副溶血性弧菌特异性核酸适配体筛选

doi:10.19586/j.2095-2341.2023.0041

生物技术进展 ›› 2023, Vol. 13 ›› Issue (4): 612-618.DOI: 10.19586/j.2095-2341.2023.0041

• 研究论文 • 上一篇

副溶血性弧菌特异性核酸适配体筛选

李洁¹(), 岳晓禹², 崔丽伟², 许文涛²^,³, 李相阳¹()

^1.北京农学院食品科学与工程学院，北京 102206
^2.河南牧业经济学院食品与生物学院，郑州 450046
^3.中国农业大学营养与健康系，北京 100083

收稿日期:2023-03-28 接受日期:2023-04-18 出版日期:2023-07-25 发布日期:2023-08-03
通讯作者: 李相阳
作者简介:李洁E-mail: lijievvvv@163.com；
基金资助:
北京市景观休闲农业创新团队项目(BAIC09-2022)

SELEX of Vibrio parahaemolyticus Specific Aptamer

Jie LI¹(), Xiaoyu YUE², Liwei CUI², Wentao XU²^,³, Xiangyang LI¹()

^1.Faculty of Food Science and Engineering，Beijing University of Agriculture，Beijing 102206，China
^2.College of Food and Bioengineering，Henan Institute of Animal Husbandry，Zhengzhou 450046，China
^3.Department of Nutrition and Health，Institute of Nutrition and Health，China Agricultural University，Beijing 100083，China

Received:2023-03-28 Accepted:2023-04-18 Online:2023-07-25 Published:2023-08-03
Contact: Xiangyang LI

摘要/Abstract

摘要：

副溶血性弧菌是水产品中常见的食源性致病菌，其快速检测方法逐渐被开发，适配体作为一种新兴的特异性识别元件受到广泛关注。以副溶血性弧菌作为靶标菌进行了12轮细胞-指数富集配体系统进化技术(cell-systematic evolution of ligands by exponential enrichment, cell-SELEX)，其中包括9轮正向筛选和3轮负向筛选，在每轮正筛中对PCR轮数和Lambada外切酶用量进行优化，筛选完成后进行核酸测序，并通过qPCR和流式细胞术多角度地对测序得到的序列性能进行验证。结果显示，F-28-10表现出较好的结合能力和特异性，且通过对PCR扩增轮数和Lambda外切酶用量优化后的cell-SELEX可以实现微生物特异性适配体的开发。研究结果为食源性致病微生物快速检测技术的开发提供了新的可利用的识别元件。

关键词: 副溶血性弧菌, 适配体, SELEX

Abstract:

As a common foodborne pathogenic bacterium in aquatic products， and a variety of rapid detection methods of Vibrio parahaemolyticus have been gradually developed. Aptamers have received widespread attention as an emerging specific identification element. In this study， 12 rounds of cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) were performed with Vibrio parahaemolyticus as the target， including 9 rounds of positive selection and 3 rounds of negative selection， and the number of PCR rounds and the dosage of Lambada exonuclease were optimized in each round of positive selection. Nucleic acid sequencing was performed after SELEX， and the obtained sequence performance was verified from multiple angles by qPCR and flow cytometry. The results showed that F-28-10 showed better binding ability and specificity. And the development of aptamers can be achieved by optimizing the number of PCR amplification rounds and the amount of Lambda exonuclease in cell-SELEX. This method provided a new usable identification element for the development of rapid detection technology of foodborne pathogenic microorganisms.

Key words: Vibrio parahaemolyticus, aptamer, SELEX

中图分类号:

TS207.4

李洁, 岳晓禹, 崔丽伟, 许文涛, 李相阳. 副溶血性弧菌特异性核酸适配体筛选[J]. 生物技术进展, 2023, 13(4): 612-618.

Jie LI, Xiaoyu YUE, Liwei CUI, Wentao XU, Xiangyang LI. SELEX of Vibrio parahaemolyticus Specific Aptamer[J]. Current Biotechnology, 2023, 13(4): 612-618.

图/表 10

表1 筛选过程中各组分加样量及反应时间

Table 1 Sample loading and reaction time of each component in the SELEX

SELEX轮数	文库加入量/pmol	菌液加入量 /μL	孵育时间 /min	洗涤次数	PCR轮数	Lambda外切酶用量/μL	BSA加入量 /μL
1	1 000	1 000	90	3	29	1	0
2	100	1 000	70	4	29	1	3
3	50	800	60	4	26	0.8	6
4	50	600	60	4	28	1.5	9
5	1 000	800	90	0	—	—	0
6	—	500	60	4	22	1.5	12
7	50	500	50	5	27	1.5	15
8	1 000	1 000	90	0	—	—	0
9	—	500	40	5	26	1.5	20
10	100	1 000	90	5	—	—	0
11	—	500	30	0	26	1.5	25
12	50	400	30	5	22	1	30

图1 副溶血性弧菌的生长曲线

Fig. 1 Growth curve of Vibrio parahaemolyticus

图2 退火温度优化结果注：M—DL2000 DNA maker。

Fig. 2 Results of annealing temperature optimization

图3 第4轮筛选PCR轮数优化结果注：M—DL2000 DNA maker。

Fig. 3 Results of PCR cycle optimization in fourth round of SELEX

图4 第3轮筛选Lambda外酶切优化结果注：M—100 bp DNA marker；1—PCR扩增后的双链DNA；红色方框中为酶切后产生的单链核酸产物。

Fig. 4 Results of Lambda exonuclease enzyme optimization in third round of SELEX

图5 10条候选适配体序列的qPCR结果

Fig. 5 Results of qPCR for 10 candidate aptamer sequences

图6 适配体与细菌结合的流式细胞术分析

Fig. 6 Flow cytometric analysis of aptamers binding to bacteria

图7 适配体F-3-2和F-28-10结合效果对比

Fig. 7 Comparison of binding effects of aptamers F-3-2 and F-28-10

图8 适配体F-28-10的亲和力研究A：不同浓度适配体扩增曲线； B: 校准曲线

Fig. 8 Affinity study of aptamer F-28-10

图9 F-28-10与副溶血性弧菌的结合解离曲线

Fig. 9 Dissociation saturation curves of F-28-10 and Vibrio parahaemolyticus

参考文献 19

1	JIANG H J, TAN R, JIN M, et al.. Visual detection of Vibrio parahaemolyticus using combined CRISPR/Cas12a and recombinase polymerase amplification[J]. Biomed. Environ. Sci., 2022, 35(6): 518-527.
2	赵颖,马琳.2018—2021年北京市大兴区由副溶血性弧菌引起的食源性疾病病原学特征及其耐药性[J].卫生研究,2022,51(4): 667-671, 679.
3	XIE T, YU Q, TANG X, et al.. Prevalence, antibiotic susceptibility and characterization of Vibrio parahaemolyticus isolates in China[J/OL]. FEMS Microbiol. Lett., 2020, 367(16): fnaa136[2020-08-01]. .
4	MARTINEZ-URTAZA J, BAKER-AUSTIN C. Vibrio parahaemolyticus[J]. Trends Microbiol., 2020, 28(10): 867-868.
5	蒋凯,宗雯琦,戴月,等.2016—2020年江苏省副溶血性弧菌食源性疾病事件流行病学分析[J].现代预防医学,2022, 49(19): 3615-3620.
6	庞梦婷,高巍,贠庆茹,等.2018—2019年包头市市售水产品中副溶血性弧菌监测结果及病原学特征分析[J].现代预防医学,2021, 48(22): 4201-4205.
7	陈建业,陈笑南,缪蔚蔚.2017年—2020年温州市瓯海区水产品中副溶血性弧菌污染情况分析[J].中国卫生检验杂志,2021, 31(20): 2534-2536.
8	LIU Z, TONG Z, LIU B, et al.. Ricin aptamer screening based on the QSAR model and construction of piezoresistive micro-cantilever aptasensor[J/OL]. Talanta, 2023, 252: 123840[2023-01-15]. .
9	CHEN K, ZHU L, DU Z, et al.. Docking-aided rational tailoring of a fluorescence- and affinity-enhancing aptamer for a label-free ratiometric malachite green point-of-care aptasensor[J/OL]. J. Hazard Mater., 2023, 447: 130798[2023-04-05]. .
10	ZHOU Z, LAN X, ZHU L, et al.. Portable dual-aptamer microfluidic chip biosensor for Bacillus cereus based on aptamer tailoring and dumbbell-shaped probes[J/OL]. J. Hazard. Mater., 2023, 445: 130545[2023-03-05]. .
11	DARMOSTUK M, RIMPELOVA S, GBELCOVA H, et al.. Current approaches in SELEX: an update to aptamer selection technology[J]. Biotechnol. Adv., 2015, 33(6): 1141-1161.
12	ELLINGTON A D, SZOSTAK J W. In vitro selection of RNA molecules that bind specific ligands[J]. Nature, 1990, 346(6287): 818-822.
13	QIAN S, CHANG D, HE S, et al.. Aptamers from random sequence space: accomplishments, gaps and future considerations[J/OL]. Anal. Chim. Acta, 2022, 1196: 339511[2022-03-01]. .
14	赵文卓,李成勋,胡作建,等.功能核酸用于致病菌检测的研究进展[J].生物技术进展,2023,13(1):30-38.
15	SIDDIQUI S, YUAN J. Binding characteristics study of DNA based aptamers for E .coli O157:H7[J/OL]. Molecules, 2021, 26(1): 204[2021-01-03]. .
16	RYCHLIK W, SPENCER W J, RHOADS R E. Optimization of the annealing temperature for DNA amplification in vitro[J]. Nucleic Acids Res., 1990, 18(21): 6409-6412.
17	杜丽.副溶血性弧菌临床检验分析[J].中国卫生标准管理,2014, 5(10): 50-51.
18	JIANG H, SUN Z, GUO Q, et al.. Microfluidic thread-based electrochemical aptasensor for rapid detection of Vibrio parahaemolyticus [J/OL]. Biosens. Bioelectron., 2021, 182: 113191[2021-01-15]. .
19	SONG S, WANG X, XU K, et al.. Visualized detection of Vibrio parahaemolyticus in food samples using dual-functional aptamers and cut-assisted rolling circle amplification[J]. J. Agric. Food Chem., 2019, 67(4): 1244-1253.

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副溶血性弧菌特异性核酸适配体筛选

SELEX of Vibrio parahaemolyticus Specific Aptamer

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