背景:许多国家已将COVID-19大流行的废水监测应用于其国家公共卫生监测措施。检测废水中SARS-CoV-2的最常用方法是定量逆转录酶-聚合酶链反应(RT-qPCR)和逆转录酶-液滴数字聚合酶链反应(RT-ddPCR)。以前的比较研究产生了相互矛盾的结果,因此,需要对这个问题进行更多的研究。
目的:本研究旨在比较RT-qPCR和RT-ddPCR检测废水中SARS-CoV-2的效果。它还旨在调查分析管道变化的影响,包括RNA提取试剂盒,RT-PCR试剂盒,和靶基因检测,关于结果。另一个目的是找到一种用于低资源设置的检测方法。
方法:我们比较了2种RT-qPCR试剂盒,TaqManRT-qPCR和定量RT-qPCR,和基于灵敏度的RT-ddPCR,阳性率,可变性,废水中SARS-CoV-2基因拷贝数与COVID-19发病率的相关性。此外,我们比较了两种RNA提取方法,基于柱状和磁珠。此外,我们评估了两种RT-qPCR的靶基因检测方法,N1和N2,以及用于ddPCRN1和E的2个靶基因测定。基于逆转录链入侵的扩增(RT-SIBA)用于定性检测废水中的SARS-CoV-2。
结果:我们的结果表明,检测废水中SARS-CoV-2的最灵敏方法是RT-ddPCR。阳性率最高(26/30),检测限最低(0.06个基因拷贝/μL)。然而,利用TaqManRT-qPCR技术,获得了废水中COVID-19发病率与SARS-CoV-2基因拷贝数的最佳相关性(相关系数[CC]=0.697,P<.001)。我们发现TaqManRT-qPCR试剂盒和QuantiTectRT-qPCR试剂盒之间的灵敏度存在显着差异,第一个具有比后者明显更低的检测限和更高的阳性率。此外,N1靶基因检测对两种RT-qPCR试剂盒最敏感,而使用RT-ddPCR在基因靶标之间没有发现显着差异。此外,当使用TaqManRT-qPCR试剂盒时,使用不同的RNA提取试剂盒会影响结果。RT-SIBA能够检测废水中的SARS-CoV-2RNA。
结论:作为我们的研究,以及以前的大多数研究,显示RT-ddPCR比RT-qPCR更敏感,应考虑将其用于SARS-CoV-2的废水监测,特别是如果在人群中传播的SARS-CoV-2的数量很低。所有的分析步骤必须优化废水监测,因为我们的研究表明,所有的分析步骤,包括RNA提取的相容性,RT-PCR试剂盒,和靶基因测定影响结果。此外,我们的研究表明,如果定性结果足够,RT-SIBA可用于检测废水中的SARS-CoV-2。
Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required.
This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings.
We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively.
Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater.
As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.