Chow V T, Yong R Y, Ngoh B L, Chan Y C
Department of Microbiology, Faculty of Medicine, National University of Singapore, Republic of Singapore.
J Clin Pathol. 1997 Apr;50(4):346-9. doi: 10.1136/jcp.50.4.346.
To apply an automated system of nucleic acid hybridisation coupled with the enzyme linked immunosorbent assay (ELISA) for the type specific detection of amplification products of dengue viruses.
Non-structural 3 (NS3) gene targets of reference strains of all four dengue and other flaviviruses, as well as dengue patient viraemic sera, were subjected to reverse transcription and polymerase chain reaction using consensus and dengue type specific primers and digoxigenin-11-dUTP label incorporation. The amplification products were detected by biotinylated type specific primers which served as ELISA capture probes bound to streptavidin coated tubes.
Significantly high spectrophotometric absorbance readings were obtained by hybridisation of the consensus and seminested amplification products of all four dengue viruses with their respective capture probes. In contrast, extremely low absorbances were observed for consensus products of Japanese encephalitis, yellow fever, and Kunjin viruses, which served as negative controls. These ELISA data correlated well with agarose gel electrophoresis of dengue type specific amplified products of diagnostic sizes.
The combination of in vitro amplification and antibody based detection offers rapid, type specific, high throughput, and gel-free detection of amplified products of dengue viruses.
应用核酸杂交自动化系统结合酶联免疫吸附测定（ELISA）对登革病毒扩增产物进行型特异性检测。
使用通用引物和登革病毒型特异性引物以及地高辛-11-dUTP标记掺入法，对所有四种登革病毒和其他黄病毒参考株的非结构3（NS3）基因靶点以及登革热患者病毒血症血清进行逆转录和聚合酶链反应。扩增产物通过生物素化的型特异性引物进行检测，这些引物用作与链霉亲和素包被管结合的ELISA捕获探针。
所有四种登革病毒的通用引物和半巢式扩增产物与其各自的捕获探针杂交后，获得了显著较高的分光光度吸收读数。相比之下，作为阴性对照的日本脑炎病毒、黄热病病毒和库京病毒的通用产物的吸光度极低。这些ELISA数据与诊断大小的登革病毒型特异性扩增产物的琼脂糖凝胶电泳结果相关性良好。
体外扩增和基于抗体的检测相结合，可对登革病毒扩增产物进行快速、型特异性、高通量且无需凝胶的检测。
