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, M.; Lakshmi, V; Vanjari, Lavanya; Priyanka, E.; Parida, Manmohan; Dash, Paban Kumar; Sharma, Shashi; Rao, P. Venkata; Reddy, Gopal

doi:10.1016/j.jviromet.2014.10.005

Cited by 32 publications

(27 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Molecular diagnosis tools for dengue is still much needed at or near POC/PON, particularly for developing countries with limited public health resources. For POC/PON dengue diagnosis, several isothermal amplification methods such as nucleic acid sequence-based amplification (NASBA), thermophilic helicase-dependent amplification (tHDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and insulated isothermal PCR (iiPCR) have been developed and reported previously as potential field-deployable tools for the detection of different microorganisms [7,10,[22][23][24][25]. Based on these technologies, various all-in-one molecular diagnostics have been made available to help minimizing risks of human errors and allowing easy molecular bio-detection at or near POC/PON in the last few years, but, to our knowledge, no reagents are available for DENV testing on these platforms.…”

Section: Introductionmentioning

confidence: 99%

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

Tsai¹,

Liu²,

Lin³

et al. 2019

PLoS ONE

View full text Add to dashboard Cite

Background The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5’ untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system. Methodology/Principal findings Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems. Conclusions/Significance With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease.

show abstract

Section: Introductionmentioning

confidence: 99%

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

Tsai¹,

Liu²,

Lin³

et al. 2019

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…However, PCR equipment is expensive, requires trained personnel, and is best suited to diagnostic reference laboratories. Other nucleic acid detection strategies based on isothermal amplification such as nucleic acid sequence-based amplification (NASBA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) have been developed to address these Diagnostic Microbiology and Infectious Disease 83 (2015) 30-36 challenges and move molecular assays closer to point-of-care use (Jittmittraphap et al, 2006;Neeraja et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we developed an RT-LAMP assay to detect all 4 serotypes of DENV in a single reaction. Other groups have explored this concept, with several assays detecting the 4 serotypes of DENV in separate reactions (Neeraja et al, 2015;Parida et al, 2005). Some groups have developed pan-serotype DENV RT-LAMP assays (Teoh et al, 2013), and others have expanded this concept to a pan-flaviviral assay that detects DENV1-4, Japanese encephalitis virus, and West Nile virus in a single reaction (Li et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus

Dauner

Mitra

Gilliland

et al. 2015

Diagnostic Microbiology and Infectious Disease

View full text Add to dashboard Cite

During dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. To facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that detects all 4 serotypes of dengue virus (DENV). We used a quencher to reduce nonspecific amplification. The assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at 63 °C. Results can be visualized using UV fluorescence, handheld readers, or lateral flow immunochromatographic tests. We report a sensitivity of 86.3% (95% confidence interval [CI], 72.7-94.8%) and specificity of 93.0% (95% CI, 83.0-98.1%) using a panel of clinical specimens characterized by DENV quantitative reverse transcription-polymerase chain reaction. This pan-serotype DENV RT-LAMP can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need.

show abstract

“…In the field of molecular diagnosis, several isothermal amplification modalities such as loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), thermophilic helicase-dependent amplification (tHDA), and recombinase polymerase amplification (RPA) have been developed for the detection of different DNA or RNA microorganisms, including those causing malaria (10,11). For molecular diagnosis of dengue, reverse transcription-LAMP (RT-LAMP) has been reported previously as a potential field-deployable POC test (12)(13)(14)(15)(16). Results of comparisons of the RT-PCR, RT-iiPCR, and RT-LAMP tests for dengue regarding the principles, equipment, primers/probes, reagent stability, time, and detection capability with respect to different DENV serotypes or related viruses are summarized in Table 1.…”

mentioning

confidence: 99%

“…First, it cannot distinguish different DENV serotypes. Given that multiple DENV serotypes have been cocirculating in regions of endemicity and that two or more DENV serotypes were reported increasingly during the same outbreak, several RT-PCR or RT-LAMP tests have been developed in multiplex format to detect and distinguish different DENV serotypes (9,(12)(13)(14). Future development of multiplex RT-iiPCR for dengue would greatly improve its clinical applications to better understand the extent and serotypes of infecting virus in the field sites.…”

mentioning

confidence: 99%

Potential Point-of-Care Testing for Dengue Virus in the Field

Wang

Gubler

2018

J Clin Microbiol

View full text Add to dashboard Cite

The four serotypes of dengue virus (DENV) cause one of the most important and rapidly emerging mosquito-borne viral diseases in humans. Of the currently available diagnostic tests for dengue, the reverse transcription-PCR (RT-PCR) assay is the most sensitive and specific, and so it is commonly used as the gold standard. However, the requirement of a sophisticated and expensive thermal cycler makes it very difficult to use as a point-of-care diagnostic test in resourcelimited regions where dengue is endemic. Tsai et al. (J Clin Microbiol 56:e01865-17, 2018, https://doi.org/10.1128/JCM.01865-17) report the analytical and clinical performances of a reverse transcription-insulated isothermal PCR (RT-iiPCR) assay with a portable nucleic acid analyzer for rapid detection of the four DENV serotypes; its reproducibility and complete agreement on clinical samples with the multiplex RT-PCR assay developed by the Centers for Disease Control and Prevention suggest that the dengue RT-iiPCR is a potential point-of-care test. Compared with other DENV RNA detection methods, the unique isothermal PCR design of RT-iiPCR, together with further improvements, would represent a promising new type of field-deployable diagnostic test for dengue. With an estimated 390 million new infections per year, the four dengue virus (DENV) serotypes are the leading causes of mosquito-borne viral infection and disease in humans (1, 2). While many DENV infections are clinically mild or inapparent, clinical disease ranges from a self-limited illness, dengue fever, to severe and potentially life-threatening disease, dengue hemorrhagic fever/dengue shock syndrome (1-3). The forms of the disease are classified as dengue, dengue with warming signs, and severe dengue according to the 2009 WHO revised case definition (3). There are approximately 3 billion people in more than 120 countries who are at risk of DENV infection (1, 2). Currently, no FDA-approved antiviral drug is available to treat dengue; the only licensed dengue vaccine, Dengvaxia, is recommended only for persons who have experienced previous DENV infection and not for dengue-naive individuals (4-6).Accurate and rapid laboratory diagnosis of dengue is critical for confirmation of clinically suspected cases to ensure timely management of severe dengue disease, especially in urban areas of resource-poor developing countries. Laboratory diagnosis for DENV infection includes virus isolation, reverse transcription-PCR (RT-PCR), nonstructural protein 1 (NS1) antigen detection, and serological tests such as IgM and IgG enzyme-linked immunosorbent assay (ELISA) and neutralization tests (2, 3). Each diagnostic method has its advantages and limitations; overall RT-PCR is the most sensitive and specific diagnostic test and has been commonly used as a gold standard for comparisons with other tests (reviewed in reference 7).The report by Tsai and colleagues in this month's issue of the Journal of Clinical Microbiology suggests that the reverse transcription-insulated isothermal PCR (RT-iiPCR) ass...

show abstract

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

Cited by 32 publications

References 30 publications

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus

Potential Point-of-Care Testing for Dengue Virus in the Field

Contact Info

Product

Resources

About