基于重组酶聚合酶扩增技术的牛源性成分快速检测方法

doi:10.19586/j.2095-2341.2023.0150

生物技术进展 ›› 2024, Vol. 14 ›› Issue (2): 278-286.DOI: 10.19586/j.2095-2341.2023.0150

• 研究论文 • 上一篇

基于重组酶聚合酶扩增技术的牛源性成分快速检测方法

王晓雪¹(), 纪艺², 余卉茹³, 徐俊锋², 彭城², 汪小福², 李玥莹¹(), 陈笑芸²()

^1.沈阳师范大学生命科学学院，沈阳 110000
^2.浙江省农业科学院农产品质量安全与营养研究所，农产品质量安全危害因子与风险防控国家重点实验室；农业农村部农业转基因生物溯源重点实验室，杭州 310021
^3.湘湖实验室，杭州 311258

收稿日期:2023-11-23 接受日期:2024-01-04 出版日期:2024-03-25 发布日期:2024-04-17
通讯作者: 李玥莹,陈笑芸
作者简介:王晓雪E-mail：1729459647@qq.com
基金资助:
科技创新2030重大项目农业生物育种重大专项(2022ZD040212);浙江省重点研发项目(2021C02059)

A Rapid Method for Bovine-derived Components Based on Recombinase Polymerase Amplification Technology

Xiaoxue WANG¹(), Yi JI², Huiru YU³, Junfeng XU², Cheng PENG², Xiaofu WANG², Yueying LI¹(), Xiaoyun CHEN²()

^1.College of Life Sciences，Shenyang Normal University，Shenyang 110000，China
^2.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products，Key Laboratory of Traceability for Agricultural Genetically Modified Organisms，Ministry of Agriculture and Rural Affairs，Institute of Quality，Safety and Nutrition of Agricultural Products Zhejiang Academy of Agricultural Sciences，Hangzhou 310021，China
^3.Xianghu Laboratory，Hangzhou 311258，China

Received:2023-11-23 Accepted:2024-01-04 Online:2024-03-25 Published:2024-04-17
Contact: Yueying LI,Xiaoyun CHEN

摘要/Abstract

摘要：

市场中肉类食品掺假的复杂多样化使得监管机构对动物源性成分鉴定的便捷性、准确性、灵敏性要求越来越高。尤其是市场上经济价值较高的牛肉类产品已成为制假的重灾区，迫切需要建立一种快速、高效的牛源性成分分子检测方法。基于此，以牛组成型表达基因β-actin为靶基因设计并筛选了几组牛特异性扩增的引物和探针组合，经过灵敏度检测和特异性验证，建立了一种牛源性成分实时荧光重组酶聚合酶扩增技术（recombinase polymerase amplification, RPA）快速检测方法。通过优化试剂配比，提高了一步法提取牛肉制品DNA的效率，同时将PRA技术与胶体金免疫试纸条显色技术相结合实现了检测的便捷化和结果的可视化。实际应用结果表明，研究建立的牛源性RPA检测方法能够特异性地检测牛源性成分，且最低检测灵敏度可达到14.8个拷贝，通过胶体金免疫试纸条得到的可视化结果的准确性和灵敏度与实时荧光RPA相同。该方法特异性强、灵敏度高，整个过程在25 min内即可完成，极大缩短了检测时间。

关键词: 牛源性成分, 重组酶聚合酶扩增技术, 胶体金免疫试纸条

Abstract:

The diversification of meat adulteration in the market has made it increasingly difficult for regulatory agencies to identify ingredients of animal origin with ease， accuracy and sensitivity. In particular， beef products with high economic value on the market have become the hardest hit areas for counterfeiting. Therefore， it is urgent to establish a fast and efficient molecular detection method for bovine components. Based on the requirement， this study designed and screened several groups of bovine specific amplified primers and probe combinations with β-actin， a reference gene expressed constitutively in cattle. Through sensitivity detection and specificity verification， recombinase polymerase amplification （RPA） method for rapid detection of bovine-derived components was established. At the same time， the efficiency of one-step DNA extraction from beef products was improved by adjusting and optimizing the reagent ratio. PRA technology was combined with colloid gold immunotest strip color development technology to realize the convenience of detection and visualization of results. Application results showed that the proposed method could detect bovine RPA specifically， with a minimum detection sensitivity of 14.8 copies， and could visualize the visualized results obtained by colloidal gold immunotest strips， and the accuracy and sensitivity of the results were the same as that of real-time fluorescence RPA. The method has strong specificity and high sensitivity， and the whole process can be completed in 25 min， greatly shortening the detection time.

Key words: bovine-derived components, recombinase polymerase amplification, colloidal gold immunoassay strips

中图分类号:

Q781

王晓雪, 纪艺, 余卉茹, 徐俊锋, 彭城, 汪小福, 李玥莹, 陈笑芸. 基于重组酶聚合酶扩增技术的牛源性成分快速检测方法[J]. 生物技术进展, 2024, 14(2): 278-286.

Xiaoxue WANG, Yi JI, Huiru YU, Junfeng XU, Cheng PENG, Xiaofu WANG, Yueying LI, Xiaoyun CHEN. A Rapid Method for Bovine-derived Components Based on Recombinase Polymerase Amplification Technology[J]. Current Biotechnology, 2024, 14(2): 278-286.

图/表 13

表1 裂解液组分

Table 1 Composition of the lysate

试剂	终浓度	体积
NaCl	100 mmol·L^-1	1 mL
Tris-HCl	10 mmol·L^-1	2 mL
EDTA, pH 8.0	25 mmol·L^-1	1 mL
SDS	0.5%	0.5 mL
Proteinase K	0.1 mg·mL^-1	1 mL

表2 RPA反应引物/探针序列

Table 2 RPA reaction primer/probe sequence

名称	序列（5'→3'）
ISO-RPA-F1	5'-TGTTTGCCTTTCTGACTAGGTGTTGCAAGCATAAA-3'
ISO-RPA-F2	5'-GCCTTTCTGACTAGGTGTTGCAAGCATAAAGTGCT-3'
ISO-RPA-F3	5'-TCTGACTAGGTGTTGCAAGCATAAAGTGCTGTGGG-3'
ISO-RPA-F4	5'-CTAGGTGTTGCAAGCATAAAGTGCTGTGGGTGTAG-3'
ISO-RPA-R1	5'-GGAAGAGATGGGCCGCGTCATACTGGGACACCACA-3'
ISO-RPA-R2	5'-AGATGGGCCGCGTCATACTGGGACACCACAAGGGG-3'
ISO-RPA-R3	5'-GGCCGCGTCATACTGGGACACCACAAGGGGCAGTC-3'
ISO-RPA-R4	5'-CGTCATACTGGGACACCACAAGGGGCAGTCGGGCC-3'
ISO-RPA-P	5'-AGGCGGCCTCGGAGTGTGTATTCAGTAXGTGCACAGTACG-3'

表3 RPA引物/探针浓度配比

Table 3 RPA primer/probe concentration ratio

编号	引物终浓度/（μmol·L^-1）	探针终浓度/（μmol·L^-1）	引物探针终浓度比
1	0.2	0.12	5:3
2	0.4	0.12	10:3
3	0.2	0.06	10:3
4	0.4	0.06	20:3

表4 RPA反应时间优化信息

Table 4 RPA reaction time optimization information

反应温度	反应时间/min	循环数
39 ℃	15	20
	21	30
	28	40
	34	50

图1 荧光RPA反应引物/探针筛选

Fig. 1 Screening of fluorescent RPA reaction primer probe

图2 RPA反应特异性检测A：F1R1引物组合；B：F3R3引物组合；C：F1R2引物组合

Fig. 2 RPA response specific detection

图3 RPA引物探针浓度比优化

Fig. 3 RPA optimization of concentration ratio of primer probe

表5 RPA检测反应体系

Table 5 RPA test reaction system

试剂	终浓度	体积
A buffer	1×	29.4 μL
10 μmol·L^-1正向引物	0.4 μmol·L^-1	2.0 μL
10 μmol·L^-1反向引物	0.4 μmol·L^-1	2.0 μL
10 μmol·L^-1探针	0.12 μmol·L^-1	0.6 μL
DNA 模板		2.0 μL
无菌水		11.5 μL
B buffer		2.5 μL
总体积		50.0 μL

图4 RPA反应的温度优化扩增曲线

Fig. 4 Temperature optimized amplification curve for RPA reaction

图5 荧光RPA反应时间优化

Fig. 5 Time optimization of RPA response

图6 RPA反应灵敏度检测

Fig. 6 RPA response sensitivity detection

图7 试纸条特异性检测

Fig. 7 Strip specificity test

图8 试纸条灵敏度检测

Fig. 8 Sensitivity test of test strip

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基于重组酶聚合酶扩增技术的牛源性成分快速检测方法

A Rapid Method for Bovine-derived Components Based on Recombinase Polymerase Amplification Technology

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