基于RPA-CRISPR/Cas12a的MSTN基因编辑猪检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2025.08.016

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 3734-3748.doi: 10.11843/j.issn.0366-6964.2025.08.016

基于RPA-CRISPR/Cas12a的MSTN基因编辑猪检测方法的建立及应用

迟顺顺¹^,²^,³(), 吴丹⁴(), 王楠²^,³, 王婉洁³, 聂雨欣³, 牟玉莲³, 刘志国³^,*(), 朱振东¹^,*(), 李奎²^,³

1. 青岛农业大学动物科技学院，青岛 266109
2. 中国农业科学院(深圳)农业基因组研究所, 岭南现代农业科学与技术广东省实验室深圳分中心, 农业农村部畜禽生物组学重点实验室，深圳 518124
3. 中国农业科学院北京畜牧兽医研究所，北京 100193
4. 天津市宁河原种猪场有限责任公司，天津 301504

收稿日期:2025-02-13 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 刘志国,朱振东 E-mail:chishunshun@163.com;190833173@qq.com;liuzhiguo@caas.cn;zzd2020@qau.edu.cn
作者简介:迟顺顺(1999-)，男，山东青岛人，硕士生，主要从事畜牧研究，Tel: 010-62813339，E-mail: chishunshun@163.com
吴丹(1987-)，女，天津宁河人，助理畜牧师，大专，主要从事畜牧兽医工作，Tel：022-69431311，E-mail: 190833173@qq.com
第一联系人：
迟顺顺与吴丹为同等贡献作者
基金资助:
农业生物育种国家科技重大专项(2022ZD04020)；中国农业科学院科技创新工程(ASTIP-IAS05)

Establishment and Application of A Detection Method for MSTN Gene-Edited Pigs Based on RPA-CRISPR/Cas12a

CHI Shunshun¹^,²^,³(), WU Dan⁴(), WANG Nan²^,³, WANG Wanjie³, NIE Yuxin³, MU Yulian³, LIU Zhiguo³^,*(), ZHU Zhendong¹^,*(), LI Kui²^,³

1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China
2. Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China
3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
4. Tianjin Ningheyuan Swine Breeding Farm Co., Ltd., Tianjin 301504, China

Received:2025-02-13 Online:2025-08-23 Published:2025-08-28
Contact: LIU Zhiguo, ZHU Zhendong E-mail:chishunshun@163.com;190833173@qq.com;liuzhiguo@caas.cn;zzd2020@qau.edu.cn

摘要/Abstract

摘要：

旨在建立一种基于重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)和成簇规律间隔短回文重复序列/CRISPR相关蛋白12a(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a，CRISPR/Cas12a)系统的MSTN基因编辑猪检测方法，以满足基因编辑动物研发、育种等过程中核酸检测以及基因型鉴定的需求。本研究构建含有猪MSTN基因突变序列和野生型序列的标准质粒并设计靶向标准质粒的crRNA，基于RPA和CRISPR/Cas12a技术建立荧光报告和胶体金试纸条检测体系，筛选特异性识别标准质粒的crRNA、优化其反应条件并评价其检测灵敏度，最后通过检测猪耳组织样品评价本方法的准确性和稳定性。研究结果表明，该方法能够特异性识别MSTN基因2 bp碱基缺失序列和MSTN基因野生型序列。荧光报告体系最低可检测到4.1×10¹ copies·μL^-1的标准质粒，胶体金试纸条体系最低可检测到4.1×10³ copies·μL^-1的标准质粒。通过对猪耳组织样本的检测，证明了该检测体系的准确性和可靠性。综上所述，本研究建立的MSTN基因编辑猪RPA-CRISPR/Cas12a检测方法具有操作简便、特异性和灵敏度高的特点，为MSTN基因编辑猪检测提供了重要技术支撑，具有广阔的应用前景。

关键词: MSTN基因编辑猪, 重组酶聚合酶扩增, 核酸检测, CRISPR/Cas12a

Abstract:

This study aimed to establish a detection method for MSTN gene-edited pigs based on recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12 (CRISPR/Cas12a)system, to meet the needs of nucleic acid testing and genotype identification in the research and breeding of gene-edited animals. Standard plasmids containing the porcine MSTN gene mutant sequence and wild-type sequence were constructed, with crRNAs targeting these standard plasmids designed. Detection systems integrating fluorescence reporter or colloidal gold test strip were established based on RPA and CRISPR/Cas12a technologies. Through systematic screening of crRNAs specifically recognizing the standard plasmids, optimization of reaction conditions, and evaluation of detection sensitivity, the method′s analytical performance was characterized. Ultimately, the accuracy and stability of this approach were validated by detecting porcine ear tissue samples. The results demonstrated that the method could specifically identify the 2 bp deletion and wild-type sequences of the MSTN gene. The fluorescence reporter system could detect as low as 4.1×10¹ copies·μL^-1 of the standard plasmid, while the colloidal gold test strip system could detect as low as 4.1×10³ copies·μL^-1. The accuracy and reliability of the system were validated by testing porcine ear tissue samples. The RPA-CRISPR/Cas12a detection method for MSTN gene-edited pigs established in this study is characterized by its simplicity, high specificity, and high sensitivity. It provides important technical support for the detection of MSTN gene-edited pigs and holds broad application prospects.

Key words: MSTN gene-edited pig, recombinase polymerase amplification, nucleic acid detection, CRISPR/Cas12a

中图分类号:

S828.2

迟顺顺, 吴丹, 王楠, 王婉洁, 聂雨欣, 牟玉莲, 刘志国, 朱振东, 李奎. 基于RPA-CRISPR/Cas12a的MSTN基因编辑猪检测方法的建立及应用[J]. 畜牧兽医学报, 2025, 56(8): 3734-3748.

CHI Shunshun, WU Dan, WANG Nan, WANG Wanjie, NIE Yuxin, MU Yulian, LIU Zhiguo, ZHU Zhendong, LI Kui. Establishment and Application of A Detection Method for MSTN Gene-Edited Pigs Based on RPA-CRISPR/Cas12a[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3734-3748.

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名称Name	引物序列(5′→3′) Primer sequences
MSTN-PCR-1F	TTGCTACTATTAACTCTTCTTTCA
MSTN-PCR-1R	TATATTATTTGTTCTTTGCCATTA
MSTN-RPA-1F	AGAAGTCAAGGTAACAGACACACCA
MSTN-RPA-1R	TACAAATTCACACTCTCCAGAGCAG

名称Name	序列(5′→3′) Sequence
GEcrRNA1	UAAUUUCUACUAAGUGUAGAUACUUUUGGAUGGGACUGGAUUAU
GEcrRNA2	UAAUUUCUACUAAGUGUAGAUACUUUUGGAUGGGACUGGAU
WTcrRNA1	UAAUUUCUACUAAGUGUAGAUAAGCUUUUGGAUGGGACUGG
WTcrRNA2	UAAUUUCUACUAAGUGUAGAUAAAAUCCACAGUUAGAGGGU
WTcrRNA3	UAAUUUCUACUAAGUGUAGAUAAAUCCACAGUUAGAGGGUA
WTcrRNA4	UAAUUUCUACUAAGUGUAGAUAGCUUUUGGAUGGGACUGGA

基因型 Genotype	样品编号 Sample ID	样品数 Number of samples	总样品数 Total number of samples
MSTN^-2/-2	1410、1412	2	23
MSTN^-2/WT	73、74、75、76、99、100、101、103、104、105、107、1380、1404	13
MSTN^WT/WT	106、3110、3111、3112、3113、3114、3116、3119	8

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基于RPA-CRISPR/Cas12a的MSTN基因编辑猪检测方法的建立及应用

Establishment and Application of A Detection Method for MSTN Gene-Edited Pigs Based on RPA-CRISPR/Cas12a

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