Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid and Real-Time Detection of Japanese Encephalitis Virus
Open accessAbstract
The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the clinical samples was 10(2) to 10(5) PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.
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LAMP and NASBA have been studied in various CNS infections and might especially be useful in laboratories with limited resources.
“LAMP and NASBA have been studied in various CNS infections and might especially be useful in laboratories with limited resources (26–31).”
From Molecular diagnostics in cerebrospinal fluid for the diagnosis of central nervous system infections (2024) · cites this paper as [28]
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“A study with 19 specific antibody-confirmed cases of JE found that LAMP targeting the E gene had a sensitivity of 68% (28).”
- Authors
- Parida, M. M., Santhosh, S. R., Dash, P. K., Tripathi, N. K., Saxena, P., Ambuj, S., Sahni, A. K., Lakshmana Rao, P. V., & Morita, K.
- Affiliations
- Defence Research and Development Establishment (IN)
- Division of Virology, Defence Research & Development Establishment, Gwalior, Madhya Pradesh 474002, India
- Nagasaki University (JP)
- Venue
- Journal of Clinical Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2006-11
- Volume
- 44
- Issue
- 11
- Pages
- 4172-4178
- ISSN
- 0095-1137 (ISSN-L)
- 1098-660X
- PMC
- PMC1698363
- OpenAlex
- W2104391647
- Cited by
- 120
- References
- 23
- Citations by year
- 2026: 3 · 2024: 2 · 2023: 5 · 2022: 10 · 2021: 6 · 2020: 5 · 2019: 4 · 2018: 3 · 2017: 11 · 2016: 5 · 2015: 4 · 2014: 6 · 2013: 7 · 2012: 9
- FWCI
- 4.35
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Journal of Clinical Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › Biosensors and Analytical Detection
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Mosquito-borne diseases and control
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- Japanese encephalitis virus, RT-LAMP, loop-mediated isothermal amplification, E gene, rapid virus detection, cerebrospinal fluid, sensitivity and specificity, primer specificity, point-of-care testing, RT-PCR comparison
- MeSH terms
- Antibodies, Viral, Cells, Cultured, Encephalitis Virus, Japanese, Humans, Immunoglobulin M, Sensitivity and Specificity, Reverse Transcriptase Polymerase Chain Reaction, Nucleic Acid Amplification Techniques
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