等特点,可用于市场和养殖场的现场快速检测。

关键词: 核酸适配体, RNA, turn-on, 非洲猪瘟

Abstract:

African swine fever virus (ASFV) is a major and prevalent pathogenic virus in livestock. Its outbreak in the recent years has seriously affected the national economic life. In this study, a specific chimeric RNA aptamer Spinach-p54 corresponding to the conserved protein p54 of ASFV, was designed and used in assembling a toehold RNA module with a truncated lightening Spinach switch. They were used to detect the p54 gene. The aptamer bound rapidly and paired specifically with the ASFV p54 target sequence and produced fluorescence with the addition of DFHBI substrate. The results showed that the limit of detection was as low as 200 nmol/L p54 fragment, under the conditions of 200 nmol/L Spinach-p54 aptamer and 37 ℃, suggesting that the current method can provide a simple, specific, sensitive, and rapid on-site detection of ASFV at the RNA level in the markets and farms.

Key words: aptamer, RNA, turn-on, African swine fever viru

韩程程, 夏凯, 龚茹莹, 吴栩涵, 张蕾, 梁新乐. 适于检测非洲猪瘟病毒的点亮Spinach-p54 RNA适配体的设计及应用[J]. 遗传, 2021, 43(12): 1170-1178.

Chengcheng Han, Kai Xia, Ruying Gong, Xuhan Wu, Lei Zhang, Xinle Liang. Design and application of lightening Spinach-p54 RNA apatmer to detect African swine fever virus[J]. Hereditas(Beijing), 2021, 43(12): 1170-1178.

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适配体名称	序列(5′→3′)
Spinach-p54-1	GATGCCGCGGATGGCGAAGGACGGGTCCAGTGCGTTCGCGCACTGTTGAGTAGAGTGTGAGCGCCAAACCGGTTGA
Spinach-p54-2	ACTGTCGTGTATGGCGAAGGACGGGTCCAGTGCGTTCGCGCACTGTTGAGTAGAGTGTGAGCGCCAGGCTCAGTCG
Spinach-p54-3	CCATAATGCCGTGGCGAAGGACGGGTCCAGTGCGTTCGCGCACTGTTGAGTAGAGTGTGAGCGCCCGGATAAACCG

RNA适配体名称	单链适配体最小自由能(kcal/mol)	与p54 RNA结合后最小自由能(kcal/mol)
Spinach-p54-1	-24.2	-52.88
Spinach-p54-2	-27.4	-51.38
Spinach-p54-3	-24.5	-46.28

适于检测非洲猪瘟病毒的点亮Spinach-p54 RNA适配体的设计及应用

Design and application of lightening Spinach-p54 RNA apatmer to detect African swine fever virus

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