基于磁珠和超滤的污水中新冠病毒的富集方法研究

Study on enrichment method of SARS-CoV-2 in sewage based on magnetic beads and ultrafiltration

目的研究一种快速和有效富集污水中新型冠状病毒的方法,为城市污水新冠病毒监测工作和疫情防控提供支持。方法利用液样微生物采集富集仪(LM Capturer-5)对加标液体样本进行富集,利用RT-qPCR检测富集前后新冠病毒核酸浓度,计算回收率,考察该方法对液体样本中新冠病毒的富集效果,比较该方法与聚乙二醇沉淀法对污水中新冠病毒富集后检测结果一致性。结果洁净液体加标样本富集后新冠病毒核酸检测结果Ct值降低3~5,污水加标样本富集后Ct值降低1~4,该方法对污水中新冠病毒的富集回收率为13.50%~44.20%,聚乙二醇沉淀法回收率为8.83%~38.85%,无显著性差异(P>0.05)。应用液样微生物采集富集仪对60份污水样本中新冠病毒进行富集,富集后检测阳性率为63.33%,与标准方法一致,并且与标准方法进行一致性检验,Kappa系数为0.785,有中高度一致性。结论本研究建立的液样微生物采集富集仪法对于污水中的新冠病毒具有较好的富集效果,且操作简便、可自动化,为我国污水中的新冠病毒监测工作提供了多一种技术储备,并且为液体样本中多种病原微生物的富集和检测研究工作提供帮助。

Objective To develop a rapid and effective method to enrich and concentrate novel coronavirus in sewage,and provide the technical support for the monitoring of SARS-CoV-2 in urban sewage and epidemic prevention and control.Methods The liquid sample microbial collection and enrichment instrument(LM Capturer-5)was used to enrich the novel coronavirus in the spiked liquid samples.RT-qPCR was used to detect the concentration of SARS-CoV-2 nucleic acid before and after enrichment.The recovery rate was calculated to assess the enrichment efficiency of the method for the virus in liquid samples.Additionally,the enrichment efficiency of the LM Capturer-5 method was compared with that of the polyethylene glycol precipitation method.Results After enrichment,the Ct values of the clean samples decreased by 3-5,while the Ct values of the sewage samples decreased by 1-4.The recovery rate of the method established in this study for enriching SARSCoV-2 in sewage ranged from 13.50%to 44.20%,while the recovery rate of the polyethylene glycol precipitation method ranged from 8.83%to 38.85%.There was no significant difference between the two methods(P>0.05).The liquid sample microbial collection and enrichment instrument(LM Capturer-5)was used to enrich SARS-CoV-2 in 60 wastewater samples.After enrichment,the positive rate detected was 63.33%,which was consistent with the standard method.The spolyethylene glycol precipitation method was used for consistency test as the standard method,and the Kappa coefficient was 0.785,indicating medium to high consistency.Conclusion This method has a good enrichment effect for SARS-CoV-2 in sewage,and is simple to operate and can be automated.It provides an additional technical reserves for the monitoring of SARS-CoV-2 in the sewage in China,and provides help for the enrichment and detection of multiple pathogenic microorganisms in liquid samples.