早期香蕉枯萎病Foc4双探针核酸纸基检测传感器研制
S668.1%S24; 为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4,Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器.该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限.基于目标序列与双探针体系的碱基互补配对形成"金标探针-目标序列-T线探针"复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测.试验结果表明,双探针纸基传感...
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| Published in | 农业工程学报 Vol. 40; no. 5; pp. 38 - 46 |
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| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
华南农业大学工程学院,广州 510642
01.03.2024
岭南现代农业科学与技术广东省实验室,广州 510642 广东海洋大学机械工程学院,湛江,524088%华南农业大学工程学院,广州 510642%华南农业大学工程学院,广州 510642 |
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| Online Access | Get full text |
| ISSN | 1002-6819 |
| DOI | 10.11975/j.issn.1002-6819.202312079 |
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| Abstract | S668.1%S24; 为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4,Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器.该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限.基于目标序列与双探针体系的碱基互补配对形成"金标探针-目标序列-T线探针"复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测.试验结果表明,双探针纸基传感器的检测限达到0.001 nmol/L,是传统纸基传感器的100倍,提高了检测灵敏度;在0.001～1 000.000 nmol/L范围内,Foc4浓度与测试线光强度峰面积呈线性关系;使用高浓度非互补探针进行试验干扰,发现非互补序列对检测效果基本无影响,表明该传感器具有较好的特异性;香蕉叶片Foc4检测的平均回收率为77.6％～102.3％,相对标准偏差为7.4％～7.7％.与传统形态学观察等检测方法相比,该研究提出的双探针核酸纸基检测传感器可以及时、快速、准确地判断早期Foc4的存在,具有良好的推广性和实际应用价值. |
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| AbstractList | S668.1%S24; 为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4,Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器.该传感器将2种不同粒径的胶体金分别与检测探针和信号增强探针结合,利用DNA探针代替抗原和抗体,形成双探针体系,通过增加信号增强探针降低检测限.基于目标序列与双探针体系的碱基互补配对形成"金标探针-目标序列-T线探针"复合物并在传感器的测试区被捕获,10 min内形成肉眼可见的目标产物,通过分析测试条带得到光强度峰面积并代入标准曲线中,实现Foc4定量检测.试验结果表明,双探针纸基传感器的检测限达到0.001 nmol/L,是传统纸基传感器的100倍,提高了检测灵敏度;在0.001～1 000.000 nmol/L范围内,Foc4浓度与测试线光强度峰面积呈线性关系;使用高浓度非互补探针进行试验干扰,发现非互补序列对检测效果基本无影响,表明该传感器具有较好的特异性;香蕉叶片Foc4检测的平均回收率为77.6％～102.3％,相对标准偏差为7.4％～7.7％.与传统形态学观察等检测方法相比,该研究提出的双探针核酸纸基检测传感器可以及时、快速、准确地判断早期Foc4的存在,具有良好的推广性和实际应用价值. |
| Abstract_FL | Banana fusarium wilt has a significant adverse effect on the banana industry.It is worth noting that fusarium oxysporum f.sp.cubense 4(Foc4)is one of the most devastating and widespread pathogens of banana fusarium wilt in the world.It is a catastrophic threat to the global banana industry.At present,there is still no technology that can cure bananas infected with banana fusarium wilt.Therefore,it is extremely important for banana planting industry to detect banana fusarium wilt in time and do a good job in prevention and control.In order to realize the early accurate detection of banana fusarium wilt race 4,this study proposed a detection method of double probe paper-based sensor based on colloidal gold,which increased the use of signal enhancement probes to increase the binding amount of colloidal gold,thereby reducing the detection limit.Two kinds of colloidal gold particles with different particle sizes were combined with the corresponding detection probes and signal enhancement probes to form a double gold label probe system.The large particle size colloidal gold was used with the detection probe,while the small particle size colloidal gold was paired with the signal enhancement probe.The sample being tested was mixed with this double gold-labeled probe system.The Foc4 target sequence in the sample would be paired with the double gold-labeled probe to form a'gold-labeled probe-target sequence-T-line probe'complex,which continued to migrate to the water-absorbing pad.The complex was captured on the test area of the sensor.Within 10 minutes,the test paper would generate a visible target product that could be observed by the naked eye at both the test line and the quality control line areas,thus achieving qualitative detection.For quantitative detection of Foc4,the light intensity of the test strip would be further analyzed using software.The light intensity peak area was obtained and substituted into the standard curve to obtain the concentration of Foc4 detected and realize quantitative detection.Experimental results demonstrate that the detection limit of the dual-probe paper-based sensor is 0.001 nmol/L,which is 100 times of that of the traditional paper-based sensor,and the detection sensitivity is improved.The sensitivity test results demonstrated a strong linear relationship between the concentration of Foc4 and the peak area of the test line light intensity within the concentration range of 0.001-1 000.000 nmol/L.This finding suggests that the sensor can be effectively employed for quantitative detection.To assess specificity,a high concentration of non-complementary probe was utilized as the target sequence for detection and compared with the detection results of samples containing Foc4.The experiment revealed the absence of a red band at the test line when the test paper was detected by the non-complementary sequence.Importantly,this observation indicated that the presence of the non-complementary sequence had minimal impact on the detection effect,thus affirming the sensor's excellent specificity.Finally,the paper-based sensor was used to detect Foc4 in banana leaves.The average recovery rate was 77.6％-102.3％,and the relative standard deviation was 7.4％-7.7％,indicating that it can be used for on-site detection of Foc4.The detection technology possesses the characteristics of being low-cost and easy to operate.In comparison to traditional detection methods,it enables timely,rapid and accurately determination of the presence of Foc4.Compared with the existing molecular detection technology,the detection can be completed without the need of expensive equipment or specialized personnel.This feature makes it highly suitable for widespread use.The dual-probe paper-based sensor realizes the combination of nucleic acid lateral chromatography detection technology and banana fusarium wilt detection.Its potential for widespread adoption in the early disease warning system of the banana industry is substantial.Furthermore,this technology can be combined with traditional polymerase chain reaction(PCR)and other techniques to facilitate on-site detection of Foc4. |
| Author | 何俊宇 欧俊辰 杨洲 张志宏 段洁利 区湘颖 罗宇康 |
| AuthorAffiliation | 华南农业大学工程学院,广州 510642;岭南现代农业科学与技术广东省实验室,广州 510642;广东海洋大学机械工程学院,湛江,524088%华南农业大学工程学院,广州 510642%华南农业大学工程学院,广州 510642;岭南现代农业科学与技术广东省实验室,广州 510642 |
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| Author_FL | YANG Zhou LUO Yukang HE Junyu ZHANG Zhihong DUAN Jieli OU Xiangying OU Junchen |
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| DocumentTitle_FL | Development of the sensor with dual-probe nucleic acid paper-based for detection of Foc4 of early banana fusarium wilt |
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| Keywords | detection sensors nucleic acid lateral chromatography 双探针 检测 胶体金 Foc4 colloidal gold 传感器 dual-probe 核酸侧向层析 |
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| Publisher | 华南农业大学工程学院,广州 510642 岭南现代农业科学与技术广东省实验室,广州 510642 广东海洋大学机械工程学院,湛江,524088%华南农业大学工程学院,广州 510642%华南农业大学工程学院,广州 510642 |
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| Snippet | S668.1%S24; 为实现对早期香蕉枯萎病4号生理小种(fusarium oxysporum f.sp.cubense 4,Foc4)的准确检测,该研究提出一种基于胶体金的双探针纸基传感器.该传感器将2种不同粒... |
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| Title | 早期香蕉枯萎病Foc4双探针核酸纸基检测传感器研制 |
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