One-step real-time RT-PCR assays for serotyping dengue virus in clinical samples

BMC Infectious Diseases

Nina Lagerqvist¹  Kerstin Ingrid Falk²  Gunnel Lindegren¹  Erik Alm¹ 

[1] Department of Microbiology, The Public Health Agency of Sweden, Solna, Sweden;Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

关键词: Serotyping;    Laboratory diagnosis;    RT-PCR;    Serotype;    Dengue virus;   

Others  :  1231029
DOI  :  10.1186/s12879-015-1226-z

 received in 2015-05-18, accepted in 2015-10-19,  发布年份 2015

【 摘 要 】

Background

Dengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.

Methods

The DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.

Results

The RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10 ²to 10 ⁶copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.

Conclusions

Our results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.

【 授权许可】

   
2015 Alm et al.

【 图 表 】

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