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

Gunesekera, Maya B; Hapugoda, Menaka D; Gunasena, S; Subasinghe, S A S C; Bandara, K B A T; Khan, Baldip K; Abeyewickreme, W

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

Date

2003-03-11

Authors

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 radiolabelled 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.

Description

The Ceylon Medical Journal.

Citation

Gunesekera MB, Hapugoda MD, Gunasena S, Subasinghe SA, Bandara KB, Khan BK, Abeyewickreme W. A novel reverse transcriptase-polymerase chain reaction based-liquid hybridisation (RT-PCR-LH) assay for early diagnosis of dengue infection. The Ceylon Medical Journal. 2003 Mar; 48(1): 17-22