建立使用非同源刺突对照检测废水中登革病毒的定量和回收率方法。

Establishment of quantitative and recovery method for detection of dengue virus in wastewater with noncognate spike control.

作者信息

Chen Kuan-Wei, Chen Tzu-Yi, Wang Sin-Tian, Hou Ting-Yu, Wang Shainn-Wei, Young Kung-Chia

机构信息

Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

出版信息

J Virol Methods. 2023 Apr;314:114687. doi: 10.1016/j.jviromet.2023.114687. Epub 2023 Feb 1.

DOI:10.1016/j.jviromet.2023.114687

PMID:36736703

Abstract

Wastewater-based epidemiology (WBE) represents an efficient approach for public pathogen surveillance as it provides early warning of disease outbreaks; however, it has not yet been applied to dengue virus (DENV), which might cause endemics via mosquito spread. In this study, a working platform was established to provide direct virus recovery and qPCR quantification from wastewater samples that were artificially loaded with target DENV serotypes I to IV and noncognate spike control viral particles. The results showed qPCR efficiencies of 91.2 %, 94.8 %, 92.6 % and 88.7 % for DENV I, II, III, and IV, respectively, and a broad working range over 6 orders of magnitude using the preferred primer sets. Next, the results revealed that the ultrafiltration method was superior to the skimmed milk flocculation method for recovering either DENV or control viral particles from wastewater. Finally, DENV-2 was loaded simultaneously with the noncognate spike control and could be recovered at comparable levels either in PBS or in wastewater, indicating the applicability of noncognate spike control particles to reflect the efficiency of experimental steps. In conclusion, our data suggest that DENV particles in wastewater could be recovered and quantitatively detected in absolute amounts, indicating the feasibility of DENV surveillance using the WBE approach.

摘要

基于废水的流行病学（WBE）是一种有效的公共病原体监测方法，因为它能对疾病暴发提供早期预警；然而，它尚未应用于登革病毒（DENV），登革病毒可能通过蚊子传播引发地方病。在本研究中，建立了一个工作平台，用于从人工接种了目标DENV血清型I至IV以及非同源刺突对照病毒颗粒的废水样本中直接回收病毒并进行qPCR定量。结果显示，DENV I、II、III和IV的qPCR效率分别为91.2%、94.8%、92.6%和88.7%，使用优选引物组时工作范围跨越6个数量级。接下来，结果表明，在从废水中回收DENV或对照病毒颗粒方面，超滤法优于脱脂牛奶絮凝法。最后，DENV - 2与非同源刺突对照同时接种，在PBS或废水中均可在相当水平上回收，这表明非同源刺突对照颗粒可用于反映实验步骤的效率。总之，我们的数据表明，废水中的DENV颗粒可以被回收并进行绝对量的定量检测，这表明使用WBE方法监测DENV是可行的。