Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses
Introduction
Dengue virus (DENV) is an enveloped single stranded positive sense RNA virus. There are four serotypes of DENV, DENV1 to DENV4, which are transmitted to humans through the bites of Aedes mosquitoes, mainly A. aegypti and A. albopictus. Dengue virus infection causes every year 50 million of an acute febrile disease in tropical and sub-tropical regions around the world: dengue fever, including more than 500 000 reported cases of the severe forms of the disease: dengue haemorrhagic fever and dengue shock syndrome.1
Laboratory diagnosis of DENV infection is based on virus isolation, detection of virus antigen or RNA and detection of DENV specific antibodies.2 Most recently, real-time RT-PCR has been developed to detect DENV RNA in human samples for all four serotypes.3, 4, 5, 6, 7 This technique is an important improvement for the rapid diagnosis of DENV infection allowing early initiation of patient management and specific preventive health measures. In the different published real-time RT-PCR, two problems arise: the lack of standardization of the method and possible false negative due to the genetic diversity in each DENV serotype and more particularly in DENV2.
We developed one system of real-time RT-PCR for the specific detection of all DENV and four systems of real-time RT-PCR allowing to serotype DENV. These diagnosis methods were validated for sensitivity, specificity, linearity and precision (repeatability and intermediate precision) on RNA transcripts.
Section snippets
Viruses and human samples
DENV1 Djibouti, DENV2 TC544, DENV3 H87 and DENV4 Dakar, Langat TP64, West Nile NY 385-99, Yellow Fever 17D, Louping Ill 369T, Japanese encephalitis Nakayama strains were used in this study. Viral titres for DENV1, DENV2, DENV3 and DENV4 were, respectively, 105.5, 107.5, 105.8, 107.1 TCID50/ml. Human serum (n = 11) and CSF (n = 2) from Thai patients and human serum (n = 79) from patients suspected of imported dengue virus infection were obtained.
Primers and probes design
Twenty-five complete sequences of DENV1, 25 complete
Development of real-time one-step RT-PCR for the detection and typing of DENV
The goal was to obtain one system able to detect specifically all DENV strains circulating in the world (pan-dengue system) and four systems detecting specifically DENV1 to DENV4, respectively. Alignments were performed with ClustalX8 using complete sequences of DENV isolated in different geographical area. The analysis showed that, depending on the region of the genome, the variability between strains of each serotype at the nucleotide level was from 7% to 23%. The most conserved regions are
Discussion
We have developed, standardized and validated five systems of real-time one-step RT-PCR allowing the detection and serotyping of DENV RNA. The originality of ours systems compare to published ones is based on an exhaustive analysis of multiple alignments of DENV strains, representing different region in the world, for the choice of primers and probes and also based on the validation of methods.3, 4, 5, 6, 7, 10 For the pan-dengue system, primers and probe were located in the most conserved
References (12)
- et al.
Rapid detection, serotyping and quantitation of dengue viruses by taqman real-time one-step RT-PCR
J Virol Methods
(2006) - et al.
Involvement of the central nervous system in patients with dengue virus infection
J Neurol Sci
(2008) - World Health Organization. Dengue and dengue haemorrhagic fever. Fact sheet; 2002,...
- et al.
Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health
J Microbiol Immunol Infect
(2005) - et al.
Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus
J Clin Microbiol
(2001) - et al.
Rapid detection and quantification of RNA of ebola and marburg viruses, lassa virus, Crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR
J Clin Microbiol
(2002)