Medicine

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Author: search.filters.author.Gunasena, S.Subject: search.filters.subject.Reverse Transcriptase Polymerase Chain Reaction-methods

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    Detection of dengue virus in Aedes albopictus mosquitoes by Reverse Transcription Polymerase-Chain Reaction-Liquid Hybridization (RT-PCR-LH) based assay.
    (Sri Lanka College of Microbiologists, 2003) Hapugoda, M.D.; Gunasekera, M.B.; de Silva, N.R.; Gunasena, S.; Prithimala, L.D.; Dayanath, M.Y.D.; Abeyewickreme, W.
    Dengue is an important public health problem. In this study an RT-PCR-LH assay was developed for the detection of dengue virus in Ae.albopictus, a vector of dengue. Laboratory bred Ae.albopictus (adults inoculated with dengue prototypes were tested by RT-PCR-LH assay. RT-PCR products of NS3 gene of 4 dengue prototypes were hybridized in liquid phase with 32P) labelled cocktail of dengue serotype-specific ologonucliotides. Semi-Nested-PCR agarose gel electrophoresis (Semi-Nested-PCR-AGE) assay with dengue type specific oligonucliotides was carried out for typing of RT-PCR products. Wild-caught Ae.albopictus (larvae (n=89 pools) and adults (n=69 pools) collected from dengue case reported stations during the period of 1999-2002 were also tested by RT-PCR-LH and typed by Nested-PCR-AGE assay). A DNA band (470bp) specific for dengue virus was observed in all pools of Ae.albopictus (inoculated with dengue prototypes in RT-PCR-LH assay. When RT-PCR products of dengue prototypes inoculated mosquitoes were typed by Semi-Nested-PCR-AGE assay, bands of 169,362, 265, 426 bp sizes corresponding to DEN1, DEN2, DEN3 and DEN4 respectively were observed. The DNA band specific for dengue virus (470bp) was also observed in 6 pools of wild-caught adults in RT-PCR-LH assay. They were found to be infected with DEN3 (265bp DEN3 specific DNA band was detected) by Semi-Nested-PCR-AGE assay. None of the wild-caught larvae showed dengue specific DNA band (470bp) in RT-PCR-LH assay). RT-PCR-LH with Semi-Nested-PCR-AGE assays are useful for the detection and typing of dengue virus in Ae.albopictus. Ae.albopictus (in Sri Lanka is competent in transmitting DEN3 and possibly other serotypes. Detection of dengue virus for the first time in Ae.albopictus in Sri Lanka confirms earlier observations that it may play an important role in transmitting dengue). Acknowledgements: Financial assistance by the International Atomic Energy Agency (Technical Co¬operation grant no SLR/ 06 / 024) and University of Kelaniya (Research grant no RP/03/04/06/01/00) is gratefully acknowledged.
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    A Comparative retrospective study of novel Reverse-Transcription Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with Polymerase Chain Reaction (PCR) amplification, virus isolation and serological techniques for early, definitive laboratory diagnosis of dengue infection
    (Malaysian Society of Parasitology and Tropical Medicine, 2007) Hapugoda, M.D.; de Silva, N.R.; Khan, B.; Gunasena, S.; Dayanath, M.Y.D.; Abeyewickreme, W.
    Dengue is an important vector borne viral infection in South East Asia. Dengue virus is responsible for dengue fever, dengue haemorrhagic fever and dengue shock syndrome. Early diagnosis of infection helps in monitoring the disease, determining when hospital admission is necessary and in reducing case fatalities. The objective of the study was to carry out a comparative retrospective study of a novel Reverse Transcription-Polymerase Chain Reaction-based Liquid Hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of Non Structural-3 gene were hybridized with a mixture of the 4 dengue type-specific Deoxyribonucleic Acid (DNA) probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 88 patients with dengue confirmed by Haemagglutination Inhibition (HAI) assay. The assay was highly specific for diagnosis of dengue infection. As an early (<5 days of fever) laboratory diagnostic method, this assay had 100% sensitivity for detection of dengue patients confirmed by HAI assay. A high analytical sensitivity of 2 fluorescent focus units of dengue virus/reaction was achieved. Novel RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection at the time during which serological methods cannot be used.
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    A Novel reverse transcriptase-polymerase chain reaction based-liquid hybridisation(RT-PCR-LH) assay for early diagnosis of dengue infection
    (Sri Lanka Medical Association, 2003) Gunasekera, M.B.; Hapugoda, M.D.; Gunasena, S.; Subasinghe, S.A.S.C.; Bandara, K.B.A.T.; Khan, K.B.; Abeyewickreme, W.
    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.
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