中国农业科学 ›› 2021, Vol. 54 ›› Issue (11): 2419-2433.doi: 10.3864/j.issn.0578-1752.2021.11.014
收稿日期:
2020-09-16
接受日期:
2020-12-21
出版日期:
2021-06-01
发布日期:
2021-06-09
通讯作者:
王毕妮
作者简介:
惠媛媛,E-mail:基金资助:
HUI YuanYuan(),PENG HaiShuai,WANG BiNi(
),ZHANG FuXin,LIU YuFang,JIA Rong,REN Rong
Received:
2020-09-16
Accepted:
2020-12-21
Online:
2021-06-01
Published:
2021-06-09
Contact:
BiNi WANG
摘要:
由于食品的多样性和食品基质的复杂性,食品安全问题已成为全社会共同关注的热点问题,其中微生物引起的食源性疾病居全球食品安全问题之首。食源性致病菌的检测是食源性疾病预防与控制的关键环节。平板计数法被评为微生物检测的金标准,但在致病菌检测过程中信号的放大需要通过单个细胞生长成菌落来实现,因此检测周期较长(3—7 d)。此外,现有的准确检测致病菌的技术有聚合酶链式反应(PCR)和酶联免疫吸附法(ELISA)等,但由于预处理时间长、操作复杂、检测结果存在假阳性等问题,不适合对致病菌进行现场快速有效的检测。核酸适配体是利用指数富集的配体系统进化技术(SELEX)从核酸分子文库中得到的寡核苷酸片段,具有良好的特异性、稳定性、易于修饰和亲和力高等特点,在毒素、抗生素、重金属和致病菌等其他小分子的检测中有很大的潜力。目前,国内外学者已开发了各种基于适配体的检测方法。本文综述了食品中常见的食源性致病菌及其传统的检测方法和近十年来用于致病菌检测的光学和电化学适配传感器,涵盖每种方法的检测策略、检测时间、检测范围和检测限等信息,也提出了适配体传感器在食品安全检测中存在的主要问题,并对其未来研究的发展趋势进行了展望,这些将为今后的研究工作提供一定的基础。
惠媛媛,彭海帅,王毕妮,张富新,刘玉芳,贾蓉,任荣. 基于适配体的光学和电化学法对食源性致病菌检测的研究进展[J]. 中国农业科学, 2021, 54(11): 2419-2433.
HUI YuanYuan,PENG HaiShuai,WANG BiNi,ZHANG FuXin,LIU YuFang,JIA Rong,REN Rong. Research Progress of Food-Borne Pathogen Detection Based on Electrochemical and Optical Aptasensors[J]. Scientia Agricultura Sinica, 2021, 54(11): 2419-2433.
表1
传统食源性病原体检测方法的基本原理、优势和局限性"
传统方法 Method | 优势 Advantage | 缺陷 Limitation |
---|---|---|
培养法 Culture method | 检测限低、可靠性强 Low detection limit and high credibility | 耗时、耗力 Time-consuming and laborious |
基于免疫的方法 Immunology-based method | 准确度高 High accuracy | 灵敏度低 Low sensitivity |
基于PCR的方法 PCR-based method | 灵敏度高 High sensitivity | 假阴性 False negative |
表2
食源性致病菌的适配体序列"
食源性致病菌 Pathogen | 菌型 Bacterial type | 筛选方法 Screening method | 靶标 Target | 适配体序列 Sequence (5′-3′) | 参考文献 Reference |
---|---|---|---|---|---|
大肠杆菌 Escherichia coli | O157:H7 | Non-SELEX | 脂多糖 LPS | TATGGCGTGGCAAGCTTGGCCCGCTTCTCAAGCATGGTTATCTAC | [ |
SELEX | 全菌 Whole bacteria | ACCAGTAGACTTTCAACTTTACTGCCATCGTGTGCCCTAA | [ | ||
Non-SELEX | 脂多糖 LPS | CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG | [ | ||
EPEC | SELEX | 全菌 Whole bacteria | ACAGTGCTCGGGATATATCAATATGTCACCTCGGCTAATG | [ | |
O55:B5 | Non-SELEX | 脂多糖 LPS | TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA | [ | |
沙门氏菌 Salmonella | 鼠伤寒 Typhimurium | Whole-cell-SELEX | 全菌 Whole bacteria | ACGGGCGTGGGGGCAATGCCTGCTTGTAGGCTTCCCCTGTGCGCG | [ |
SELEX | 全菌 Whole bacteria | TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG | [ | ||
SELEX | IVB型菌毛 IVB pili | GGGAACAGUCCGAGCCUCACUGUUAUCCGAUAGCAGCGCGGGAUGAGGGUCAAUGCGUCAUAGGAUCCCGC | [ | ||
O8 | Whole-cell-SELEX | 全菌 Whole bacteria | GATCCGGGCCTCATGTCGAACACCCCCCAACTAAAACAACAAAACACCACCGCCATTGAGCGTTTATTCTGAGCTCCCA | [ | |
SELEX | 全菌 Whole bacteria | TGATCCGGGCCTCATGTCGAACCCACACCCCACAACCACCCAGCCCCAGCCCGCTATTGAGCGTTTATTCTGAGCTCCCA | [ | ||
金黄色葡萄球菌 Staphylococcus aureus | SELEX | 磷壁酸 Teichoic acid | GGGAGUUUUGAUACGGCUUCAUGCAGUAAUGUUUUUAU | [ | |
SELEX | 肠毒素C1 Enterotoxin C1 | AGCAGCACAGAGGTCAGATGTATACTTCTAAAATTTGTTTGTATCTACGATGTTCTTCGTCCTATGCGTGCTACCGTGAA | [ | ||
SELEX | 肠毒素B Enterotoxin B | AGCAGCACAGAGGTCAGATGTATACTTCTAAAATTTGTTTGTATCTACGATGTTCTTCGTCCTATGCGTGCTACCGTGAA | [ | ||
SELEX | 肠毒素B Enterotoxin B | TGCAGGATCCGGTATCCGTGGACGGTGTGCAGGATCCGGTATCCGTGGGCACGAGAATTCCTCCGTTGCG | [ | ||
SELEX | 肠毒素A Enterotoxin A | TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC | [ | ||
单核增生李斯特菌 Listeria monocytogenes | Whole-cell-SELEX | 全菌 Whole bacteria | TGGGAGCTCAGAATAAACGCTCAACTTTGTTCTTCTTTGCTTTTTTTTTCTTTTTTTGTTCGACATGAGGCCCGGATCA | [ | |
阪崎肠杆菌 Cronobacter sakazakii | Whole-cell-SELEX | 全菌 Whole bacteria | ATAGGAGTCACGACGACCAGGCCGCCCGGGGACGGGGGCGGCGGGGAGGAGGGCGGCGGGTATGTGCGTCTACCTCTTGA | [ | |
蜡样芽孢杆菌 Bacillus cereus | SELEX | 芽孢 Spore | CATCCGTCACACCTGCTCGGTGCAGACCCATAGGGGGGGCGTGCGGATGTAGGAGTAGGGTGTTGGCTCCCGTATC | [ | |
铜绿假单胞菌 Pseudomonas aeruginosa | Whole-cell-SELEX | 全菌 Whole bacteria | CCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTGC TTCCTTTCTTG | [ | |
副溶血性弧菌 Vibrio parahemolyticus | Whole-cell-SELEX | 全菌 Whole bacteria | ATAGGAGTCACGACGACCAGAATCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACTTATGTGCGTCTACCTCTTGACTAAT | [ |
表3
用于食源性致病菌检测的传感器性能比较"
传感器类型 Sensor type | 方法 Method | 策略 Strategy | 检测范围 Detection range (cfu·mL-1) | 检测限 LOD (cfu·mL-1) | 参考文献 Reference |
---|---|---|---|---|---|
电化学 Electrochemical | 将适配体固定在电极表面,靶标与适配体的结合导致电极表面阻抗增大,通过检测电化学信号的变化检测靶标 The aptamer is modified on the surface of the electrode, and the combination of the target and the aptamer leads to an increase in the surface impedance of the electrode. The target is detected by detecting the change of electrochemical signal | rGO-TiO2复合纳米材料信号放大 RGO -TiO2 composite nanomaterial signal amplification | 101—108 | 10 | [ |
rGO-CNT复合纳米材料信号放大 rGO-CNT composite nanomaterial signal amplification | 101—108 | 10 | [ | ||
聚[吡咯-co-3-羧基吡咯]共聚物信号放大 poly[pyrrole-co-3-carboxyl-pyrrole]copolymer signal amplification | 102—108 | 3 | [64] | ||
三明治法 Sandwich assay | 适配体-靶标-MB@MI纳米材料 Apt-target-MB@MI | 102—108 | 32 | [ | |
抗体/磁性纳米颗粒-靶标-适配/脲酶复合物 Antibody/magnetic nanoparticles - target - aptamer/urease complex | 101—104 | 10 | [ | ||
三明治法 Sandwich assay | 滚环扩增 Rolling cycle amplification | 2.2—2.2×108 | 2 | [ | |
比色 Colorimetric | NaCl-AuNPs | 高浓度的盐会引起AuNPs聚集,颜色从酒红色变为蓝色 high-salt induce AuNPs aggregation from red to blue | _ | 105 | [ |
102—107 | 56 | [ | |||
105—108 | 7.2×105 | [ | |||
过氧化氢氧化法 H2O2 oxidation | 7-四链体-heminDNA酶可催化四甲基联苯胺(TMB)-过氧化氢(H2O2)体系发生反应,颜色从无色变为蓝色 G4/Hemin DNAzyme can catalyze TMB-H2O2 reaction system, and colour of the substrates changing from colourless to blue | 102—107 | 10 | [ | |
纳米模拟酶可催化TMB-H2O2 体系发生反应,颜色从无色变为蓝色 Nanozymes can catalyze TMB-H2O2 reaction system, and colour of the substrates changing from colourless to blue | 11—1.1×105 | 11 | [ | ||
102—106 | 10 | [ | |||
荧光 Fluorescent | 荧光的产生 Fluorescence generation | TPE-OH 能在牛血清白蛋白(BSA)微球中聚集,产生明亮的荧光信号 TPE-OH aggregates in bovine serum albumin (BSA) microspheres producing bright fluorescent signal | 101—106 | 10 | [ |
荧光的猝灭 Fluorescence quenching | MoS2纳米材料对于荧光素(FAM)的猝灭 Quenching of MoS2 nanomaterials to fluorescein (FAM) | 102—105 | 10 | [ | |
CIP@MWCNT纳米材料对于荧光素(FAM)的猝灭 Quenching of CIP@MWCNT nanomaterials to fluorescein (FAM) | 7.15—103 | 3.15×102 | [ | ||
荧光染料AccuBlue? Fluorescent dye AccuBlue? | 靶标与适配体的结合会改变荧光染料的荧光信号 The binding of target and aptamer changed the fluorescence signal of fluorescent dye | 50—106 | 25 | [ | |
荧光染料罗丹明B Fluorescent dye Rhodamine B | 1530—96938 | 464 | [ | ||
化学发光 Chemiluminescence | 化学发光共振能量转移 Chemiluminescence resonance energy transfer | Co2+增强/ABEI功能化花状纳米金和滚环扩增技术 Co2+ enhanced /ABEI functionalized flower-like nanogold and roll ring amplification techniques | 32—3.2 ×106 | 10 | [ |
50—1.5×105 | 15 | [ | |||
表面增强拉曼散射 SERS | 将适配体修饰在SERS基地上,当靶标与适配体结合后会产生拉曼信号变化,通过对拉曼变化信号的检测进而检测靶标菌 The aptamer is modified on the SERS base. When the target and the aptamer are combined, the Raman signal will change, and the target bacteria can be detected by detecting the Raman change signal | 适配体和罗丹明 B 同时修饰的金纳米棒作为拉曼信号探针 The gold nanorods modified by aptamer and rhodamine B was used as Raman signal probe | 101—104 | 3 | [ |
适配体和拉曼分子修饰的金纳米颗粒作为拉曼信号探针 The nanoparticles modified by aptamer and raman molecules was used as Raman signal probe | 102—107 | 35/15 | [ | ||
适配体和罗丹明修饰的Au@Ag 核壳纳米材料作为拉曼信号探针 The Au@Ag core-shell nanomaterials modified by ROX and aptamer was used as Raman signal probe | 15—1.5×105 | 15 | [ | ||
表面等离子体共振 Surface plasmon resonance | 将适配体修饰在等离子共振装置表面上。当靶标菌存在时共振装置折射率、反射光等会发生变化,通过对光变化信号的检测可以检测靶标 The aptamer is modified on the surface of the plasmon resonance device. When the target bacteria are present, the refractive index and reflected light of the resonance device will change, and the target can be detected by detecting the light change signal | 使用Ω形光纤探针 Using a novel Ω-shaped ?ber-optic probe | 5.0×102— 1.0×108 | 5 | [ |
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