基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立

doi:10.11843/j.issn.0366-6964.2023.09.020

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (9): 3803-3811.doi: 10.11843/j.issn.0366-6964.2023.09.020

基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立

杨芷翊¹, 王新凯^1,2, 史玉婷¹, 付思源¹, 张钰炘¹, 曹琛福², 贾伟新^1*

1. 华南农业大学兽医学院国家禽流感专业实验室(广州)/广东省人兽共患病防控制剂工程实验室/人兽共患病防控制剂国家地方联合工程实验室/农业农村部人畜共患病重点实验室/广东省动物源性人兽共患病防控重点实验室, 广州 510642;
2. 深圳海关动植物检验检疫技术中心, 深圳 518045

收稿日期:2022-11-15 发布日期:2023-09-22
通讯作者: 贾伟新,主要从事重大动物疫病和人兽共患病防控技术研究,E-mail:jiaweixin@scau.edu.cn;曹琛福,主要从事动物及动物产品检验检疫工作及技术研究,E-mail:caochenfu@163.com
作者简介:杨芷翊(1998-),女,广东广州人,研究生,主要从事动物疫病检测与诊断方法研究,E-mail:401770240@qq.com;王新凯(1998-),男,山东潍坊人,研究生,主要从事动物疫病检测与诊断方法研究,E-mail:813176709@qq.com
基金资助:
广东省科技计划项目(2021B1212030015);国家现代农业产业技术体系(CARS-41)

Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA

YANG Zhiyi¹, WANG Xinkai^1,2, SHI Yuting¹, FU Siyuan¹, ZHANG Yuxin¹, CAO Chenfu², JIA Weixin^1*

1. National Avian Influenza Para-Reference Laboratory(Guangzhou), Guangdong Engineering Laboratory for Medicament of Zoonosis Prevention and Control, National Local Joint Engineering Laboratory of Zoonosis Prevention and Control Agents, Key Laboratory of Zoonoses of Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory for Prevention and Control of Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center, Shenzhen 518045, China

Received:2022-11-15 Published:2023-09-22

摘要/Abstract

摘要： 采用逆转录-重组酶介导等温核酸扩增技术(RT-RAA)和成簇的规律间隔的短回文重复序列(CRISPR)检测系统,建立一种快速、高灵敏度和高特异性的禽流感病毒(AIV)H5 亚型核酸检测新方法。通过分析 342 条禽流感病毒H5 亚型的 HA 基因序列,选取保守性区域设计具有强特异性的 CRISPR RNA(crRNA) 序列以及 RT-RAA 引物,并对检测体系中的主要成分 LwaCas13a蛋白、crRNA、TaqMan探针、T7 RNA Polymerase、NTP Buffer Mix 含量进行优化,建立了基于 RT-RAA 的AIV H5 亚型核酸 CRISPR-Cas13a 检测方法。取 10⁵~1 copies·μL^-1含有目的基因的质粒标准品和其他亚型禽流感病毒以及其他禽病病毒分别验证和评价该方法的灵敏性和特异性。结果表明,本文设计的 RT-RAA 引物与 crRNA 特异且灵敏,其最佳反应体系中主要成分用量分别为 LwaCas13a蛋白(60 μg·mL^-1)2.0 μL、crRNA(100 μmol·L^-1)3.2 μL、TaqMan探针(50 μmol·L^-1)1.28 μL、T7 RNA Polymerase 0.25 μL、NTP Buffer Mix 2.0 μL。该方法检测灵敏度可达 1 copy·μL^-1,且与 AIV H3、H6、H7、H9、H10 亚型以及 NDV、IBV、IBDV、DTMUV 无交叉反应。对 56 份已知的临床样品进行检测,检测结果与荧光定量 RT-PCR 符合率为98.2%。综上,本研究通过 RT-RAA 等温扩增技术结合 CRISPR-Cas13a 基因编辑技术建立了针对 AIV H5 亚型的快速检测平台,该方法从样本的核酸抽提到获得检测结果的时间控制在 1 h 20 min 以内,为 AIV H5 亚型高灵敏度和高特异性的临床快速检测提供了新的技术手段,具有良好的应用前景。

关键词: H5亚型禽流感病毒, CRISPR, 等温扩增, 核酸检测

Abstract: A new rapid, highly sensitive and highly specific nucleic acid detection method for avian influenza virus(AIV) H5 subtype was established by using reverse transcription-recombinase aided amplification(RT-RAA) and clustered regularly interspaced short palindromic repeats(CRISPR). By analyzing the HA gene sequences of 342 avian influenza virus H5 subtype genes, the conserved regions were selected to design highly specific CRISPR RNA(crRNA) sequences and RT-RAA primers. The contents of main components in the detection system were optimized, including LwaCas13a protein, crRNA, TaqMan probe, T7 RNA Polymerase and NTP Buffer Mix.Thus, the RT-RAA based detection method of AIV H5 subtype nucleic acid CRISPR-Cas13a was established.The sensitivity and specificity of the method were evaluated by taking 10⁵~1 copies·μL^-1 of standard plasmid containing the target gene and other subtypes of avian influenza viruses and other avian disease viruses. The results showed that the RT-RAA primer and crRNA designed in this paper were specific and sensitive. The main components in the optimal reaction system were LwaCas13a protein (60 μg·mL^-1) 2.0 μL, crRNA (100 μmol·L^-1) 3.2 μL, TaqMan probe (50 μmol·L^-1) 1.28 μL, T7 RNA Polymerase 0.25 μL and NTP Buffer Mix 2.0 μL, respectively. The detection sensitivity of this method was up to 1 copy·μL^-1, and there was no cross reaction with AIV H3, H6, H7, H9, H10 subtypes, neither with NDV, IBV, IBDV or DTMUV. The method established in this study and fluorescence quantitative RT-PCR were used to detect 56 clinical samples, and the coincidence rate of the two methods was 98.2%. In conclusion, a rapid detection method for AIV H5 subtype was established by using RT-RAA isothermal amplification technology combined with CRISPR-Cas13a gene editing technology in this study. The results were obtained within 1 h and 20 min. It provides a new technique for the rapid clinical detection of AIV H5 subtype with high sensitivity and high specificity, and has a good application prospect.

Key words: avian influenza virus H5 subtype, CRISPR, isothermal amplification, nucleic acid testing

中图分类号:

S852.65

杨芷翊, 王新凯, 史玉婷, 付思源, 张钰炘, 曹琛福, 贾伟新. 基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立[J]. 畜牧兽医学报, 2023, 54(9): 3803-3811.

YANG Zhiyi, WANG Xinkai, SHI Yuting, FU Siyuan, ZHANG Yuxin, CAO Chenfu, JIA Weixin. Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3803-3811.

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基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立

Establishment of Nucleic Acid Detection Methods for Avian Influenza H5 Subtype Based on CRISPR-Cas13a and RT-RAA

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