Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification.

BACKGROUND: Human Cytomegalovirus virus (HCMV) is a worldwide virus that causes no serious symptoms in most adults. However, HCMV infection during pregnancy, it may lead to a series of serious complications, such as hearing loss, mental retardation, visual impairment, microcephaly and developmental retardation. AIM: The aim of this study was to develop a simple, low dependence on equipment and accurate method for HCMV detection based on the recombinase polymerase amplification (RPA) and lateral flow chromatography strip (LFS) reading. METHODS: In order to meet the feasibility of HCMV early screening, three pairs of RPA primers were designed based on the UL123 gene encoding IE1, which was expressed immediately in the early stage of HCMV. In order to improve the specificity of the reaction and satisfy the visual detection, a specific probe was designed to insert THF site between upstream and downstream primers, fluorescein isothiocyanate (FITC) and C3spacer were used to modify the 5' end and the 3' end respectively, and Biotin was used to modify the 5' end of the reverse primer. HCMV standard strain AD169 was enriched by ARPE-19 cells culture, and its genome was extracted. The primers and probes were screened by RPA-LFS test, and the optimal reaction temperature and time were determined The specificity was verified in different viruses, bacteria and parasites. The standard curve was drawn based on the constructed recombinant plasmid of pMD18T-HCMV-UL123 and used for HCMV genomic DNA quantification and determination of the detection sensitivity. Urine samples from artificial HCMV contamination or clinical collection were prepared to evaluate the consistency with the results of real-time quantitative PCR. RESULTS: The results showed that the primers and probes for HCMV RPA-LFS detection based on UL123 gene were successfully screened, the amplification of HCMV genomic DNA with as low as 30 copies could be completed at 37 °C within 15 min, it did not react with Human herpesvirus 1, Streptococcus pyogenes, Candida albicans, Listeria monocytogenes, Y. enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginnolyfificus, Vibrio parahaemolyticus, S. typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Trichomonas vaginalis. The positive rate of PCR was 96.67 % in 30 simulated urine samples and 100 % in 127 clinical urine samples with the same UL123 gene detection. CONCLUSIONS: To sum up, we developed a diagnostic method for HCMV based on UL123 gene combined with RPA and LFS, which is low dependent on equipment, fast, sensitive and specific, provide reference for point-of-care testing HCMV in grass-roots laboratories and remote areas.

Paeonol alleviates placental inflammation and apoptosis in preeclampsia by inhibiting the JAK2/STAT3 signaling pathway.

Preeclampsia (PE) is a multisystemic and placental inflammatory disease that causes maternal and infant health issues. As one of the active components in peony root extract, paeonol (Pae) exerts anti-apoptosis and anti-inflammatory effects. Nonetheless, the protective role of Pae in PE has not yet been characterized. A mouse model of PE was constructed through tail vein injection of 1 mg/d phosphatidylserine/dioleoyl-phosphatidycholine suspension. The levels of inflammatory cytokines in the placenta were examined via enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines (TNF-α, IL-6, IFN-γ, and IL-4) and apoptosis markers (Bax, Bcl-2, and caspase-3) were tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot analysis was performed to detect the protein levels of apoptosis markers and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway-related molecules. Here, Pae repressed the inflammatory response in the placenta of PE-like mouse models, as demonstrated by the decreased concentrations and mRNA levels of TNF-α, IL-6, and IFN-γ and the increased concentrations and mRNA levels of IL-4. Apoptosis in the placentas of PE-like mouse models was attenuated by Pae, as manifested by the downregulated mRNA and protein levels of Bax and cleaved-caspase-3 and the upregulated Bcl-2. Administration of Pae inhibited the phosphorylation of JAK2 and STAT3 in the placental tissues of PE mice. The JAK2/STAT3 pathway agonist (SC-39100) reversed Pae treatment-mediated suppression of placental inflammation and apoptosis in PE mice. Overall, Pae inhibits the JAK2/STAT3 signaling pathway to attenuate placental inflammation and apoptosis in PE mice.