A Novel reverse transcriptase-polymerase chain reaction based-liquid hybridisation(RT-PCR-LH) assay for early diagnosis of dengue infection

Abstract

BACKGROUND: Early definitive laboratory diagnosis of dengue is difficult with the tests in routine use at present. OBJECTIVE: To develop a reverse transcriptase-polymerase chain reaction based liquid hybridisation (RT-PCR-LH) technique for the rapid and early diagnosis of dengue. RESEARCH DESIGN: RT-PCR products of the NS3 gene of dengue virus prototypes and of a few positive sera for dengue virus by culture, were allowed to hybridise in liquid phase with a mixture of dengue specific radio-labelled oligonucleotides. The products were separated by PAGE and visualised by autoradiography. 78 suspected dengue sera were also tested by RT-PCR-LH method, and by IgM-ELISA and HAI tests, for comparison. RESULTS: Two DNA bands (approximately equal to 470 bp and approximately equal to 455 bp) specific to dengue virus, were observed. RT-PCR-LH assay takes only 24 h. Of the 78 suspected dengue acute sera tested, 45/78 were positive by RT-PCR-LH, 31/78 were positive by IgM-ELISA, and 14/78 had a HAI titre > or = 2560. Duration of fever was known in 72 cases, and infection was detected by RT-PCR-LH in 11/22 of cases with < 5 d fever and by IgM-ELISA in 1/22. In cases with 5 to 15 d fever RT-PCR-LH and IgM-ELISA/HAI titre > or = 2560 detected infection in 30/50 and 27/50 respectively. The 10 sera which were negative by RT-PCR-LH, but were positive by either IgM-ELISA or HAI titre > or = 2560 were all > 5 d fever cases. RT-PCR-LH together with IgM-ELISA were capable of detecting dengue infection in 56/78 of the suspected cases. CONCLUSION: RT-PCR-LH assay developed in this study appears to have an advantage over other diagnostic techniques for the early detection of dengue.