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

ABSTRACT: 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.

SUBMITTER: Dauner AL

PROVIDER: S-EPMC7126901 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Dauner Allison L AL Mitra Indrani I Gilliland Theron T Seales Sajeewane S Pal Subhamoy S Yang Shih-Chun SC Guevara Carolina C Chen Jiann-Hwa JH Liu Yung-Chuan YC Kochel Tadeusz J TJ Wu Shuenn-Jue L SJ

Diagnostic microbiology and infectious disease 20150515 1

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 reactio ...[more]

PMID: 26032430

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Project description:Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens causing infectious diseases. However, mismatches between primers (especially in the 3'-end) and templates significantly reduced the amplification efficiency of LAMP, and limited its application to genetically diverse viruses. Here, we reported a novel mismatch-tolerant LAMP assay and its application in the detection of dengue viruses (DENV). The novel method features the addition of as little as 0.15 U of high-fidelity DNA polymerase to the standard 25 μl LAMP reaction mixture. This amount was sufficient to remove the mismatched bases at the 3'-end of primers, thereby resulting in excellent tolerance for various mismatches occurring at the 3'-end of the LAMP primers during amplification. This novel LAMP assay has a markedly improved amplification efficiency especially for the mutants forming mismatches with internal primers (FIP/BIP) and loop primers (FLP/BLP). The reaction time of the novel method was about 5.6-22.6 min faster than the conventional LAMP method regardless of the presence or absence of mismatches between primers and templates. Using the novel method, we improved a previously established pan-serotype assay for DENV, and demonstrated greater sensitivity for detection of four DENV serotypes than the previous one. The limit of detection (LOD) of the novel assay was 74, 252, 78, and 35 virus RNA copies per reaction for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. Among 153 clinical samples from patients with suspected DENV infection, the novel assay detected 94.8% samples being DENV positive, higher than that detected by the commercial NS1 antigen assay (92.2%), laboratory-based RT-PCR method (78.4%), and the conventional RT-LAMP assay (86.9%). Furthermore, the novel RT-LAMP assay has been developed into a visual determination method by adding colorimetric dyes. Because of its simplicity, all LAMP-based diagnostic assays may be easily updated to the newly improved version. The novel mismatch-tolerant LAMP method represents a simple, sensitive and promising approach for molecular diagnosis of highly variable viruses, and it is especially suited for application in resource-limited settings.

Dataset Information

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

Publications

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

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