G-三链体可视化核酸传感器用于四环素的检测

doi:10.13560/j.cnki.biotech.bull.1985.2021-1172

摘要/Abstract

摘要：

四环素因其广谱活性和低成本，在畜牧业和水产养殖中被广泛用于预防细菌感染和提高生长速度，四环素的过度使用导致抗生素在食品中残留，严重威胁人类的健康。硫代黄素T（ThT）可以嵌入到特殊核酸结构中，从而荧光强度得到强激发实现信号输出。本文中四环素适配体可与富G序列结合形成双链，但四环素存在时优先与适配体结合，经解除结合后的富G序列可自身折叠形成G-三链体结构，增强了ThT的荧光强度，依据靶标浓度与ThT荧光强度良好的线性关系，实现对四环素的定量检测。本方法在四环素浓度为1 nmol/L-1 μmol/L范围内有良好线性，R²高达0.99，检测限为0.07 nmol/L，并且在25 min内可以完成检测。因此，一种基于G-三链体的双链竞争内劈裂可视化方法被开发出来并实现了四环素的简便、快速、灵敏、特异、低成本的检测。

关键词: 硫代黄素T, 四环素, G-三链体, 适配体, 功能核酸

Abstract:

Tetracycline is widely used in animal husbandry and aquaculture to prevent bacterial infection and improve growth rate because of its broad-spectrum activity and low cost. Excessive use of tetracycline leads to antibiotic residues in food，which seriously threatens human health. Thiophenin T（ThT）can be embedded into a special nucleic acid structure，so that the fluorescence intensity is strongly excited to achieve signal output. In this paper，tetracycline aptamer can bind with G-rich sequence to form a double chain，but tetracycline preferentially binds with the aptamer when it exists. The G-rich sequence after unbound can fold itself to form a G-triplex structure，which enhances the fluorescence intensity of ThT. According to the good linear relationship between the target concentration and the fluorescence intensity of ThT，the quantitative detection of tetracycline can be acheieved. The method offers good linearity in the range of tetracycline concentration from 1 nmol/L-1 μmol/L 1 μ m，R² is as high as 0.99，detection limit is 0.07 nmol/L，and detection can be completed within 25 min. Therefore，a visualization method based on G-triplex for double-stranded competitive internal cleavage has been developed and simple，rapid，sensitive，specific and low-cost detection of tetracycline is achieved.

Key words: thiophenin T, tetracycline, G-triplex, aptamer, functional nucleic acid

刘宁宁, 王鑫昕, 兰欣悦, 褚华硕, 陈旭, 常世敏, 李腾飞, 许文涛. G-三链体可视化核酸传感器用于四环素的检测[J]. 生物技术通报, 2022, 38(10): 106-114.

LIU Ning-ning, WANG Xin-xin, LAN Xin-yue, CHU Hua-shuo, CHEN Xu, CHANG Shi-min, LI Teng-fei, XU Wen-tao. G-Triplex Visualization Nucleic Acid Sensor for the Detection of Tetracycline[J]. Biotechnology Bulletin, 2022, 38(10): 106-114.

图/表 8

表1 适配体及G-三链体序列表

Table 1 Sequence list of aptamers and G-triplex

名称 Name	序列Sequence	碱基数 Number of bases/nt
G01	5'-GGGCACCACCAGGGTTAGGG-3'	20
G02	5'-GGGCACCACAGGGTTAGGG-3'	19
G03	5'-GGGCACCAAGGGTTAGGG-3'	18
G04	5'-GGGCACCAGGGTTAGGG-3'	17
G05	5'-GGGCACAGGGTTAGGG-3'	16
G06	5'-GGGCAAGGGTTAGGG-3'	15
G07	5'-GGGCAGGGTTAGGG-3'	14
Apt1	5'-CGGTGGTGCCC-3'	11

图1 G3-Apt1检测四环素原理图

Fig. 1 Schematic diagram of detecting tetracycline by G3-Apt1

图2 适配体及序列优化图 A：不同条件下胶体金的颜色图，1：阴性对照，2 ：阳性对照，3-5：分别加入四环素1、10和100 μmol/L；B：G01-G07序列与适配体结合加入靶标前后ThT荧光强度

Fig. 2 Aptamer and sequence optimization diagram A：Color diagram of colloidal gold under different conditions，1 is negative control，2 is positive control，and 3-5 refers to adding tetracycline 1，10，and 100 μmol/L respectively. B：ThT fluorescence intensities before and after adding target while G01-G07 sequences bound with aptamers

图3 不同条件下ThT荧光强度 A：不同离子存在下ThT荧光强度（1：Tris-HCl+CaCl2，2：Tris-HCl+MgCl2，3：Tris-HCl+KCl，4：H2O，5：CaCl2，6：MgCl2，7：KCl；pH=7.4）；B：不同浓度KCl溶液对ThT荧光强度的影响

Fig. 3 ThT fluorescence intensities under different conditions A: ThT fluorescence intensity in the presence of different ions（1: Tris-HCl+CaCl2，2: Tris-HCl+MgCl2，3: Tris-HCl+KCl，4: H2O，5: CaCl2，6: MgCl2，and 7: KCl；PH=7.4）；B: effect of KCl solution with different concentration on the ThT fluorescence intensity

图4 时间的优化图 A：靶标与适配体孵育0-30 min内ThT荧光强度变化；B：0-30 min内ThT荧光强度的变化

Fig. 4 Time optimization A: the change of ThT fluorescence intensity during 0-30 min incubation between target and aptamer；B: changes of ThT fluorescence intensity within 0-30 min

图5 G3-Apt1传感器的建立图 A：不同浓度四环素存在下ThT荧光强度；B：标准曲线

Fig. 5 Establishment of G3-Apt1 sensor A：ThT fluorescence intensity in the presence of tetracycline with different concentrations；B：standard curve

图6 特异性验证图 1：四环素；2：盐酸四环素；3：土霉素；4：氯霉素；5：链霉亲和素；6：林可霉素

Fig. 6 Specificity verification chart 1：Tetracycline. 2：Tetracycline hydrochloride. 3：Oxytetracycline. 4：Chloramphenicol. 5：Streptavidin. 6：Lincomycin

表2 牛奶样品中四环素添加回收表

Table 2 Addition and recovery table of tetracycline in milk samples

添加浓度Adding concentration/（nmol·L^-1）	荧光强度 Fluorescence intensity/（a.u.）			回收浓度 Recovery concentration/（nmol·L^-1）			平均浓度 Mean concentration/（nmol·L^-1）	回收率 Recovery rate/%
0	115.2	114.1	115.1	0.01	0	0.03	0.01	101.33
1	17.0	173.2	172.5	1.08	1.10	1.05	1.08	107.67
10	210	208	209	11.09	9.78	10.42	10.43	104.3
100	245	245.6	246	99.89	104.41	106.35	103.55	103.55
1 000	282	281.1	281.5	1 019.66	1 120.36	988.14	1 042.72	104.27

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