RESUMO
Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/µL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.
Assuntos
RNA Viral , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Sensibilidade e Especificidade , Humanos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/virologia , RNA Viral/genética , Lactente , Pyrococcus furiosus/genética , Pyrococcus furiosus/isolamento & purificação , Proteínas Argonautas/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Limite de Detecção , Nasofaringe/virologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Pré-EscolarRESUMO
Microspheres (MSs) are ideal candidates as biological scaffolds loading with growth factors or cells for bone tissue engineering to repair irregular alveolar bone defects by minimally invasive injection. However, the high initial burst release of growth factor and low cell attachment limit the application of microspheres. The modification of microspheres often needs expensive experiments facility or complex chemical reactions, which is difficult to achieve and may bring other problems. In this study, a sol-grade nanoclay, laponite XLS is used to modify the surface of MSs to enhance its affinity to either positively or negatively charged proteins and cells without changing the interior structure of the MSs. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is used as a representation of growth factor to check the osteoinduction ability of laponite XLS-modified MSs. By modification, the protein sustained release, cell loading, and osteoinduction ability of MSs are improved. Modified by 1% laponite XLS, the MSs can not only promote osteogenic differentiation of MC3T3-E1 cells by themselves, but also enhance the effect of the rhBMP-2 below the effective dose. Collectively, the study provides an easy and viable method to modify the biological behavior of microspheres for bone tissue regeneration.