Simultaneous detection of dengue virus serotypes using a facile gel-free nucleic acid based lateral flow assay

Timely and accurate diagnosis of dengue virus serotypes is crucial for optimal management of patients and control of outbreaks. Diagnostic methods currently available in clinical settings, such as ELISA based tests detect overall DENV infection in patients. In addition, RT-PCR based tests that require specialized equipment and expertise are utilized to detect all four serotypes of DENV. This study focussed on the development of gel free PCR based lateral flow strip assay to detect all four serotypes of DENV on site. The assay uses anti-biotin/streptavidin colloidal gold conjugates with florescent/enzymatic tagged DENV serotype specific antibodies for direct detection on a nitrocellulose membrane using anti-IgG as control line in the DENV infected serum samples. The detection limit of the assay was upto 105 dilutions (10 copies) of initial DENV cDNA. DENV RT-PCR performed to compare the results of the lateral flow assay revealed 100% similarity with the lateral flow results. Results demonstrated 100% sensitivity and specificity for dengue detection and serotype identification.

及时精准鉴定登革病毒血清型，对于患者的优化诊疗及疫情防控均具有关键意义。当前临床可用的诊断方法中，基于酶联免疫吸附试验（ELISA）的检测仅能检出患者体内的登革病毒（DENV）整体感染情况；此外，需依托专用设备与专业技术的逆转录聚合酶链反应（RT-PCR）检测方法，则可用于检出全部4种登革病毒血清型。本研究旨在开发无需凝胶电泳的基于聚合酶链反应的侧流层析试纸条检测技术，以实现登革病毒血清型的现场检出。该检测技术采用抗生物素/链霉亲和素胶体金偶联物，搭配带有荧光/酶标记的登革病毒血清型特异性抗体，可在硝酸纤维素膜上直接检测登革病毒感染的血清样本，并以抗IgG作为质控线。该检测方法的检出限可达初始登革病毒互补DNA（cDNA）的10^5倍稀释度（即10个拷贝）。以逆转录聚合酶链反应（RT-PCR）的检测结果作为对照进行比对，发现本侧流层析检测技术的结果与之完全一致。实验结果表明，该技术在登革病毒检测及血清型鉴定方面均具备100%的灵敏度与特异性。