Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay

Abstract

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

FIG. 1.

Schematic of the PCR-LDR assay for the identification of DENV serotypes. Serotype-specific PCR primers amplify one region of gene C and one region of gene E (for clarity, only the gene E amplicon is shown). Within each PCR amplicon, LDR primers are designed to identify and differentiate the four different DENV serotypes. The LDR primers target two locations in gene C and three locations in gene E. The 5′ upstream LDR primers bear zip-code complements, while the 3′ downstream LDR primers have either a FAM or a Cy3 fluorescent label. Ligation of the LDR primers results in fluorescently labeled products of different lengths that are then detected either by CE or on a universal array. NTR, nontranslated region.

FIG. 2.

Reconstructed CE images of representative DENV isolates. Ligation products were labeled with FAM, separated by CE on a 3730 DNA analyzer, and sized using a LIZ-labeled internal standard. Each serotype produces a distinct pattern depending on the number and sizes of the ligation products obtained. The approximate sizes of the ligation products, which ranged from 73 to 92 bases, are indicated on each side. The CE data shown are reconstructed images produced by Gelrender software (34). Negative samples (n = 7) and samples from other viruses yielded no observable products. For other flaviviruses, lanes are as follows: 1, West Nile virus; 2, St. Louis encephalitis virus; 3, Japanese encephalitis virus; 4, Kunjin virus; 5, Murray Valley virus; 6, Powassan virus; 7, yellow fever virus.

FIG. 3.

Detection of DENV by the universal array. (A) Universal microarray layout of expected positive signals (filled circles) for each DENV serotype. Zip-codes were spotted in duplicate. Open circles indicate addresses available for the detection of other viruses as the assay is further developed. (B) Representative universal array detection of DENV-1 to -4 using the RT-PCR-LDR assay. Each serotype generates at least four or five unique signals that permit identification. DENV-1 and DENV-3 produce two common signals, as described in the text.

FIG. 4.

LOD of the PCR-LDR assay. Microarray images show the LOD for DENV-1. The number of PFU used in each PCR is given. Fluorescent signals ≥10-fold higher than the background negative signals were deemed positive.