High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v1
High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v1
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使用数字 RT-PCR v1 对沉降固体中的 SARS-COV-2、PMMOV 和 BCoV 进行高通量定量
DOI：
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Alexandria B Boehm
中科院分区：
文献类型：
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作者：
Aaron Topol (Verily Life Sciences);marlene.wolfe not provided;Brad White (Verily Life Sciences);K. Wigginton;Alexandria B Boehm
本过程说明描述了使用三重逆转录酶液滴数字聚合酶链反应定量分析SARS-CoV-2核酸的步骤靶向N基因的RT-ddPCR测定，S基因和ORF 1a，以及针对群体水平SARS-CoV的固体废水样品中提取和纯化的RNA样品中的牛冠状病毒疫苗（BCoV）和辣椒温和斑驳病毒（PMMoV）的双重检测-2社区监控RT-ddPCR是常规RT-PCR工作流程的修改版本，其涉及在热循环之前将反应混合物分离成许多分区（~ 20，000），这允许靶RNA分子的直接绝对定量。该方案使用该方案提取的RNA：高回收量RNA提取和沉淀固体的PCR抑制剂去除用于SARS-CoV-2 RNA的废水监测。该RNA是从进行预分析步骤的样品中产生的：用于SARS-CoV-2 RNA分析的废水沉降固体的高通量预分析处理。该方案描述了2个单独的PCR反应，一个含有靶向三种SARS-CoV-2靶标的引物/探针混合物，一个含有靶向BCoV和PMMoV的引物/探针混合物。在核酸提取前将BCoV加标到样品中，并用作过程控制以及PCR抑制的指示剂。PMMoV是一种包膜病毒，在人粪便中含量丰富，可作为数据标准化的内源性对照。PMMoV RNA在废水样品中以如此高的水平丰富，以至于样品在定量之前必须稀释100倍。该测定的读数是提取的RNA样品中每个靶标的浓度（拷贝/uL）。适用范围本过程说明适用于使用Bio-Rad AutoDG Droplet Digital PCR系统（由AutoDG Automated Droplet Generator和QX 200液滴读取器组成），通过ddPCR定量分析固体废水样品中SARS-CoV-2 RNA的核酸。
This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.