Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy

Abstract

Dengue virus (DENV) infection causes sudden fever along with several nonspecific signs and symptoms and in severe cases, death. DENV is transmitted to people by Aedes aegypti and Ae. albopictus mosquitoes, whose populations increase during rainy season. West Nile Virus (WNV), Rickettsia spp. and Leptospira spp. are fever-causing pathogens that share many of the initial symptoms of DENV infection and also thrive in the rainy season. Outbreaks in some regions may be due to any of these pathogens that can co-circulate. Plus, they are clinically indistinguishable until severe symptoms appear, even though these diseases should be treated differently. An effective differential diagnosis would help clinicians and vector control departments to make right decisions for control and treatment of these diseases. Therefore, we developed four different SYBR green (®) -based reverse transcription quantitative PCR (RT-qPCR) assays for simultaneous detection of DENV, WNV, Rickettsia spp. and Leptospira spp. The assay has been optimized to yield results in less than 1 h; and in order to reduce contamination risk, all reagents were premixed and lyophilized on 96 well plates and thus only requires the addition of water and total nucleic acids from the sample. Sensitivities of the assays were less than 100 copies of nucleic acid targeted for these four pathogens. Assays did not show cross reactivity with any of the four pathogens nor to human nucleic acids. We are presenting a sensitive and selective kit that detects four relevant pathogens from tropical regions, that is quick, cost-effective and easy to use.

Keywords: Dengue; Leptospira; Real time PCR; Rickettsia; West Nile Virus.

Fig. 1

Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV (blue), WNV (violet), Rickettsia spp. (red) and Leptospira spp. (green). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [light-colored wells with a “(−)” sign] and another for the Universal Positive Control (dark-colored wells with “UPC”)

Fig. 2

Blood samples were obtained from patients presenting fever during up to 5 days and were left at room temperature (RT) for 15 min for clotting. Samples were then transported from the hospital to the research center in a refrigerated container. Samples were centrifuged at 2000 rpm for 10 min in a cold centrifuge and recovered from the tubes inside a vertical laminar flow cabinet Class II Type A2 in BSL2 facilities. Lysis reagent of MagNA Pure LC total nucleic acid isolation kit was added to each sample and incubated for 15 min inside the laminar flow cabinet. 300 μL of inactivated serum were transferred into 32 well-plates for Roche MagNA Pure LC instrument. Total nucleic acids extractions were stored at −20 °C until RT-qPCR were performed

Fig. 3

Linearity ranges of the assays. Standard curves of the amplification plots are shown for the assays of Dengue virus (a), west Nile virus (b), Rickettsia (c), and Leptospira (d). Serial dilutions of 10⁶–10⁰ molecules of each positive control were used as a template for RT-qPCR reactions. The reactions were carried out in duplicate and Cycle threshold (C_t), efficiency, and slope were determined according to the Fix Points algorithm included in the Lightycler 480 II^® software

Fig. 4

Amplification plots of the lyophilization assay. Fresh positive control showed a Cycle threshold (C_t) of 22, trehalose 0 % C_t of 24, trehalose 2.5 % C_t of 26, trehalose 5 % C_t of 29 and trehalose 10 % C_t of 33