(24,25) These compounds can influence the efficiency of RT-qPCR amplification and the subsequent detection and quantification. (21,22) In addition, RT-qPCR amplification may be affected by wastewater samples that often contain inhibitors, such as pharmaceuticals, personal care products, household detergents, industrial effluents, and metals. (19) Variations in protocols, reagents, sample quality, instruments (e.g., ultraviolet spectrophotometers, PCR platforms, and sample homogenizers), data analysis, software, and interpretation within and across laboratories can lead to the inaccurate and unreliable quantification of SARS-CoV-2 RNA in wastewater.
CUSTOM QPCR AND DPCR PBES MICROSYNTH 2019 SERIES
(6,21,22) However, several factors can result in quantification bias, as RT-qPCR assays rely on calibration (i.e., standard) curves constructed using a dilution series of the defined number of target molecules (i.e., plasmid DNA, PCR amplicons, synthetic nucleic acid, genomic DNA, and cDNA), typically having five or six dilution points (10-fold each) with at least three replicates per point. Reverse transcription-quantitative PCR (RT-qPCR) has been employed as a gold standard method for detecting and quantifying SARS-CoV-2 RNA in both clinical and environmental samples. (19−21) Trace detection, the reliable detection of minuscule amounts of RNA in wastewater, is crucial for regions with few COVID-19 cases and for jurisdictions in which elimination is a management strategy. (6,8,18) These procedures require careful optimization to maximize their sensitivity for detecting traces of SARS-CoV-2 in wastewater, especially in the absence of standard methods. (6−17) This approach uses a series of complex environmental microbiology procedures ranging from wastewater sampling to the detection and quantification of viral RNA primarily via polymerase chain reaction (PCR)-based or loop-mediated isothermal amplification (LAMP) assays. (1−5) Wastewater surveillance of SARS-CoV-2 RNA has been employed in at least 55 countries to monitor the presence and support management of COVID-19 in various communities. The emergence and spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have highlighted the utility of wastewater surveillance for early identification of infection in communities and guidance of public health measures to reduce transmission risks. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR. The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029 N2, p = 0.0003) and pellet (N1, p = 0.0015 N2, p = 0.0067) samples. During sample processing, the endogenous ( n = 96) and exogenous ( n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The ALODs using RT-dPCR were approximately 2–5 times lower than those using RT-qPCR. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays.
0 kommentar(er)
