生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 81-89.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0698
侯炜辰1(), 叶柯1, 李洁2, 张洋子3, 许文涛3, 朱龙佼3, 李相阳1()
收稿日期:
2023-07-19
出版日期:
2023-12-26
发布日期:
2024-01-11
通讯作者:
李相阳,男,副教授,研究方向:食品安全;E-mail: lxy2002cn@163.com作者简介:
侯炜辰,女,硕士研究生,研究方向:食品加工与安全;E-mail: houweichen007@163.com
基金资助:
HOU Wei-chen1(), YE Ke1, LI Jie2, ZHANG Yang-zi3, XU Wen-tao3, ZHU Long-jiao3, LI Xiang-yang1()
Received:
2023-07-19
Published:
2023-12-26
Online:
2024-01-11
摘要:
大肠杆菌O157: H7(Escherichia coli O157:H7)是一种重要的与公共卫生相关的食源性病原体。抗体分子是体液免疫应答中最重要的效应分子,具有多种生物学活性,最主要的生物学功能是与相应抗原特异性结合。适配体是体外合成的较短DNA序列,可通过识别特定区域和靶标特异性结合。利用抗体和适配体的特异性识别功能及免疫磁珠的捕获作用和磁分离,并通过多克隆抗体和裁剪得到的适配体搭建生物传感器,使用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR)可以实现对靶标在(8×103)-(8×106) CFU/mL范围内的定量检测,检测限为800 CFU/mL。
侯炜辰, 叶柯, 李洁, 张洋子, 许文涛, 朱龙佼, 李相阳. 基于抗体-适配体夹心生物传感器检测大肠杆菌O157: H7[J]. 生物技术通报, 2023, 39(12): 81-89.
HOU Wei-chen, YE Ke, LI Jie, ZHANG Yang-zi, XU Wen-tao, ZHU Long-jiao, LI Xiang-yang. Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor[J]. Biotechnology Bulletin, 2023, 39(12): 81-89.
名称 Name | 序列 Sequence(5'-3') |
---|---|
S1 | CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT ATG GGT GGT GGA TGA GTG TGT GGC TCG CAC TGC TCC TGA ACG TAC |
S1-1 | CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT A TCG CAC TGC TCC TGA ACG TAC |
S1-2 | CAG GTC CAT CGA GTG GTA GGA TGG GTG GTG GAT GAG TGT GTG GC TCG CAC TGC TCC TGA ACG TAC |
S1-3 | CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TGG GTG GT TCG CAC TGC TCC TGA ACG TAC |
S1-3-1 | CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TCG CAC TGC TCC TGA ACG TAC |
FP | CAG GTC CAT CGA GTG GTA GGA |
RP | GTA CGT TCA GGA GCA GTG CGA |
表1 实验所用的核酸序列
Table 1 Nucleic acid sequences used in the experiment
名称 Name | 序列 Sequence(5'-3') |
---|---|
S1 | CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT ATG GGT GGT GGA TGA GTG TGT GGC TCG CAC TGC TCC TGA ACG TAC |
S1-1 | CAG GTC CAT CGA GTG GTA GGA TGG TCG TGG TGA GGT GCG TGT A TCG CAC TGC TCC TGA ACG TAC |
S1-2 | CAG GTC CAT CGA GTG GTA GGA TGG GTG GTG GAT GAG TGT GTG GC TCG CAC TGC TCC TGA ACG TAC |
S1-3 | CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TGG GTG GT TCG CAC TGC TCC TGA ACG TAC |
S1-3-1 | CAG GTC CAT CGA GTG GTA GGA GAG GTG CGT GTA TCG CAC TGC TCC TGA ACG TAC |
FP | CAG GTC CAT CGA GTG GTA GGA |
RP | GTA CGT TCA GGA GCA GTG CGA |
图2 适配体裁剪可行性分析 A:原始菌液-适配体和梯度稀释后的扩增曲线;B:原始菌液-适配体和梯度稀释后的变化趋势
Fig. 2 Feasibility analysis of adaptor clipping A:Amplification curve of original bacterial solution adaptor and gradient dilution.B:Change trend of original bacterial solution adaptor and gradient dilution
图3 裁剪后适配体和原始适配体比较 A:S1和S1-1扩增曲线;B:S1和S1-2扩增曲线;C:S1和S1-3扩增曲线;D:S1和S1-3-1扩增曲线
Fig. 3 Comparison of tailored adaptor and original adaptor A: S1 and S1-1 amplification curves; B: S1 and S1-2 amplification curves; C: S1 and S1-3 amplification curves; D: S1 and S1-3-1 amplification curves
图5 生物传感器可行性分析 A:IMB-靶标-适配体、IMB-适配体和IMB-靶标3种复合物扩增曲线;B:菌液4次10倍梯度稀释后扩增曲线
Fig. 5 Feasibility analysis for biosensor A: Amplification curves of three complexes of IMB target adaptor, IMB adaptor, and IMB target. B: Amplification curves after four times ten fold gradient dilution of bacterial solution
图6 条件优化结果 A:适配体裁剪结合缓冲液类型优化;B:适配体裁剪缓冲液pH优化;C:适配体裁剪孵育温度优化;D:生物传感器IMB-靶标孵育时间优化
Fig. 6 Condition optimization results A: Optimization of the type of adapted body scissor binding buffer; B: optimization of the pH of adapted body scissor buffer; C: optimization of adapted body scissor incubation temperature; D: optimization of sensor IMB target incubation time
图8 大肠埃希菌 O157∶H7 检测方法的特异性 A:裁剪适配体特异性;B:传感器属间特异性;C:传感器种间特异性
Fig. 8 Specificity of detection method for E. coli O157∶H7 A: Tailor adaptor specificity; B&C: interspecific specificity of sensors
样品 Sample | 传统培养法Traditional cultivation method /(CFU·mL-1) | 检测量Detection quantity/ (CFU·mL-1) | 回收率 Rate of recovery/% | RSD/% |
---|---|---|---|---|
1 | 3×103 | 2.89×103 | 96.3 | 2.15 |
2 | 3×104 | 3.11×104 | 103 | 2.07 |
3 | 3×105 | 3.26×105 | 108 | 1.35 |
表2 实际样品检测
Table 2 Actual sample testing
样品 Sample | 传统培养法Traditional cultivation method /(CFU·mL-1) | 检测量Detection quantity/ (CFU·mL-1) | 回收率 Rate of recovery/% | RSD/% |
---|---|---|---|---|
1 | 3×103 | 2.89×103 | 96.3 | 2.15 |
2 | 3×104 | 3.11×104 | 103 | 2.07 |
3 | 3×105 | 3.26×105 | 108 | 1.35 |
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