Reverse Transcriptase Droplet Digital PCR Research Articles

Analytical validation of a semi-automated methodology for quantitative measurement of SARS-CoV-2 RNA in wastewater collected in northern New England.

Wastewater-based epidemiology (WBE) can be used to monitor the community presence of infectious disease pathogens of public health concern such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral nucleic acid has been detected in the stool of SARS-CoV-2-infected individuals. Asymptomatic SARS-CoV-2 infections make community monitoring difficult without extensive and continuous population screening. In this study, we validated a procedure that includes manual pre-processing, automated SARS-CoV-2 RNA extraction and detection workflows using both reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR). Genomic RNA and calibration materials were used to create known concentrations of viral material to determine the linearity, accuracy, and precision of the wastewater extraction and SARS-CoV-2 RNA detection. Both RT-qPCR and RT-ddPCR perform similarly in all the validation experiments, with a limit of detection of 50 copies/mL. A wastewater sample from a care facility with a known outbreak was assessed for viral content in replicate, and we showed consistent results across both assays. Finally, in a 2-week survey of two New Hampshire cities, we assessed the suitability of our methods for daily surveillance. This paper describes the technical validation of a molecular assay that can be used for long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.IMPORTANCEThis paper describes the technical validation of a molecular assay that can be used for the long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

Wastewater-Based SARS-CoV-2 Surveillance in Northern New England.

ABSTRACTSARS-CoV-2 viral RNA is shed in the stool of 55–70% of infected individuals and can be detected in community wastewater up to 7 days before people present with COVID-19 symptoms. The detection of SARS-CoV-2 RNA in wastewater may serve as a lead indicator of increased community transmission. Here, we monitored viral concentrations in samples collected from nine municipal wastewater facilities in New Hampshire (NH) and Vermont (VT).Twenty-four-h composite primary influent wastewater samples were collected from nine municipal wastewater treatment facilities twice per week for 5 months (late September 2020 to early February 2021). Wastewater was centrifuged for 30 min at 4600 × g, then the supernatant was frozen until further analysis. Once thawed, samples were concentrated, extracted, and tested for SARS-CoV-2 RNA using reverse transcriptase-quantitative PCR (RT-qPCR) and reverse transcriptase-droplet digital PCR (RT-ddPCR) detection methods. Active case counts for each municipality were tracked from the NH and VT state COVID-19 dashboards. We received a total of 283 wastewater samples from all sites during the study period. Viral RNA was detected in 175/283 (61.8%) samples using RT-qPCR and in 195/283 (68.9%) samples using RT-ddPCR. All nine sites showed positivity in the wastewater, with 8/9 (88.8%) sites having over 50% of their samples test positive over the course of the study. Larger municipalities, such as Nashua, Concord, and Lebanon, NH, showed that SARS-CoV-2 positivity in the wastewater can precede spikes in active COVID-19 case counts by as much as 7 days. Smaller municipalities, such as Woodsville, NH and Hartford, VT, showed sporadic SARS-COV-2 detection and did not always precede a rise in active case counts. We detected SARS-CoV-2 RNA in samples from all 9 municipalities tested, including cities and small towns within this region, and showed wastewater positivity as an early indicator of active case count increases in some regions. Some of the smaller rural municipalities with low case counts may require more frequent sampling to detect SARS-CoV-2 in wastewater before a case surge. With timely collection and analysis of wastewater samples, a community could potentially respond to results by increasing public health initiatives, such as tightening mask mandates and banning large indoor gatherings, to mitigate community transmission of SARS-CoV-2.IMPORTANCE Despite vaccination efforts, the delta and omicron variants of SARS-CoV-2 have caused global surges of COVID-19. As the COVID-19 pandemic continues, it is important to find new ways of tracking early signs of SARS-CoV-2 outbreaks. The manuscript outlines how to collect wastewater from treatment facilities, concentrate the virus in a dilute wastewater sample, and detect it using two sensitive PCR-based methods. It also describes important trends in SARS-CoV-2 concentration in wastewater of a rural region of the United States from Fall 2020 – Winter 2021 and demonstrates the utility of wastewater monitoring as a leading indicator of active SARS-CoV-2 cases. Monitoring changes in concentration of SARS-CoV-2 virus in wastewater may offer an early indicator of increased case counts and enable appropriate public health actions to be taken.

Improving correlation of wastewater SARS-CoV-2 gene copy numbers with COVID-19 public health cases using readily available biomarkers.

The COVID-19 pandemic has highlighted the potential role that wastewater-based epidemiology can play in assessing aggregate community health. However, efforts to translate SARS-CoV-2 gene copy numbers obtained from wastewater samples into meaningful community health indicators are nascent. In this study, SARS-CoV-2 nucleocapsid (N) genes (N1 and N2) were quantified weekly using reverse transcriptase droplet digital PCR from two municipal wastewater treatment plants for seven months. Four biomarkers (ammonium, biological oxygen demand (BOD), creatinine, and human mitochondrial gene NADH dehydrogenase subunit 5) were quantified and used to normalize SARS-CoV-2 gene copy numbers. These were correlated to daily new case data and one-, two-, and three-week cumulative case data. Over the course of the study, the strongest correlations were observed with a one-day case data lag. However, early measurements were strongly correlated with a five-day case data lag. This indicates that in the early stages of the pandemic, the wastewater samples may have indicated active COVID-19 cases before clinical indications. Mitochondrial and creatinine normalization methods showed the strongest correlations throughout the study, indicating that human-specific biomarkers were better at normalizing wastewater data than ammonium or BOD. Granger causality tests supported this observation and showed that gene copies in wastewater could be predictive of new cases in a sewershed.

The effect of influenza A (H1N1) pdm09 virus infection on cytokine production and gene expression in BV2 microglial cells

Acute influenza infection has been reported to be associated with neurological symptoms such as influenza-associated encephalopathy (IAE). Although the pathophysiology of this condition remain unclear, neuroinflammation and associated alterations in the central nervous system (CNS) are usually induced. Microglia (MGs), CNS-resident macrophages, are generally the first cells to be activated in response to brain infection or damage. We performed reverse transcriptase droplet digital PCR (RT-ddPCR) and luminex assays to investigate virus proliferation and immune reactions in BV2 MGs infected with influenza A(H1N1)pdm09 virus. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to investigate the dynamic change in the protein expression profile in BV2 MGs to gain insight into the CNS response to influenza A (H1N1) pdm09 infection. Our results showed that the influenza A(H1N1)pdm09 virus was replicative and productive in BV2 MG cells, which produced cytokines such as interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. The expression of osteopontin (OPN) in the influenza A (H1N1) pdm09-infected BV2 MGs was upregulated at 16 and 32 h post-infection (hpi) compared to that in the control group, resulting in aggravated brain damage and inflammation. Our study indicates that OPN signalling might provide new insights into the treatment of CNS injury and neurodegenerative diseases in IAE.

Endogenous human retrovirus-K is not increased in the affected tissues of Japanese ALS patients

Activation of human endogenous retrovirus-K (HERV-K) is one of the proposed risk factors for amyotrophic lateral sclerosis (ALS). The HERV-K envelope protein has been reported to show neurotoxicity, and development of therapy with reverse transcriptase inhibitors is being investigated. On the other hand, some reports have failed to show HERV-K activation in ALS. In this study, we analyzed the expression of HERV-K mRNA in the motor cortex and spinal cord of 15 Japanese patients with sporadic ALS and 19 controls using reverse transcriptase droplet digital PCR. This revealed no significant increase of HERV-K expression in ALS-affected tissues, suggesting that the association between ALS and HERV-K remains questionable.

Development and Validation of a Gamma Globin RT-Ddpcr Exploratory Biomarker for Sickle Cell Phase 1 Clinical Trial Fis 002-2020

FTX-6058, a selective and potent binder of embryonic ectoderm development protein (EED), is being investigated in Sickle Cell Disease (SCD). In-vitro and pre-clinical Townes mouse studies show FTX-6058 upregulates expression of the gamma globulin gene leading to increased levels of fetal hemoglobin (HbF). In human clinical trials, as an early indicator of FTX-6058 induced gamma globulin expression, a reverse transcriptase droplet digital PCR (RT-ddPCR) assay was developed and validated by Precision for Medicine. Versus RT-qPCR, RT-ddPCR gives absolute copy number, does not require a standard curve, and is more precise and reproducible for low expressed targets 1,2. Precision for Medicine developed and validated RT-ddPCR assays for the target globin genes α, β, and γ and for the housekeeping genes TFRC and OAZ1. RNA isolated from 3 healthy and 3 SCD affected individuals was used for development and validation of the assay. The upper and lower limits of quantification were 120,000 and 10.5 copies/20μL reaction, respectively. For samples with >50 copies/20μL reaction, the CV for technical replicates was <20% for all transcripts. Furthermore, Precision for Medicine demonstrated SCD affected individuals express approximately 15-fold more γ globulin versus non affected individuals. Fulcrum utilized the globin RT-ddPCR assay validated by Precision for Medicine in the FTX-6058 phase 1 clinical trial FIS 002-2020.1 Zhao Y, Xia Q, Yin Y, Wang Z (2016), Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri. PLoS ONE 11(7): e0159004. doi:10.1371/journal.pone.01590042 Taylor SC, Laperriere G, Germain H (2017), Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports 7(2409): DOI:10.1038/s41598-017-02217-x DisclosuresRoth: Fulcrum Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Yang: Fulcrum Therapeutics, Inc.: Consultancy. Tsavachidou: Fulcrum Therapeutics, Inc.: Consultancy. Davis: Fulcrum Therapeutics, Inc.: Consultancy.

Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study

During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

Development and application of a reverse transcriptase droplet digital PCR (RT-ddPCR) for sensitive and rapid detection of Japanese encephalitis virus

Japanese encephalitis (JE) is one of the most common zoonoses caused by Japanese encephalitis virus (JEV). Droplet digital PCR (ddPCR) is a novel sensitive, accurate method that enables absolute quantitation without the need for calibration curves. The aim of this study was to develop a RT-ddPCR method to detect JEV, and to compare its sensitivity with real-time TaqMan RT-PCR by analysis of clinical samples. The methods of JEV real-time RT-PCR and RT-ddPCR were established and optimal reaction conditions were confirmed. Each method was evaluated for linearity, limit of detection and specificity. A total of 103 porcine samples were analysed by both methods and the detection rate was calculated. Both methods showed a high degree of linearity and positive correlation for standards (R2≥0.999). The assays indicated that the detection limit for RT-ddPCR was approximately 2 copies/20μL well, a 100-fold greater sensitivity than TaqMan real-time RT-PCR. The detection results for clinical samples showed that the positive detection rate of RT-ddPCR (27.2%) was higher than that of TaqMan real-time RT-PCR (16.5%). The cross-reaction was performed with other porcine pathogens, and negative amplification of the cross-reaction assay demonstrated the high specificity of this method. The novel JEV RT-ddPCR assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swineries and would be beneficial for public health security.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.

BackgroundDetection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR.ResultsTransfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR.ConclusionsThis study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-014-0042-6) contains supplementary material, which is available to authorized users.