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Detection and typing of dengue virus by one-step RT-PCR-based high-resolution melting assay

Abstract

Dengue is a rapidly spreading arboviral disease that can be transmitted through any of the four types of dengue virus (DENV) serotypes. Previous studies have observed that individuals who have a pre-existing secondary infection due to a different dengue serotype, experience severe forms of this disease. During a DENV outbreak, a time-sensitive preliminary diagnosis of the origin of DENV might be useful in controlling the epidemic. Here, we developed a rapid and accurate one-step TB Green RT-PCR-based high-resolution melting (HRM) assay to identify and serotype DENV using serotyping primers based on the alignment with the E gene. This assay had a detection limit of 7.7 × 102 (DENV 1), 3.8 × 102 (DENV 2), 6.2 × 102 (DENV 3), and 1.2 × 103 (DENV 4) RNA copies/mL. No cross-reactivity with the Chikungunya, Zika, and Japanese encephalitis viruses was observed. The feasibility of using this assay for clinical diagnosis was evaluated in DENV-positive patient sera. The HRM assay and the RT-qPCR had complete matched results derived from DENV detection, including 51 serum positive and 20 serum negative. Additionally, eight DENV 2 strains in the same serotype were successfully differentiated by an HRM assay. Thus, this assay facilitated accurate detection and serotyping of DENV, along with the time-sensitive identification of the infectious focus of different DENVs.

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Funding

This study was supported by the Medical Science and Technology Program of Zhejiang, China (Grant No. 2020KY092), the Hangzhou City Medical Science and Technology Platform Project (Grant No. OO20190025), Natural science foundation of Zhejiang (Grant No. LGF20H260003), and the Key Laboratory of Zhejiang Provincial Public Health Emergency Detection Technology.

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Authors and Affiliations

Authors

Contributions

Conceptualization: RT, HY, and ZY; Methodology and software: RT and HY; validation: YJ, LL, and AW; resources: HY; data curation: ZY; writing original draft preparation: RT and HY; writing review and editing: HY; supervision: KY; and project administration: KY. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Zhangnv Yang or Kuangming Yu.

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Conflict of interest

The authors declare no conflict of interest.

Informed consent

Informed consent was obtained from all subjects involved in the study.

Institutional review board statement

The study of DENV 1 was approved by the Institutional Ethical Committee of Shangcheng Center for Disease Control and Prevention, Hangzhou City. A written informed consent was obtained from each participant. The detection study of four serotypes of DENV was approved by Institutional Ethical Committee of Provincial Centers for Disease Control and Prevention (CDC) of Zhejiang with approval number 2020-12.

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Tian, R., Yan, H., Jiang, Y. et al. Detection and typing of dengue virus by one-step RT-PCR-based high-resolution melting assay. Virus Genes 58, 319–326 (2022). https://doi.org/10.1007/s11262-022-01906-8

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  • DOI: https://doi.org/10.1007/s11262-022-01906-8

Keywords

  • Dengue virus
  • E gene
  • High-resolution melting
  • Serotype
  • RT-PCR