title: Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.
abstract: Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.
authors.affiliation: Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad 500082, Andhra Pradesh, India. Electronic address: mamidineeraja@yahoo.co.in.
authors.forename: M
authors.initials: M
authors.lastname: Neeraja
authors.affiliation: Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad 500082, Andhra Pradesh, India. Electronic address: lakshmi57vemu@gmail.com.
authors.forename: V
authors.initials: V
authors.lastname: Lakshmi
authors.affiliation: Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad 500082, Andhra Pradesh, India. Electronic address: vanjari.lavanya@gmail.com.
authors.forename: Vanjari
authors.initials: V
authors.lastname: Lavanya
authors.affiliation: Dept. of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad 500082, Andhra Pradesh, India. Electronic address: nagapriyanka.e@gmail.com.
authors.forename: E N
authors.initials: EN
authors.lastname: Priyanka
authors.affiliation: Department of Virology, Defense R&D Establishment, DRDE, DRDO, Ministry of Defence, Jhansi Road, Gwalior 474002, Madhya Pradesh, India. Electronic address: paridamm@rediffmail.com.
authors.forename: M M
authors.initials: MM
authors.lastname: Parida
authors.affiliation: Department of Virology, Defense R&D Establishment, DRDE, DRDO, Ministry of Defence, Jhansi Road, Gwalior 474002, Madhya Pradesh, India. Electronic address: pabandash@rediffmail.com.
authors.forename: P K
authors.initials: PK
authors.lastname: Dash
authors.affiliation: Department of Virology, Defense R&D Establishment, DRDE, DRDO, Ministry of Defence, Jhansi Road, Gwalior 474002, Madhya Pradesh, India. Electronic address: shashibiotech@gmail.com.
authors.forename: Shashi
authors.initials: S
authors.lastname: Sharma
authors.affiliation: DRDO-BU Center for Life Sciences, Coimbatore, India. Electronic address: pvlrao@rediffmail.com.
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
authors.affiliation: Dept. of Microbiology, Osmania University, Hyderabad 500007, Andhra Pradesh, India. Electronic address: gopalred@hotmail.com.
authors.forename: Gopal
authors.initials: G
authors.lastname: Reddy
mesh_terms: D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007194:India; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D012367:RNA, Viral / Q000235:genetics; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D065288:Serogroup; D062606:Tertiary Care Centers; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins
keywords: DENV serotypes; Genie(®) II flourometer; NS 1 antigen; RT-LAMP; RT-PCR; Serotyping