Improved Molecular Detection of Dengue Virus Serotype 1 Variants

Abstract

Vorndam, J. Clin. Microbiol. 30:545-551, 1992) is used worldwide for diagnosis and surveillance. However, it failed to detect DENV-1 variants in Cambodia due to a point mutation. We describe an improvement of the method that allows the detection of additional DENV-1 strains, including potential variants.Dengue virus (Flaviviridae, genus Flavivirus) serotypes 1 to 4 are transmitted to humans mainly by Aedes aegypti mosquitoes. Human infection leads to a clinical picture ranging from asymptomatic infection or mild dengue fever to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome. Dengue is a major public health problem of increasing concern, as evidenced by the estimate of 50 million infections annually, including 400,000 DHF cases, and 2.5 billion individuals at risk (16). Southeast Asia, including Cambodia, is a an area of dengue hyperendemicity, where the four serotypes are circulating simultaneously and DHF cases are reported each year. Since no vaccine is available to prevent the disease, its control relies mainly on surveillance, vector control, and case management (16).Laboratory diagnosis of dengue infection includes molecular techniques such as reverse transcription (RT)-PCR, for which more than 16 different protocols have been published (9). One protocol, developed by Lanciotti et al. (10), a seminested RT-PCR, targets the C and pre-M region. It uses universal outer dengue primers D1 and D2, followed by a subsequent serotype-specific seminested PCR combining primer D1 with one of the following internal primers: TS1, TS2, TS3, or TS4. This assay is known to exceed the sensitivity of virus isolation when aliquots of the same sample are used (8) and is widely used for diagnosis and surveillance of dengue, particularly in Southeast Asian countries (2-5, 7, 13, 15).In Cambodia, our laboratory surveillance of dengue utilizes hemagglutination inhibition and immunoglobulin M capture enzyme-linked immunosorbent assays, virus isolation on AP61 and Vero E6 cells (12), and seminested RT-PCR by the method of Lanciotti et al. (10) with slight modifications. Briefly, 100 l of serum was extracted by using Trizol LS reagent (Life Technologies, Gaithersburg, Md.), according to the manufacturer's instructions. The resulting RNA pellet was resuspended in 30 l of RNase-free water and incubated for 10 min at 55°C, and then 5 l was used in an RT reaction in a 15-l mixture containing 500 M deoxynucleoside triphosphate (dNTP), 0.25 M D2 primer, 20 U of RNasin (Promega, Madison, Wis.), 5 U of avian myeloblastosis virus reverse transcriptase (Promega), 5ϫ RT buffer, and RNase-free water. RT was carried out at 42°C for 1 h. The first-round PCR was carried out in a 50-l volume containing 2.5 mM MgCl 2 , 0.2 mM (each) dNTPs (Boehringer Mannheim, Mannheim, Germany), 0.2 M (each) D1 and D2 outer primers, 1ϫ Taq buffer, 2 U of Taq polymerase (Promega), 5 l of the RT product, and PCR-grade water. The PCR program started with 5 min of denaturation at 95°C, followed by 35 cycles consisting of 94°C for 1 min, 55°C for 1 min, an...

Under-recognition and reporting of dengue in Cambodia: a capture–recapture analysis of the National Dengue Surveillance System

“…Only acute-phase sera of participants who were positive for anti-DENV IgM in the convalescent sample were tested for DENV using molecular methods. DENV ribonucleic acid amplification, detection and serotyping were performed using reverse transcriptase-polymerase chain reaction (RT-PCR) according to Lanciotti et al [17] as modified by Reynes et al [18]. Overall, the median age of participants was 7 years and 52% were males.…”

Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses

“…The SYBR green detection method was adapted for the C-prM and 3ЈNC amplimer sets. The C-prM region protocol was chosen because it is the most widely used protocol and has been modified and adapted in various laboratories to serotype dengue viruses (8,14,19,20). Mismatching between viral RNA sequences and D1, D2, or TS sequences resulting in falsenegative PCR results has previously been observed (8,20; C. Chin, personal communication).…”

“…The C-prM region protocol was chosen because it is the most widely used protocol and has been modified and adapted in various laboratories to serotype dengue viruses (8,14,19,20). Mismatching between viral RNA sequences and D1, D2, or TS sequences resulting in falsenegative PCR results has previously been observed (8,20; C. Chin, personal communication). We addressed this observation by modifying the D1 amplimer (mD1) and replacing the DENV-1, DENV-2, and DENV-4 type-specific amplimers with redesigned TS1 (rTS1) and TS4 (rTS4) and modified TS2 (mTS2) after an extensive search using the DENV sequences available in the GenBank database ( Table 1).…”

“…The C-prM protocol utilizes a DENV consensus sequence for outer amplimers D1 and D2 in an initial RT-PCR, followed by a subsequent serotype-specific heminested PCR, combining D1 with one or more of the following serotype-specific internal amplimers: TS1, TS2, TS3, and TS4. In spite of this, several authors have reported falsenegative PCR results using this protocol due to a mismatch between the dengue viral RNA sequence and the D1, D2, or TS sequence (8,20; C. Chin, personal communication).…”

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay

“…Such viral genetic heterogeneity has implications for the design of molecular assays. False-negative PCR results due to sequence mismatches between DENV RNA and the primers used in realtime PCR assays have been reported, necessitating assay revisions (6,16,36). In the design of our assay, we used several primer pairs with degeneracies to accommodate possible failures of amplification due to sequence mismatches.…”