Teng Yongyong, Mo Qiuhua, Wang Qi, Tang Minghui, Zhao Dejian, Tan Hua, Tu Chengning, Yang Ze, Chen Qing, Sun Hong
Department of Epidemiology, Southern Medical University, Guangzhou, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2013 May;33(5):724-7.
To establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.
Primers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.
The 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.
The established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.
建立一种基于同源标签辅助非二聚体系统（HAND）的一步四重逆转录聚合酶链反应（RT-PCR）方法，用于同时检测轮状病毒、星状病毒、诺如病毒和札如病毒这4种腹泻病毒。
根据这4种病毒的保守基因组序列设计引物，并在5′端添加同源尾序列。通过优化通用标签引物和基因组特异性同源尾引物的浓度等PCR参数，构建多重RT-PCR体系。系统评价该方法的特异性、稳定性和敏感性。
成功建立了基于HAND系统的4种多重RT-PCR方法。特异性分析表明，这4种腹泻病毒之间无交叉反应。敏感性分析表明，轮状病毒、星状病毒、诺如病毒和札如病毒的检测限分别为每个反应48、1.92、9.6和48 pg。
所建立的基于HAND系统的多重RT-PCR检测方法简便、快速、特异、灵敏、稳定，能低成本检测4种常见腹泻病毒，适用于普通医学实验室应用。
