Research Article: Internally Controlled, Generic Real-Time PCR for Quantification and Multiplex Real-Time PCR with Serotype-Specific Probes for Serotyping of Dengue Virus Infections

Date Published: December 5, 2011

Publisher: Hindawi Publishing Corporation

Author(s): Sandra Menting, Khoa T. D. Thai, Tran T. T. Nga, Hoang L. Phuong, Paul Klatser, Katja C. Wolthers, Tran Q. Binh, Peter J. de Vries, Marcel Beld.

http://doi.org/10.1155/2011/514681

Abstract

Dengue has become a global public health problem and a sensitive diagnostic test for early phase detection can be life saving. An internally controlled, generic real-time PCR was developed and validated by testing serial dilutions of a DENV positive control RNA in the presence of a fixed amount of IC with results showing a good linearity (R2 = 0.9967) and a LOD of at least 1.95 × 104 copies/mL. Application of the generic PCR on 136 patient samples revealed a sensitivity of 95.8% and specificity of 100%. A newly developed multiplex real-time PCR with serotype-specific probes allowed the serotyping of DENV for 80 out of 92 (87%) generic real-time PCR positive patients. Combined these real-time PCRs offer a convenient diagnostic tool for the sensitive and specific quantification of DENV in clinical specimens with the possibility for serotyping.

Partial Text

Mosquito-borne flavivirus infections such as dengue have rapidly spread and are the most significant and dreaded infectious diseases in the world, in terms of morbidity and mortality [1, 2]. Recent estimates indicate that over 3.5 billion people (∼55%) of the world population live in areas at risk for dengue [3]. Worldwide there are 50–100 million cases of dengue infections per year, which result in 25,000 deaths. Dengue has become a major international public health problem due to the expanding geographic distribution of the vector in tropical and subtropical countries [1]. Increased international travel accompanied with increasing transmission or re-emergence and changing epidemiology of dengue in various (sub) tropical countries contribute to a steady rise in confirmed dengue among ill-returned travelers [4]. Dengue is caused by an RNA virus (DENV). DENV is primarily transmitted through bites of the infected Aedes aegypti mosquito vector. The majority of DENV infections, with any of the four different virus serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), are mildly asymptomatic and often difficult to recognize in the early phase of infection because signs and symptoms are nonspecific and resemble other febrile illnesses. Only a small number of DENV infections (~5%) will result in severe forms of the disease [5, 6]. The most used diagnostic tools for confirmation of DENV infections are based on detection of antibodies (Ab) or, recently, NS1 antigen (Ag) detection [7]. However, both Ab and Ag detection methods do not distinguish the respective DENV serotypes. Tests based on detection and serotyping of dengue virus offer the possibility to look better at the association between complications of the life threatening DHF and DSS, serotype, and sequential infections [8, 9]. Other real-time PCR assays for DENV infections are based upon type-specific primers but all without internal control (IC) [8, 10, 11]. For reliable detection and to rule out false negative results the use of an IC is essential [12]. Serotyping of DENV is important because the lack of cross-reactive immunity for the four different DENV serotypes may lead to a life threatening complication [9]. In this study we developed and validated an internally controlled generic real-time PCR for detection and quantification of all known DENV serotypes and a multiplex PCR for discrimination of serotypes based upon serotype-specific probes.

DENV infections have become a major international public health problem due to the increasing geographic distribution of the vector [1], the increased international travel accompanied with increasing confirmed DENV infection among ill-returned travelers [4], and the lack of cross-reactive immunity for the four different DENV serotypes and hyperendemic circulation of the four serotypes in the same regions. These all are factors that play a significant role in the threat posed by DENV.

 

Source:

http://doi.org/10.1155/2011/514681

 

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