Yamazaki Yasuko, Tanaka Riku, Castillo Gladys, Macalanda Adrian Miki C, Talactac Melbourne R, Yamazaki Wataru
Center for Southeast Asian Studies, Kyoto University, 46 Shimoadachi-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Center for Southeast Asian Studies, Kyoto University, 46 Shimoadachi-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan; College of Veterinary Medicine and Biomedical Sciences, Cavite State University, Indang, Cavite 4122, Philippines.
J Virol Methods. 2025 Jan;331:115059. doi: 10.1016/j.jviromet.2024.115059. Epub 2024 Nov 1.
When diagnosing viral infections in humans and animals, the presence of virus in a sample in trace amounts that are below the analytical sensitivity of the detection system may cause false negative results and inaccurate diagnosis. We previously reported the development of a simple virion concentration technique using 12 ml large-volume samples that can dramatically improve diagnostic sensitivity by increasing analytical sensitivity by 100-fold over conventional methods. The present study was conducted to further improve the simplicity and versatility of this method. We constructed a simple and highly sensitive method for the detection of SARS-CoV-2 in human saliva after concentration using a magnetic nanoparticle conjugated with polyethylene glycol (PEG).
Performance of the method was evaluated by comparing a combination of automated nucleic acid extraction and RT-qPCR or triplex RT-LAM detection in a spiked sample of 20 ml saliva collected from healthy humans. The method theoretically achieved 300-fold concentration of spiked SARS-CoV-2 in saliva, enabling 10- to 1000-fold higher analytical sensitivity for detection compared to conventional RNA extraction methods.
This newly developed method allows for easy and reliable concentration of the virion in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. Further, the method allows for easy and reliable enrichment of the virus in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. This method is easily used for highly sensitive virus detection from a variety of human oral fluid samples and may also be applied to rapid and labor-saving screening tests by pooling a large number of samples.
在诊断人类和动物的病毒感染时，样本中病毒含量处于检测系统分析灵敏度以下的痕量水平，可能会导致假阴性结果和诊断不准确。我们之前报道了一种使用12毫升大体积样本的简单病毒粒子浓缩技术，与传统方法相比，该技术可将分析灵敏度提高100倍，从而显著提高诊断灵敏度。本研究旨在进一步提高该方法的简便性和通用性。我们构建了一种简单且高度灵敏的方法，用于在使用与聚乙二醇（PEG）偶联的磁性纳米颗粒浓缩后检测人类唾液中的SARS-CoV-2。
通过在从健康人采集的20毫升唾液加标样本中比较自动核酸提取与RT-qPCR或三重RT-LAM检测的组合，对该方法的性能进行了评估。该方法理论上可实现唾液中加标SARS-CoV-2的300倍浓缩，与传统RNA提取方法相比，检测的分析灵敏度提高了10至1000倍。
这种新开发的方法能够在不到60分钟的时间内轻松、可靠地浓缩病毒粒子，提高SARS-CoV-2检测的分析灵敏度。此外，该方法能够在不到60分钟的时间内轻松、可靠地富集病毒，提高SARS-CoV-2检测的分析灵敏度。该方法易于用于从各种人类口腔液样本中进行高灵敏度病毒检测，也可通过合并大量样本应用于快速且省力的筛查测试。
