Detection of infectious dengue virus by selective real-time quantitative polymerase chain reaction

The dengue virus (DENV) is a single-stranded positivesense RNA virus that belongs to the family Flaviviridae (Gubler, 2002), and has four serotypes, DENV1–DENV4, which are transmitted via Aedes aegypti and Aedes albopictus (Rodriguez-Roche and Gould, 2013). It has been reported that more than 50 million dengue infections occur each year (Guzman et al., 2010), and a serious outbreak occurred in the Southern Provinces of China in the summer of 2014. The clinical presentations of dengue infection range from mild febrile illness to severe dengue characterized by dengue hemorrhagic fever and shock syndrome, which make the accurate laboratory confirmation of the diagnosis challenging but crucial. Currently, serological assays and real-time quantitative polymerase chain reaction (qPCR) techniques are most commonly applied in the diagnosis of dengue infection (Guzman et al., 2010; Bäck and Lundkvist, 2013; Guzman and Harris, 2015). However, the current methods have some major drawbacks, such as falsepositive results owing to cross-reactivity with other arthropod-borne flaviviruses, and inability to determine the infectiousness in individual patients (Schwartz et al., 2000). Therefore, a simple and accurate method is needed to detect the virus, especially its associated infectious particles. Propidium monoazide (PMA) is a fluorescent, photoreactive dye with a high affinity for nucleic acids. Previous studies have indicated that PCR combined with PMA can discriminate between viable and inactivated cells, because of the reduction in PCR signal from DNA of dead cells (Nocker et al., 2006). The azide group of the compound generates highly reactive nitrene intermediates that readily form covalent nitrogen-carbon bonds with nucleic acids. The nucleic acids modified by PMA cannot be used as templates to be transcribed or amplified. However, the compound does not penetrate intact cell walls or membranes, and only reacts with DNA or RNA exposed after such barriers are damaged (Nocker et al., 2006). Hence, the compound may be used to selectively amplify templates from structurally intact organisms. The PMA-qPCR method has been widely applied to detect and quantify viable particles of the echovirus (Parshionikar et al., 2010), bacteriophage T4 (Fittipaldi et al., 2010), influenza virus (Graiver et al., 2010), and norovirus (Kim and Ko, 2012). In this study, we investigated the feasibility and reliability of selective qPCR to detect DENV2. Cytopathogenic DENV2 was obtained from the BSL-3 Laboratory at the Southern Medical University, China, and was propagated and assayed in BHK-21 cells. Semipurified stocks were subsequently produced from these cultures. Briefly, the cells were lysed by repeated freezethaw cycles at –80 °C (Low-temperature Refrigerator, SANYO, Osaka, Japan); the lysates were cleared by centrifugation for 15 min at 4000 rpm at 4 °C, and then stored at –80 °C. The infectious titers were measured as a 50% tissue culture infectious dose in eight replicates. Clustal W was used to align the specific conserved cDNA fragment from 20 DENV2 strains isolated from different geographical areas at different times. The primers for this fragment were designed by using the Primer 5.5, and assessed by using the Oligo 7.0 program (Supplementary Table S1). The fragment was also synthesized, inserted into the plasmid pMD18-T (TAKARA Co., Dalian, China) to construct the standard plasmid pMD-DENV2 (Figure 1A). The plasmids were propagated in Escherichia coli DH5α and purified by using the TAKARA MiniBEST plasmid purification kit. A standard curve was generated by amplifying 10-fold dilutions of the standard plasmid (Supplementary Figure S1A), starting with a stock concentration of 279.1 ng/μL, and then the cycle threshold (Ct) values were plotted against the concentrations of the template [Supplementary Figure S1B, Y = −3.258 × Log (X) ± 9.79, Eff. = 102.7%]. For pretreatment with PMA, a 20 mmol/L stock solution was prepared by dissolving 1 mg of PMA (Biotium, Inc., Hayward, USA) in 98 μL of distilled water (dH2O), and the stock was stored at −20 °C until it was needed. The viral preparations were incubated in 50 μmol/L PMA for 5 min in the dark at 20 °C, with occasional mixing. Then, the samples were crosslinked by exposing them to blue light-emitting diode (LED) light (LedActive Blue, Philips, Holland) for 15 min on ice. The RNA