Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses

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Abstract

Background

Dengue virus, transmitted by mosquitoes, causes every year 50 million cases of dengue fever. A standardize method for early diagnosis is still needed for clinical diagnosis and epidemiological studies.

Objective

To develop and validate for sensitivity, specificity, linearity and precision real-time one-step RT-PCR for the detection of dengue viruses.

Study design

Multiple alignments of dengue virus sequence for each serotype were done and used to develop five systems of real-time RT-PCR to detect all dengue virus strains and then identify the serotype. These systems were validated on synthetic RNA transcripts for specificity, sensitivity, precision and linearity and then applied on series of human samples.

Results

The specificity of each system was determined by sequence alignments and experimentally tested on different flaviviruses. Methods precision and linearity were statistically validated. Each of these systems allowed the detection of less than one infectious particle and was able to detect and serotype quickly dengue virus in human samples where infectious virus cannot be isolated anymore.

Conclusions

These systems are valuable tools for dengue virus diagnosis and epidemiological studies. Standardization and validation of these methods allow an easy transfer to diagnostic laboratories.

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

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