Yang Jiayi, Liu Mingwei, Li Huijie, Gao Yunhua, Dong Lianhua
Center for Advanced Measurement Science, National Institute of Metrology, Beijing, 100029, China.
Shenzhen Institute for Technology Innovation, NIM, Shenzhen, 518132, China.
Anal Bioanal Chem. 2025 May;417(12):2513-2523. doi: 10.1007/s00216-024-05690-2. Epub 2024 Dec 16.
The rotavirus (RV) is the predominant causative pathogen associated with acute gastroenteritis in children aged below 5 years, leading to an annual mortality rate of 200,000 infants globally. Despite the development of a vaccine, it exacerbates the medical burden around the world. Here, we have developed reverse transcription digital PCR (RT-dPCR) methods for precise and absolute quantification of nucleic acid in rotavirus G3P8 and G9P8. The pseudovirus reference material (RM) contained the RNA fragment encoding VP4 and VP7. The assigned values with expanded uncertainty were determined as (2432 ± 510) copies/μL and (3406 ± 613) copies/μL. The RM and RT-dPCR methods were employed to validate various digital platforms, revealing the inadequate performance of platform III, which could potentially result in "false-negative" outcomes. The application of RT-dPCR techniques and pseudovirus RM confers advantages in the diagnosis of RV-induced diseases, thereby enhancing the survival rate of young children suffering from acute gastroenteritis.
轮状病毒（RV）是5岁以下儿童急性胃肠炎的主要致病病原体，全球每年导致20万婴儿死亡。尽管已研发出疫苗，但它仍加剧了全球的医疗负担。在此，我们开发了逆转录数字PCR（RT-dPCR）方法，用于精确和绝对定量轮状病毒G3P8和G9P8中的核酸。伪病毒参考物质（RM）包含编码VP4和VP7的RNA片段。扩展不确定度下的赋值确定为（2432±510）拷贝/μL和（3406±613）拷贝/μL。使用RM和RT-dPCR方法验证了各种数字平台，发现平台III性能不足，可能导致“假阴性”结果。RT-dPCR技术和伪病毒RM的应用在RV引起疾病的诊断中具有优势，从而提高了患急性胃肠炎幼儿的存活率。
