长引物快速PCR结合核酸试纸条法可视化检测四种病原体

目的建立一种基于病原体核酸分子的血液安全性检定方法,可视化区分血液中乙肝病毒（HBV）、丙肝病毒（HCV）、艾滋病毒（HIV）和梅毒螺旋体（TP）。方法利用长引物PCR扩增四种病原体核酸,循环步骤由传统的三步简化为两步。将修饰有小分子抗原的扩增产物点样于固定有相应抗体的纳米颗粒试纸条上,肉眼判读显色结果。优化了长引物PCR对于四种病原体扩增的退火和变性温度,并评价了该方法用于病原体阳参及血清样本的检测准确性。结果四种病原体核酸扩增的退火温度分别为76℃、78℃、76℃和78℃;变性温度为84℃、90℃、84℃和88℃;本方法能成功区分四种病原体的阴阳性样本,准确性高;扩增时间仅为40 min/40个循环,检测为10 min。结论该方法能够用于四种病原体核酸的可视化检测,与传统的检测方法相比,检测速度快,成本低,无需昂贵的仪器,易于广大基层医院普及。

Development of a Long-Primer Fast PCR Coupled with Visual Nucleic Acid Dipstick Assay for Detection of Four Pathogens

Objective To develop a blood-safety test method based on detecting nucleic acids of a pathogen and visually to determine which kind of pathogens （HBV, HCV, HIV, or TP） in the blood. Methods The nucleic acids of four pathogens were amplified with long-primer PCR. The steps of PCR cycles were simplified from tra- ditional three steps to two steps. PCR products labeled with antigens were applied to the gold-nanoparticles dip- stick immobilized with antibodies. The developing results were determined by naked eyes. The annealing tem- peratures and denaturing temperatures for the four pathogens were optimized and the accuracy of the method for detecting the stimulated and the serum samples of four pathogens were evaluated. Results The annealing tem- peratures of four pathogens were 76℃ , 78℃ , 76℃ and 78℃; the denaturing temperatures of them were 84℃, 90℃, 84℃ and 88℃. The assay was successfully developed for discriminating the positive and negative samples with high accuracy. The amplification time was only 40 min/40 cycles and the detection time was 10 rain. Conclusion The assay which developed in this study can be used for detecting nucleic acids of four pathogens visually. In comparison with traditional methods, the assay has the advantages of the rapidity, low cost and no requirements of special instruments and holds a great promise for wide applications in small hospitals.