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

ABSTRACT: BACKGROUND: Early 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. METHODS: A 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). RESULTS: Acute 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 (??=?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. CONCLUSION: The 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.

SUBMITTER: Teoh BT

PROVIDER: S-EPMC3846474 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Teoh Boon-Teong BT Sam Sing-Sin SS Tan Kim-Kee KK Johari Jefree J Danlami Mohammed Bashar MB Hooi Poh-Sim PS Md-Esa Rafi R AbuBakar Sazaly S

BMC infectious diseases 20130821

<h4>Background</h4>Early 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.<h4>Methods</h4>A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for ...[more]

PMID: 23964963

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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

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

Publications

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

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