Development of multiplex PCR-ligase detection reaction assay for detection of West Nile virus

Abstract

We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

FIG. 1.

Schematic of the multiplex RT-PCR/LDR assay for WNV. Multiple PCR primer pairs are designed to amplify three distinct regions of the WNV cDNA (in black). Each PCR primer contains between one and three degenerate positions to accommodate minor sequence variation at the primer binding sites. For simplicity, only one PCR amplicon is shown. The presence of each PCR amplicon is detected by LDR primer pairs specific for three regions within each amplicon. The 5′ upstream LDR primers bear zip code complements, while the 3′ LDR downstream primers bear a 6-carboxyfluorescein (FAM) fluorescent label. Ligation of an LDR primer pair results in fluorescently labeled products of different lengths that can be detected either by CE or by hybridization to a universal DNA microarray. NTR, nontranslated region.

FIG. 2.

Representative CE profiles of ligation products from the PCR/LDR. (A to C) CE profiles from a uniplex PCR/LDR for each of the three regions of WNV. (A) Peaks at 72, 79, and 80 bases represent LDR products from regions WNV939, WNV1021, and WNV831, respectively. (B) Peaks at 79, 86, and 88 bases represent LDR products from regions WNV5548, WNV5427, and WNV5341, respectively. (C) Peaks at 80, 82, and 84 bases represent LDR products from regions WNV6244, WNV6168, and WNV6094, respectively. (D) CE cumulative profile obtained after a multiplex PCR/LDR when all three regions are amplified and detected together. Note that the peaks at 79 and 80 bases merge together to give a single peak at 80 bases and that the peak at 86 bases merges with the peak at 88 bases, giving a small peak at around 86 bases. Fluorescence intensity is indicated on the y axis, and the number of bases is indicated on the x axis.

FIG. 3.

Determination of the sensitivity of the one-step multiplex PCR/LDR/CE assay. CE profiles were obtained after RNA extraction and one-step multiplex RT-PCR/LDR of the WNV load panel. CE signals below a threshold of 200 fluorescence units were considered negative. Fluorescence intensity is indicated on the y axis, and the number of bases is indicated on the x axis.

FIG. 4.

Representative WNV detection with a universal DNA microarray. (A) Schematic of the microarray. The green box includes the area where the nine WNV-specific zip codes were spotted in duplicate (E4 to E12 and F4 to F12), with red, green, and blue spots indicating the three sets of zip codes corresponding to LDR products for amplicon regions 1, 2, and 3, respectively. Other zip codes on the array are designated for detecting dengue virus (yellow spots) and other hemorrhagic fever viruses (white spots). (B) LDR products from a representative WNV-positive mosquito pool sample revealing hybridization of LDR products from each region to the correct zip codes. The colors represent the fluorescence intensity at each spot, white being the strongest and blue being the weakest. (C) LDR products from a WNV negative control mosquito pool sample. (D) Hybridization of a carboxy-X-rhodamine-labeled fiducial complement internal control to verify uniform spotting of zip codes.