利用双标记PCR产物的量子点检测副溶血性弧菌

Development of Quantum Dot by Double Labeling PCR Products for Detection of Vibrio parahemolyticus

副溶血弧菌是一种能引起食源性疾病的重要病原菌,为了对该细菌进行检测,研制一种快速、灵敏、简便的量子点免疫层析试纸条。通过磁珠富集DNA后用双标记的特异性引物（上游引物5’用地高辛标记,下游引物5’用生物素标记）扩增不耐热溶血毒素基因（TLH）,进行量子点免疫试纸条的检测。结果表明,荧光定量PCR检测磁珠（Si-MNPs）的捕获率在浓度102~106 CFU/m L均在50%以上,浓度为102 CFU/m L时达到最高捕获率（64%）。在纯培养物及模拟检测中试纸条的检测限均为4×100 CFU/m L,但在模拟检测中T线的显色强度弱于纯培养物的检测,说明鱼肉糜样本的复杂成分对检测灵敏度有一定的影响,但添加高浓度杂菌（阪崎肠杆菌、志贺氏菌）不影响该方法的特异性。方法具有特异性强、灵敏度高,并且操作简便、检测成本低的特点,2 h即可完成,为副溶血弧菌的快速检测提供了有效手段。

Vibrio parahemolyticus is an important pathogen which can cause food-borne illness.A rapid,sensitive and simple quantum dot immunity test strip was developed in order to detect bacteria.Genomic DNA was extracted with silica-coated magnetic nanoparticles,and a species-specific gene（thermolabile hemolysin,TLH） was amplified by PCR using digoxigenin/biotin-labeled primers,and used for immunochromatographic test strip（ICTS）.The results showed that in the range of 10~2~10~6 CFU/mL,the extraction ratio of DNA of V parahemolyticus maintained above 50%,and the highest recovery（64%） by Si-MNP took place in 102 CFU/mL.The limit of detection for V parahemolyticus was 4 ×10° CFU/mL in pure culture and fish paste samples,respectively,which was not affected by those non-V parahemolyticus strains.However,detection color intensity is weaker in fish paste samples than in pure culture,indicating that the complex composition of fish meat sample has little influence s for the detection sensitivity.The method has high specificity,high sensitivity,and easy to operate,low detection cost,accomplished in two hour,and provides an effective means for rapid detection of V parahaemolyticus.