基于微流控芯片电泳的番茄黄化曲叶病毒快速检测

doi:10.3864/j.issn.0578-1752.2014.17.008

摘要/Abstract

摘要： 【目的】探索微流控芯片电泳方法在PCR产物检测方面的效果，并建立针对番茄黄化曲叶病毒（Tomato yellow leaf curl virus，TYLCV）的微流控芯片电泳检测方法，弥补琼脂糖凝胶电泳方法在试剂消耗、所用时间、安全性方面的缺陷。【方法】通过对TYLCV的基因组进行分析后，在该基因组中相对稳定的位置设计引物，同时兼顾引用已被研究者研究过的引物，对这些选择的引物进行特异性、稳定性、灵敏度等方面的验证，筛选出用于后续试验的引物。选择DNA标准物φX174/BsuR I(Hae Ⅲ) marker，分别进行琼脂糖凝胶电泳和微流控芯片电泳，对二者在耗材、耗时和灵敏度方面进行比较，确定微流控芯片电泳在核酸检测方面的应用价值。利用筛选出的其中1对引物对番茄叶片的实际样品进行PCR扩增，随后通过微流控芯片电泳对其进行检测，以此探讨微流控芯片电泳在病毒检测方面的检测效果。【结果】共筛选出14对TYLCV备用引物，其中2对引自文献，12对为本文设计,每对引物均可满足微流控芯片检测要求。选择其中1对引物TYLCV-T作为随后的研究对象。利用琼脂糖凝胶电泳和微流控芯片电泳对DNA标准物检测，结果表明微流控芯片电泳在耗时方面不足琼脂糖凝胶电泳的1/10，约为13 min，试剂消耗为琼脂糖凝胶电泳的1/8，检测灵敏度方面至少比琼脂糖凝胶电泳高103倍，根据DNA标准物原液浓度计算可知，微流控芯片电泳至少可准确检测到浓度为5×10-6 μg?μL-1的核酸样品。利用微流控芯片电泳对TYLCV-T扩增的TYLCV PCR产物进行检测，将检测峰值图与DNA标准物的峰值图时间比较，就可判断出产物峰的大小范围。【结论】筛选出的关于TYLCV的备用引物可作为进一步研究微流控芯片技术在该病毒检测方面的基础；通过将琼脂糖凝胶电泳和微流控芯片电泳进行比较，确立了后者在核酸检测方面的应用价值；通过微流控芯片电泳对TYLCV PCR产物的检测，建立了基于微流控芯片电泳的TYLCV快速检测方法，为TYLCV的快速检测提供新的技术支持。

关键词: 番茄黄化曲叶病毒 , 微流控芯片电泳 , 琼脂糖凝胶电泳 , 快速检测

Abstract: 【Objective】The objectives of this study are to explore the effect of microfluidic electrophoresis on detection of PCR products, build a detection method for Tomato yellow leaf curl virus (TYLCV) by electrophoresis in microfluidic chip, and to remedy the defect in reagent consumption, long time, lack of safety of agarose gel electrophoresis.【Method】Primers in relatively stable positions of TYLCV genome were designed, some primers in references were taken into account, and these selected primers were verified. TYLCV primers were screened out based on the criterion of specificity, stability, and sensitivity. DNA standards φX174/BsuR I (Hae Ⅲ) marker was subjected to agarose gel electrophoresis and microfluidic electrophoresis, and the two methods were compared in supplies, time-consumption and sensitivity to confirm the value of microfluidic electrophoresis in nucleic acid detection. In order to evaluate the value of microfluidic electrophoresis in virus detection, the PCR amplification products of one pair of selected primers on the actual samples were processed by microfluidic electrophoresis.【Result】Fourteen pairs of TYLCV primers were screened out, 2 pairs came from the literatures and the other 12 pairs were designed in this study. Each pair of primers could meet the requirement for microfluidic detection. TYLCV-T was chosen from these primers for the subsequent study. By comparison of agarose gel electrophoresis and microfluidic electrophoresis, the time consumption and reagent consumption of microfluidic electrophoresis were 1/10 and 1/8 of those of agarose gel electrophoresis, respectively. The detection sensitivity of microfluidic electrophoresis was at least 103 times higher than that of agarose gel electrophoresis, which could detect accurately 5×10-6 μg?μL-1 of nucleic acid according to calculation of the DNA standards concentration. By comparing the microfluidic electrophoresis peak of the sample with that of DNA marker, the size of the nucleic acid could be determined.【Conclusion】Primers of TYLCV were screened, which can be used for further study of virus detection by microfluidic technology. Microfluidic electrophoresis was more dominant than agarose gel electrophoresis in sensitivity, effectiveness, reagent consumption and time saving. Microfluidic electrophoresis detection was initially established, which provides a new rapid detection approach for TYLCV.

Key words: Tomato yellow leaf curl virus (TYLCV) , microfluidic electrophoresis , agarose gel electrophoresis , rapid detection

余明芬1;2;曾洪梅2;钟润涛3;赵小明3;邱德文1;2. 基于微流控芯片电泳的番茄黄化曲叶病毒快速检测[J]. 中国农业科学, 2014, 47(17): 3405-3413.

YU Ming-fen;ZENG Hong-mei; ZHONG Run-tao; ZHAO Xiao-ming; QIU De-wen;. Microfluidic Electrophoresis Detection of Tomato yellow leaf curl virus (TYLCV)[J]. Scientia Agricultura Sinica, 2014, 47(17): 3405-3413.

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