RESUMO
BACKGROUND: Dengue infection represents a global health issue of growing importance. Dengue non-structural protein 1 (NS1) plays a central role in the early detection of the disease. The most common method for NS1 detection is testing by lateral flow immunoassays (LFIAs) with varying sensitivity. In this study, we present a highly sensitive magneto-enzyme LFIA for prompt diagnosis of dengue. METHODS: We have demonstrated the development of a magneto-enzyme LFIA combining super-paramagnetic nanoparticles as labels and Biotin-Streptavidin signal amplification strategy to detect dengue NS1. Factors affecting the test performance including antibody pair, super-paramagnetic nanoparticle size, nitrocellulose membrane type, amounts of detection and capture antibodies, and amounts of Streptavidin-polyHRP were optimized. Analytical sensitivity and cross-reactivity were determined. Clinical performance of the novel assay was evaluated using a panel of 120 clinical sera. RESULTS: This newly developed assay could detect NS1 of all four serotypes of dengue virus (DENV). The limit of detection (LOD) was found to be as low as 0.25 ng ml-1 for DENV-1 and DENV-3, 0.1 ng ml-1 for DENV-2, and 1.0 ng ml-1 for DENV-4. The LOD for DENV-2 was a 50-fold improvement over the best values previously reported. There was an absence of cross-reactivity with Zika NS1, Hepatitis B virus, Hepatitis C virus, and Japanese encephalitis virus. The sensitivity and specificity of the novel assay were 100% when tested on clinical samples. CONCLUSIONS: We have successfully developed a magneto-enzyme LFIA, allowing rapid and highly sensitive detection of dengue NS1, which is essential for proper management of patients infected with DENV.
RESUMO
PEG1 (or MEST) is an imprinted gene located on human chromosome 7q32 that is expressed predominantly from the paternal allele. In the mouse, Peg1/Mest is associated with embryonic growth and maternal behavior. Human PEG1 is transcribed from two promoters; the transcript from promoter P1 is derived from both parental alleles, and the transcript from P2 is exclusively from the paternal allele. We characterized the P1 and P2 transcripts in various normal and neoplastic tissues. In the normal tissues, PEG1 was transcribed from both promoters P1 and P2, whereas in six of eight neoplastic tissues, PEG1 was transcribed exclusively from promoter P1. Bisulfite sequencing demonstrated high levels of CpG methylation in the P2 region of DNA from a lung tumor. In the region between P1 and P2, we identified a novel transcript, PEG1-AS, in an antisense orientation to PEG1. PEG1-AS is a spliced transcript and was detected as a strong 2.4-kilobase band on a Northern blot. PEG1-AS and PEG1 P2-sense transcript were expressed exclusively from the paternal allele. Fragments of DNA from within the 1.5-kilobase region between PEG1-AS and the P2 exon were ligated to a pGL3 luciferase reporter vector and transfected into NCI H23 cells. This DNA exhibited strong promoter activity in both the sense and antisense directions, indicating that PEG1-AS and P2 exon share a common promoter region. Treatment of the transfected DNA fragments with CpG methylase abolished the promoter activity. Of interest, PEG1-AS was expressed predominantly in testis and in mature motile spermatozoa, indicating a possible role for this transcript in human sperm physiology and fertilization.
