G-Triplex Visualization Nucleic Acid Sensor for the Detection of Tetracycline

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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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