RNasin和gp32用于城市污水中SARS-CoV-2 PCR检测的效能评估

doi:10.12183/j.scjpm.2026.0827

摘要/Abstract

摘要： 目的建立一种适用于城市污水中新型冠状病毒（SARS-CoV-2）监测的双靶标数字PCR（dPCR）检测方法,并评估核糖核酸酶抑制剂（RNasin）和T4噬菌体基因32编码蛋白（gp32）对缓解污水样本中PCR抑制效应的作用。方法合成靶向SARS-CoV-2 ORF1ab和N基因的引物探针。采用梯度稀释的SARS-CoV-2 RNA标准品,评估该dPCR方法的线性范围、精密度、空白限（LoB）和检出限（LoD）。使用SARS-CoV-2假病毒标准品配制城市污水模拟样品,优化RNasin工作浓度,并系统比较添加RNasin或gp32前后,dPCR和实时定量PCR（qPCR）的检测效能差异。结果该dPCR方法检测ORF1ab与N基因的线性关系良好（R²分别为0.999 9和0.999 8）;对高、中浓度样本重复性好（均CV<10%）,低浓度样本CV则大于10%。ORF1ab和N基因LoB均为0 copies/μL,LoD分别为0.89 copies/μL和0.78 copies/μL。RNasin的最佳工作浓度为4.8 U/μL。在污水模拟样品检测中,dPCR的检出浓度显著高于qPCR（P<0.05）。qPCR体系添加RNasin或gp32后,模拟样品的病毒检出浓度均显著提升（P<0.05）。dPCR体系中,添加RNasin对检测结果无显著影响（P>0.05）;添加gp32则在部分浓度下对扩增反应表现出抑制作用（P<0.05）。结论本研究成功建立了一种适用于城市污水SARS-CoV-2监测的双靶标dPCR检测方法。与qPCR相比,dPCR方法对城市污水基质中的抑制物具有更强的耐受性。在对污水样本进行qPCR检测时,添加RNasin或gp32可有效缓解扩增抑制,提高检测灵敏度;而dPCR检测则无需添加此类增强剂,添加gp32反而可能产生负面影响。

关键词: 核糖核酸酶抑制剂, T4噬菌体基因32编码蛋白, 数字PCR, 实时定量PCR, 新型冠状病毒

中图分类号:

R373.9

狄宇, 许文倩, 贺靖, 陈静芳, 杨学文. RNasin和gp32用于城市污水中SARS-CoV-2 PCR检测的效能评估[J]. 华南预防医学, 2026, 52(7): 827-832.

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