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J Clin Microbiol. 2015 Mar;53(3):830-7. doi: 10.1128/JCM.02648-14. Epub 2015 Jan 7.

Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification.

Author information

1
Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
2
TwistDx, Ltd., Minerva Building, Babraham Research Campus, Cambridge, United Kingdom.
3
Department of Public Health and Clinical Medicine, Epidemiology and Global Health, Umeå University, Umeå, Sweden Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.
4
Arbovirus and Imported Viral Diseases, National Centre for Microbiology, Institute of Health Carlos III, Madrid, Spain.
5
Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia sazaly@um.edu.my.

Abstract

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.

PMID:
25568438
PMCID:
PMC4390637
DOI:
10.1128/JCM.02648-14
[Indexed for MEDLINE]
Free PMC Article

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