生物技术通报 ›› 2020, Vol. 36 ›› Issue (11): 245-258.doi: 10.13560/j.cnki.biotech.bull.1985.2019-1273
王琦1(), 颜春蕾1, 高洪伟2, 吴薇1(), 杨庆利1()
收稿日期:
2019-12-29
出版日期:
2020-11-26
发布日期:
2020-11-20
作者简介:
王琦,女,硕士研究生,研究方向:食品危害物的快速检测;E-mail: 基金资助:
WANG Qi1(), YAN Chun-lei1, GAO Hong-wei2, WU Wei1(), YANG Qing-li1()
Received:
2019-12-29
Published:
2020-11-26
Online:
2020-11-20
摘要:
核酸适配体是可以识别并与靶标分子结合的单链DNA或RNA分子。由于适配体具有良好的特异性、稳定性、选择性、易于修饰和亲和力高等特点,在病原检测和其他小分子识别方面具有很大的潜力。综述了适配体的筛选技术以及适配体应用技术,包括比色法、可见光谱法、荧光法、电化学法、表面增强拉曼法和胶体金试纸条法等在食源性致病菌检测中的应用。目前适配体传感器在检测食品致病菌领域取得了显著进展。然而核酸适配体应用方面的挑战和缺陷仍需克服。
王琦, 颜春蕾, 高洪伟, 吴薇, 杨庆利. 基于核酸适配体传感器检测食品致病菌的研究进展[J]. 生物技术通报, 2020, 36(11): 245-258.
WANG Qi, YAN Chun-lei, GAO Hong-wei, WU Wei, YANG Qing-li. Research Progress of DNA Aptasensors for Foodborne Pathogen Detection[J]. Biotechnology Bulletin, 2020, 36(11): 245-258.
图1 利用SELEX筛选食源性致病菌适配体的示意图 SELEX分为5个步骤:1. 合成体外DNA随机文库;2. DNA文库和靶标病原菌一起孵育;3. 未结合核酸序列的分离;4. 与靶标结合的核酸序列的洗脱;5. 目标结合核酸序列作为下一次PCR扩增的模板。扩增后的产物作为新的DNA文库用于下一轮的筛选
靶标 | 菌型 | 筛选方法 | 序列(5'-3') | 参考文献 |
---|---|---|---|---|
大肠杆菌 | 大肠杆菌O157:H7 | 全细胞-SELEX | ACCAGTAGACTTTCAACTTTACTGCCATCGTGTGCCCTAA | [25,32] |
Non-SELEX | TATGGCGTGGCAAGCTTGGCCCGCTTCTCAAGCATGGTTATCTAC | [33] | ||
大肠杆菌O111:B4 | 磁珠-SELEX | ATCCGTCACCCCTGCTCTCGTCGCTATGAAGTAACAAAGATAGGAGCAAT CGGGTGGTGTTGGCTCCCGTAT | [34] | |
大肠杆菌O111 | 全细胞-SELEX | ATCCGTCACACCTGCTCTACTGGCCGGCTCAGCATGACTAAGAAGGAAGT TATGTGGTGTTGGCTCCCGTAT | [35] | |
大肠杆菌O55:B5 | Non-SELEX | TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA | [36] | |
沙门氏菌 | 鼠伤寒沙门氏菌 | 全细胞-SELEX | ACGGGCGTGGGGGCAATGCCTGCTTGTAGGCTTCCCCTGTGCGCG | [37] |
SELEX | TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG | [38] | ||
沙门氏菌O8 | 全细胞-SELEX | GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACC ACCGCCATTGAGCGTTTATTCTGAGCTCCCA | [39] | |
SELEX | TGATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACAC CACCGCCATTGAGCGTTTATTCTGGCTCCCA | [40] | ||
肠炎沙门氏菌 | 磁珠-SELEX | GGGUUCACUGCAGACUUGACGAAGCUUGAGAGAUGCCCCCUGAUGTGC AUUCUUGUUGUGUUGCGGCAAUGGAUCCACAUCTACGAAUUC | [41] | |
金黄色葡萄球菌 | 金黄色葡萄球菌 | 全细胞-SELEX | GCGCCCTCTCACGTGGCACTCAGAGTGCCGGAAGTTCTGCGTTAT | [42] |
肠毒素 | SELEX | TTTGGTATTGAGGGTCGCATCCACTGGTCGTTGTCTGTTGTCTGTTAT GTTGTTTCGTGATGGCTCTAACTCTCCTCT | [43] | |
磷壁酸 | SELEX | GGGAGUUUUGAUACGGCUUCAUGCAGUAAUGUUUUUAU | [31] | |
肠毒素B | SELEX | AGCAGCACAGAGGTCAGATGTATACTTCTAAAATTTGTTTGTATCTAC GATGTTCTTCGTCCTATGCGTGCTACCGTGAA | [44] | |
肠毒素A | SELEX | TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC | [45] | |
副溶血弧菌 | 全细胞-SELEX | ATAGGAGTCACGACGACCAGAATCTAAAAATGGGCAAAGAAACAGT GACTCGTTGAGATACTTATGTGCGTCTACCTCTTGACTAAT | [46] | |
单核细胞增生李斯特菌 | 全细胞-SELEX | TGGGAGCTCAGAATAAACGCTCAACTTTGTTCTTCTTTGCTTTTTTTTT CTTTTTTTGTTCGACATGAGGCCCGGATCA | [47] | |
蜡样芽孢杆菌 | 芽孢 | SELEX | CATCCGTCACACCTGCTCGGTGCAGACCCATAGGGGGGGCGTGCGGA TGTAGGAGTAGGGTGTTGGCTCCCGTATC | [48] |
志贺氏菌 | 痢疾志贺氏菌 | 全细胞-SELEX | CGGAACTAGCGTTTAAATGCCAGGACTGAAGTAGGCAGGG | [22] |
宋内志贺菌 | 全细胞-SELEX | ATTATGTTGTCTGAACTGCCATGGTCCCTCGTGTTTATTATGTTGTCT GACTGGCTGAGATTGCACTTACTATCT | [49] | |
绿脓杆菌 | 全细胞-SELEX | CCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTGC TTCCTTTCTTG | [50] |
表1 不同筛选方法得到的食源性致病菌的适配体
靶标 | 菌型 | 筛选方法 | 序列(5'-3') | 参考文献 |
---|---|---|---|---|
大肠杆菌 | 大肠杆菌O157:H7 | 全细胞-SELEX | ACCAGTAGACTTTCAACTTTACTGCCATCGTGTGCCCTAA | [25,32] |
Non-SELEX | TATGGCGTGGCAAGCTTGGCCCGCTTCTCAAGCATGGTTATCTAC | [33] | ||
大肠杆菌O111:B4 | 磁珠-SELEX | ATCCGTCACCCCTGCTCTCGTCGCTATGAAGTAACAAAGATAGGAGCAAT CGGGTGGTGTTGGCTCCCGTAT | [34] | |
大肠杆菌O111 | 全细胞-SELEX | ATCCGTCACACCTGCTCTACTGGCCGGCTCAGCATGACTAAGAAGGAAGT TATGTGGTGTTGGCTCCCGTAT | [35] | |
大肠杆菌O55:B5 | Non-SELEX | TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA | [36] | |
沙门氏菌 | 鼠伤寒沙门氏菌 | 全细胞-SELEX | ACGGGCGTGGGGGCAATGCCTGCTTGTAGGCTTCCCCTGTGCGCG | [37] |
SELEX | TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG | [38] | ||
沙门氏菌O8 | 全细胞-SELEX | GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACC ACCGCCATTGAGCGTTTATTCTGAGCTCCCA | [39] | |
SELEX | TGATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACAC CACCGCCATTGAGCGTTTATTCTGGCTCCCA | [40] | ||
肠炎沙门氏菌 | 磁珠-SELEX | GGGUUCACUGCAGACUUGACGAAGCUUGAGAGAUGCCCCCUGAUGTGC AUUCUUGUUGUGUUGCGGCAAUGGAUCCACAUCTACGAAUUC | [41] | |
金黄色葡萄球菌 | 金黄色葡萄球菌 | 全细胞-SELEX | GCGCCCTCTCACGTGGCACTCAGAGTGCCGGAAGTTCTGCGTTAT | [42] |
肠毒素 | SELEX | TTTGGTATTGAGGGTCGCATCCACTGGTCGTTGTCTGTTGTCTGTTAT GTTGTTTCGTGATGGCTCTAACTCTCCTCT | [43] | |
磷壁酸 | SELEX | GGGAGUUUUGAUACGGCUUCAUGCAGUAAUGUUUUUAU | [31] | |
肠毒素B | SELEX | AGCAGCACAGAGGTCAGATGTATACTTCTAAAATTTGTTTGTATCTAC GATGTTCTTCGTCCTATGCGTGCTACCGTGAA | [44] | |
肠毒素A | SELEX | TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC | [45] | |
副溶血弧菌 | 全细胞-SELEX | ATAGGAGTCACGACGACCAGAATCTAAAAATGGGCAAAGAAACAGT GACTCGTTGAGATACTTATGTGCGTCTACCTCTTGACTAAT | [46] | |
单核细胞增生李斯特菌 | 全细胞-SELEX | TGGGAGCTCAGAATAAACGCTCAACTTTGTTCTTCTTTGCTTTTTTTTT CTTTTTTTGTTCGACATGAGGCCCGGATCA | [47] | |
蜡样芽孢杆菌 | 芽孢 | SELEX | CATCCGTCACACCTGCTCGGTGCAGACCCATAGGGGGGGCGTGCGGA TGTAGGAGTAGGGTGTTGGCTCCCGTATC | [48] |
志贺氏菌 | 痢疾志贺氏菌 | 全细胞-SELEX | CGGAACTAGCGTTTAAATGCCAGGACTGAAGTAGGCAGGG | [22] |
宋内志贺菌 | 全细胞-SELEX | ATTATGTTGTCTGAACTGCCATGGTCCCTCGTGTTTATTATGTTGTCT GACTGGCTGAGATTGCACTTACTATCT | [49] | |
绿脓杆菌 | 全细胞-SELEX | CCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTGC TTCCTTTCTTG | [50] |
传感器 | 适用场所 | 灵敏度CFU/mL | 优缺点 | 靶标 | LOD | 检测范围 | 参考 文献 |
---|---|---|---|---|---|---|---|
比色适配体传感器 | 直观快速检测复杂的食品基质 | 10 | 结果直观,适用于现场检测,但结果难以量化 | 副溶血性弧菌 | 10 CFU/mL | 102-107CFU/mL | [57] |
鼠伤寒沙门氏菌 | 11 CFU/mL | 11-1.10×105CFU/mL | [58] | ||||
可见光适配体传感器 | POC测试与商业检测 | 100 | 检测快速、易于操作、成本低、仪器便携,但灵敏度较差,难以精确量化 | 伤寒沙门菌 | 103CFU/mL | 103-107 CFU /mL | [59] |
金黄色葡萄球菌 | 3.17 μmol/L | 8-23 μmol/L | [60] | ||||
荧光适配体传感器 | 各种高通量的样品 | 103 | 特异性高,试剂和仪器成本高 | 嗜水气单胞菌和迟缓爱德华菌 | 1.5 CFU/mL和 67 CFU/mL | 0.1-1.3×105CFU/mL和1.3-1.3×104CFU/mL | [61] |
电化学适配体传感器 | 痕量检测 | 金黄色葡萄球菌 | 1 CFU/mL | 10-1×106CFU/mL | [62] | ||
O157:H7型大肠杆菌 | 10 CFU/mL | - | [56] | ||||
表面增强拉曼散射适配体传感器 | 痕量靶标的无损检测 | 5 | 灵敏度高,无标签,专业性要求高,仪器贵重 | 金黄色葡萄球菌 | 1.5 CFU/mL | 10-107 CFU/mL | [63] |
测流适配体传感器 | 直观快速检测 | 10 | 方便携带、易于操作、成本低,但难以量化 | 副溶血性弧菌 | 5.6 CFU/mL | - | [19] |
表2 用于食源性致病菌检测的传感器的性能比较
传感器 | 适用场所 | 灵敏度CFU/mL | 优缺点 | 靶标 | LOD | 检测范围 | 参考 文献 |
---|---|---|---|---|---|---|---|
比色适配体传感器 | 直观快速检测复杂的食品基质 | 10 | 结果直观,适用于现场检测,但结果难以量化 | 副溶血性弧菌 | 10 CFU/mL | 102-107CFU/mL | [57] |
鼠伤寒沙门氏菌 | 11 CFU/mL | 11-1.10×105CFU/mL | [58] | ||||
可见光适配体传感器 | POC测试与商业检测 | 100 | 检测快速、易于操作、成本低、仪器便携,但灵敏度较差,难以精确量化 | 伤寒沙门菌 | 103CFU/mL | 103-107 CFU /mL | [59] |
金黄色葡萄球菌 | 3.17 μmol/L | 8-23 μmol/L | [60] | ||||
荧光适配体传感器 | 各种高通量的样品 | 103 | 特异性高,试剂和仪器成本高 | 嗜水气单胞菌和迟缓爱德华菌 | 1.5 CFU/mL和 67 CFU/mL | 0.1-1.3×105CFU/mL和1.3-1.3×104CFU/mL | [61] |
电化学适配体传感器 | 痕量检测 | 金黄色葡萄球菌 | 1 CFU/mL | 10-1×106CFU/mL | [62] | ||
O157:H7型大肠杆菌 | 10 CFU/mL | - | [56] | ||||
表面增强拉曼散射适配体传感器 | 痕量靶标的无损检测 | 5 | 灵敏度高,无标签,专业性要求高,仪器贵重 | 金黄色葡萄球菌 | 1.5 CFU/mL | 10-107 CFU/mL | [63] |
测流适配体传感器 | 直观快速检测 | 10 | 方便携带、易于操作、成本低,但难以量化 | 副溶血性弧菌 | 5.6 CFU/mL | - | [19] |
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