Research on rapid detection of Staphylococcus aureus by fluorescent biosensor based on DNAzyme
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摘要:目的
为解决传统方法检测金黄色葡萄球菌耗时长、操作复杂等问题，研发一种基于脱氧核酶(DNAzyme)的荧光生物传感器，实现金黄色葡萄球菌的快速检测。
方法将特异性DNAzyme和互补链Subatrate相结合制成荧光生物传感器，并对荧光生物传感器进行生物材料浓度和溶液pH优化，并对金黄色葡萄球菌、大肠埃希菌、芽孢杆菌、无乳链球菌、变形杆菌等进行特异性检测；最后对牛奶样品进行基于DNAzyme的荧光生物传感器的试验验证。
结果基于DNAzyme的荧光生物传感器在pH为 6.8时，3 min内可以实现对金黄色葡萄球菌的检测，线性范围为1~1×107 cfu·mL−1，最低检测限为1 cfu·mL−1。
结论基于DNAzyme的荧光生物传感器解决了传统检测方法耗时长、操作复杂等问题，实现了对金黄色葡萄球菌的快速检测，具有重要的应用价值。
Abstract:ObjectiveTo solve the problems of time-consuming and complex operation in traditional methods of detecting Staphylococcus aureus, a fluorescent biosensor based on deoxyribonuclease (DNAzyme) was developed to achieve rapid detection of S. aureus.
MethodThe fluorescence biosensor was made by combining DNAzyme with specific complementary chain substrate, and optimized for the concentration of biomaterials and pH of solution. Then, the specific detection of five different bacteria such as S. aureus, Escherichia coli, Bacillus, Streptococcus agalactiae and Proteus was performed. Finally, the DNAzyme-based fluorescent biosensor was tested and verified on milk samples.
ResultThe fluorescent biosensor based on DNAzyme can detect S. aureus within 3 min at a pH of 6.8, with a linear range of 1−1×107 cfu·mL−1 and a minimum detection limit of 1 cfu·mL−1.
ConclusionThe fluorescent biosensor based on DNAzyme solves the problems of time-consuming and complex operation of traditional detection methods, and realizes the rapid detection of S. aureus in milk, which has significant practical value.
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Keywords:
- Staphylococcus aureus /
- DNAzyme /
- Molecular beacon /
- Fluorescence biosensor
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图 1 不同浓度SA-substrate与SA-DNAzyme等浓度等体积结合前后的光密度变化
“*”“****”分别表示在P<0.05和P<0.000 1水平差异显著(t检验)
Figure 1. Optical density changes of different concentrations of SA-substrate before and after binding to the same concentration and volume of SA-DNAzyme
“*” and “****” indicate significant differences at P<0.05 and P<
0.0001 levels, respectively (t test)![]()
图 2 不同浓度生物敏感材料与金黄色葡萄球菌结合后光密度变化
Figure 2. Optical density changes of different concentrations of biosensitive materials after combining with Staphylococcus aureus
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图 3 溶液pH对荧光生物传感器相应信号的影响
“*”“**”“****”分别表示在P<0.05、P<0.01和P<0.000 1水平差异显著(t检验)
Figure 3. Effect of pH on the corresponding signal of fluorescent biosensor
“*” “**” and “****” indicate significant differences at P<0.05、P<0.01 and P<
0.0001 levels, respectively (t test)![]()
图 4 荧光生物传感器检测细菌随时间变化的光密度
相同时间柱子上方的不同小写字母表示处理间差异显著 (P<0.05，Duncan’s 法)
Figure 4. Optical density changes of bacteria detected by fluorescent biosensor over time
Different lowercase letters on columns of the same time indicate significant differences (P<0.05，Duncan’s method)
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图 5 荧光生物传感器检测不同细菌后的光密度变化
1：空白，2：大肠埃希菌，3：芽孢杆菌，4：金黄色葡萄球菌，5：无乳链球菌，6：变形杆菌；柱子上方的不同小写字母表示处理间差异显著 (P<0.05，Duncan’s 法)
Figure 5. Optical density changes after detection of different bacteria by fluorescent biosensors
1: Blank, 2: Escherichia coli, 3: Bacillus, 4: Staphylococcus aureus, 5: Streptococcus agalactiae, 6: Proteus; Different lowercase letters on bars indicate significant differences among treatments (P<0.05, Duncan’s method)
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图 6 不同浓度荧光生物传感器溶液随时间变化的光密度
Figure 6. Optical density changes of different concentrations of fluorescence biosensor solutions over time
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图 7 不同菌落数量金黄色葡萄球菌的光密度(A)及线性关系(B)
图A中，柱子上方的不同小写字母表示处理间差异显著 (P<0.05，Duncan’s 法)；菌落数量0~8分别代表0、1×100、1×101、1×102、1×103、1×104、1×105、1×106和1×107 cfu·mL−1
Figure 7. Optical densities of different colony amounts of Staphylococcus aureus and their linear relationship
In figure A, different lowercase letters on bars indicate significant differences among treatments (P<0.05, Duncan’s method); The colony amounts from zero to eight are 0, 1×100, 1×101, 1×102, 1×103, 1×104, 1×105, 1×106 and 1×107 cfu·mL−1, respectively
表 1 试验DNA序列
Table 1 DNA sequences in the experiments
名称
Name核酸序列(5′→3′)
Nucleic acid sequence特异性互补链
SA-substrateCTAATGAGTACCTACTGTCTCTGGATGATCCTATGAACTGACQ/rA/FGACCTCACTACCAAG 脱氧核酶
SA-DNAzymeATGCCATCCTACCAACCACGAAGTACATTTCAAACTCATAACAATCCATCGGTTAGGTCCTGGTTGGAGCTCTGAACTCGAGACAGTAGGTACTCATTAG 
下载: 导出CSV
表 2 荧光生物传感器对含有金黄色葡萄球菌牛奶样品的回收率结果
Table 2 Recovery rate results of the fluorescent biosensor to milk samples containing Staphylococcus aureus
菌落数量/(cfu·mL−1) Colony amount 相对标准
偏差/%
RSD回收率/%
Recovery
rate真实
Actual平板计数法检测
Detected by counting method荧光生物传感器检测
Detected by fluorescent biosensors1×103 (0.973±0.052)×103 (0.903±0.069)×103 7.64 92.8 1×104 (1.024±0.059)×104 (1.059±0.046)×104 4.34 97.7 1×105 (1.046±0.049)×105 (1.083±0.061)×105 5.63 103.5 1×106 (0.997±0.073)×106 (1.032±0.059)×106 5.72 103.5 1×107 (1.037±0.055)×107 (1.076±0.057)×107 5.30 103.8
下载: 导出CSV
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1. 王亚琴. 食品中致病微生物的检测方法研究. 食品安全导刊. 2025(11): 131-133 . 
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