Rapid Identification of Chikungunya and Dengue Virus by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Method

Lü, Xi; Li, Xiaobo; Mo, Ziyao; Jin, Fang; Wang, Boliang; Zhao, Hongbo; Shan, Xiaoxiao; Shi, Lei

doi:10.4269/ajtmh.2012.11-0721

Cited by 44 publications

(41 citation statements)

References 37 publications

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“…The RT-LAMP assay developed here differs from those reported earlier in at least two major areas [36-38]. Parida et al (2005) developed a four-tube RT-LAMP assay which employed serotype-specific primers targeting the proximal half of the 3′UTR of DENV genome [36].…”

Section: Discussionmentioning

confidence: 99%

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

et al. 2013

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BackgroundEarly and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.MethodsA single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).ResultsAcute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.ConclusionThe RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

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Section: Discussionmentioning

confidence: 99%

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

et al. 2013

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“…The RT-LAMP assay is being increasingly used by various investigators for rapid detection and typing of emerging viruses (Chan and Fox, 1999;Mori et al, 2001;Parida et al, 2005). These earlier reports, however, evaluated their RT-LAMP assays for the detection of DENV infection with a small clinical sample size (<100) and using the C-prM gene (Lu et al, 2012) or serotype-specific regions of the 3 untranslated region (UTR) (Parida et al, 2005;Li et al, 2011;Sahni et al, 2013). The C-prM gene, however, was relatively less conserved among all the four DENV serotypes (inter-serotype) in comparison to the 3 UTR (Teoh et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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

Neeraja

Lakshmi

Vanjari

et al. 2015

Journal of Virological Methods

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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.

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“…Reports of RT‐LAMP assays have tended to focus on qualitative assays in which a fluorescence signal is evaluated at the reaction endpoint; real‐time RT‐LAMP protocols that monitor fluorescence in real‐time throughout the reaction have received less attention. One research team has used solution turbidity as a real‐time indicator of RT‐LAMP reaction progress, and it appears that turbidity also exhibits exponential growth during the initial phase of the isothermal process; this suggests that the math model of Equation may be suitable. As compared to the Erwinia LAMP assay, the reverse transcription step in RT‐LAMP may impose an additional temporal delay as the reaction proceeds; such a delay could be accounted for in the model of Equation through addition of a delay term.…”

Section: Discussionmentioning

confidence: 99%

Mathematical modeling of a real‐time isothermal amplification assay for Erwinia amylovora

Gordon

Klemer²,

Fuller

et al. 2019

Engineering Reports

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A general mathematical model that describes the temporal behavior of a real‐time isothermal process used for nucleic acid amplification is derived. A monotonically‐increasing fluorescence signal s(t) generated and measured during the amplification reaction can be modeled in the form of a logistic function of time that is completely described by three parameters (k, t50, and Smax), which may be readily estimated from experimentally acquired s(t) data. Experimental data obtained from a real‐time loop‐mediated isothermal amplification (LAMP) assay for the infectious pathogen Erwinia amylovora (E. amylovora) are used to illustrate and validate the mathematical model. Implementation of such a modeling approach can allow for the extraction of quantitative information from real‐time LAMP data through parameter estimation techniques; this is demonstrated experimentally using real‐time amplification data acquired using the real‐time E. amylovora assay.

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Rapid Identification of Chikungunya and Dengue Virus by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Method

Cited by 44 publications

References 37 publications

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

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

Mathematical modeling of a real‐time isothermal amplification assay for Erwinia amylovora

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