HIV专一快速荧光可视化检测研究

With the aid of virus specific sequence screening technology, HIV specific sequence can be acquired from the Gene bank through quantitative calculation and simulation, and then, its corresponding complementary sequence can be tagged by fluorescent probe FAM through a soft carbon chain linkage, which can be employed as HIV detection sequence (shorted as FAMH). After loaded on the surface of GO to get FAMH/GO complex, the fluorescence of FAM is completely quenched. Then the FAMH/GO can be added into the target sample, and not any fluorescence increasing can be observed for normal ones. In comparison, significant fluorescence enhancement of FAM can be observed in the case of HIV samples, due to the formation of the double chain between FAM labeled detection sequence and HIV, leading to the release of FAM labeled HIV detection sequence from the surface of GO and the recovery of the typical FAM emission. To figure out the concentration of the virus, probe 1 designed with typical double chain intercalation moieties of pyrene (shorted as Py) and Ru(bpy)2dppzCl2 (shorted as Ru) can be added into the above mentioned system, then the ratiometric emission of the two components can be measured simultaneously, and the non-virus interference can be easily deduced by those before addition of FAMH/GO. The HIV concentration can be acquired according to the calibration cure established between IRu/IPy and the reference HIV blood samples from the national AIDS reference laboratory. The proposed method can be processed into portable kits, after addition of only one drop of the target blood, whether it is infected by HIV or not can be easily checked in no more than 10 min, under irradiation of a laser pen according to the fluorescence changes in the solution by naked eye. All these can be operated easily and quite suitable for not only normal check-ups and out-patient testing for HIV infected person in early stage, but also for the self-test of the suspected HIV infected person. Furthermore, the method can be developed into a universal qualitative and quantitative detection method just replacing the HIV specific sequence with the corresponding specific sequence of other viruses, all these would definitely broaden its applicability to some extent.

利用病毒特异序列筛查技术筛选出HIV不同亚型的同源特异序列，在其互补序列与FAM之间插入适当长度碳链得到探测序列后，借助柔性桥连赋予二者良好的空间相对自由度。检测前先将FAM负载到GO上进行荧光淬灭，再加入到待测样本中。正常样本无荧光变化，而HIV样本中探测序列与病毒杂交成双链后可释放FAM荧光。设计与所有双链DNA及RNA二级结构扦插的双波长发射检测分子1，在测试时扣减FAM加入前后的双波长发射光强比值，可有效排除非HIV病毒干扰。建立双波长发射比例与国家艾滋病参比实验室HIV标准品浓度曲线，可对HIV定量。进一步加工成试剂盒后，只需加入一滴血样，便可借助激光笔照射在10分钟内判断样本是否感染HIV。此法操作简便，适用于常规门诊的体检化验、HIV感染人群的初步筛查及HIV疑感人群的自我检测。将HIV特异序列更换成其它病毒的特异序列并考察其普遍适用性后，可建立相应的病毒荧光可视化检测方法。

病毒的早期筛查、诊断不仅可以为有效治疗赢得宝贵时间，大大提高治愈率，而且有利于相关部门及人群采取措施，阻止病毒的传播扩散。然而，病毒常用检测手段血清学检测和荧光PCR技术，易出现假阴性结果，操作复杂，周期较长，不适用于常规的门诊筛查。为此，本项目基于荧光团标记的病毒特异核酸序列为探针，利用不同猝灭手段，建立了具有普适性的病毒荧光可视化检测方法，可用于人类免疫缺陷病毒、非洲猪瘟、新冠病毒等检测，并利用构建的基于钌（II）联吡啶络合物的三发射、三比例、自校准的特异性检测方法对阳性样本中的病毒进行定量检测。构建了多种荧光可视化、电化学等检测方法并应用于相关疾病的诊断：开发了基于牛血清白蛋白和方酸菁染料具有荧光“开-关”变化的自组装体，能够快速、灵敏地检测胃蛋白酶，可用于诊断胃食管反流病；开发了一种硝基还原酶探针，用于检测大肠杆菌和金黄色葡萄球菌；开发了一种丙二醛（MDA）特异性检测探针，该探针从细胞和动物水平追踪了内源性MDA（活性氧产生的有毒物质之一），相关方法的建立有助于心血管疾病的诊断和治疗；开发了新型的超薄金属有机框架杂化材料，通过电化学信号变化检测原发性肝癌的生物标志物甲胎蛋白。本项目建立的检测方法可为相关疾病的诊断和治疗提供关键技术与思路。