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

• Published in: Journal of virological methods 2015-01, Vol.211, p.22-31
• Main Authors: Neeraja, M., Lakshmi, V., Lavanya, Vanjari, Priyanka, E.N., Parida, M.M., Dash, P.K., Sharma, Shashi, Rao, P.V. Lakshmana, Reddy, Gopal
• Format: Article
• Language: English
• Subjects: antibodies; antigens; dengue; Dengue - diagnosis; Dengue - virology; Dengue virus; Dengue Virus - classification; Dengue Virus - genetics; Dengue Virus - isolation & purification; DENV serotypes; enzyme-linked immunosorbent assay; genes; Genie® II flourometer; hospitals; Humans; immunoglobulin G; immunoglobulin M; India; Molecular Sequence Data; monitoring; NS 1 antigen; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - standards; patients; quantitative polymerase chain reaction; rapid methods; reverse transcriptase polymerase chain reaction; Reverse Transcription; reverse transcription loop-mediated isothermal amplification; RNA, Viral - genetics; RT-LAMP; RT-PCR; Sensitivity and Specificity; Sequence Analysis, DNA; Serogroup; serotypes; Serotyping; silver; Tertiary Care Centers; Time Factors; Viral Nonstructural Proteins - genetics
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2014.10.005

•This research article highlights the development of an improved, inexpensive, faster, sensitive and specific RT-LAMP assay for detection and serotyping of DENV, targeting NS1 region using Genie II fluorometer.•RT-LAMP or CDC Real time assay when used in combination with NS1 antigen and anti Dengue IgG and IgM Elisa increased the diagnostic coverage of febrile patients to 96%.•Tm values and anneal curves displayed at the end of the reaction eliminates the need for gel electrophoresis or turbidity detection and allows for a closed-tube system thus reducing the cost of the assay. 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.