title: Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification.
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.
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Boon-Teong
authors.initials: BT
authors.lastname: Teoh
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Sing-Sin
authors.initials: SS
authors.lastname: Sam
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kim-Kee
authors.initials: KK
authors.lastname: Tan
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Mohammed Bashar
authors.initials: MB
authors.lastname: Danlami
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Meng-Hooi
authors.initials: MH
authors.lastname: Shu
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Jefree
authors.initials: J
authors.lastname: Johari
authors.affiliation: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Poh-Sim
authors.initials: PS
authors.lastname: Hooi
authors.affiliation: TwistDx, Ltd., Minerva Building, Babraham Research Campus, Cambridge, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Brooks
authors.affiliation: TwistDx, Ltd., Minerva Building, Babraham Research Campus, Cambridge, United Kingdom.
authors.forename: Olaf
authors.initials: O
authors.lastname: Piepenburg
authors.affiliation: TwistDx, Ltd., Minerva Building, Babraham Research Campus, Cambridge, United Kingdom.
authors.forename: Oliver
authors.initials: O
authors.lastname: Nentwich
authors.affiliation: 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.
authors.forename: Annelies
authors.initials: A
authors.lastname: Wilder-Smith
authors.affiliation: Arbovirus and Imported Viral Diseases, National Centre for Microbiology, Institute of Health Carlos III, Madrid, Spain.
authors.forename: Leticia
authors.initials: L
authors.lastname: Franco
authors.affiliation: Arbovirus and Imported Viral Diseases, National Centre for Microbiology, Institute of Health Carlos III, Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Tenorio
authors.affiliation: 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.
authors.forename: Sazaly
authors.initials: S
authors.lastname: AbuBakar
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D042241:Early Diagnosis; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D055815:Young Adult
chemical_list: D012367:RNA, Viral