Japhet Margaret Oluwatoyin, Bankole Adeogo Timilehin, Omotade Temiloluwa Ifeoluwa, Adeoye Oyelola Eyinade, Famurewa Oladiran, Adesina Simeon K
Department of Microbiology, Faculty of Science, Obafemi Awolowo University, Ile-Ife 220103, Osun State, Nigeria.
Department of Pharmaceutical Sciences, College of Pharmacy, Howard University, Washington, DC 20059, USA.
Methods Protoc. 2025 Jul 17;8(4):81. doi: 10.3390/mps8040081.
Every year, diarrhoea is responsible for >1 million deaths in children with ages from 0 to 5 years, with rotavirus as the leading cause. The regions most affected lack routine rotavirus diagnosis due to high cost, lack of necessary equipment and shortage of trained-personnel for Enzyme-Link-Immunosorbent-Assay (ELISA) and molecular methods. We report the development and evaluation of a cheap, nanoparticle-based immunoassay for routine machine-free rotavirus diagnosis. In this work, optimal conditions for oxidation of cotton swabs and aldehyde production for kit development was confirmed by Fourier-Transform Infrared Spectroscopy (FTIR). Lactoferrin (LF) needed to bind the virus to the cotton swab was immobilised on activated cotton swabs, followed by the capture of commercial rotavirus antigen on LF-immobilised swabs. This was dipped in coloured nanobeads covalently coupled to rotavirus-group-specific monoclonal antibody for visual rotavirus detection. Subsequently, rotavirus detection by nanoassay, commercial ELISA and quantitative reverse transcription PCR were compared using same set of 186 stool samples and subjected to statistical analyses. Optimal oxidisation condition was observed using 48 mg/mL NaIO in 0.1 M sodium acetate buffer at 35 °C for 9 h. Rotavirus detection was confirmed visually by blue colour retention on swabs after several washings. Sensitivity, specificity, positive-predictive-value and negative-predictive-value of ELISA in rotavirus detection were 60%, 84%, 53% and 88%, respectively, while our immunoassay showed performance at 88%, 94%, 82% and 96%. This immunoassay will provide effective rotavirus public health interventions in low-and-middle-income countries with high morbidity/mortality.
每年，腹泻导致0至5岁儿童死亡人数超过100万，其中轮状病毒是主要病因。受影响最严重的地区因成本高昂、缺乏必要设备以及酶联免疫吸附测定法（ELISA）和分子方法的专业人员短缺而无法进行常规轮状病毒诊断。我们报告了一种用于常规无机器轮状病毒诊断的廉价、基于纳米颗粒的免疫测定方法的开发和评估。在这项工作中，通过傅里叶变换红外光谱（FTIR）确定了用于试剂盒开发的棉签氧化和醛生成的最佳条件。将与病毒结合所需的乳铁蛋白（LF）固定在活化的棉签上，随后在固定有LF的棉签上捕获商业轮状病毒抗原。将其浸入与轮状病毒组特异性单克隆抗体共价偶联的彩色纳米珠中以进行可视化轮状病毒检测。随后，使用同一组186份粪便样本比较了纳米测定法、商业ELISA和定量逆转录PCR对轮状病毒的检测，并进行了统计分析。在35°C下，于0.1 M醋酸钠缓冲液中使用48 mg/mL NaIO观察到最佳氧化条件9小时。经过几次洗涤后，通过棉签上保留蓝色在视觉上确认轮状病毒检测。ELISA在轮状病毒检测中的敏感性、特异性、阳性预测值和阴性预测值分别为60%、84%、53%和88%，而我们的免疫测定法的性能分别为88%、94%、82%和96%。这种免疫测定法将在发病率/死亡率高的低收入和中等收入国家提供有效的轮状病毒公共卫生干预措施。
