Droplet Digital RT-PCR Research Articles

Droplet Digital RT-PCR (dd RT-PCR) Detection of SARS-CoV-2 in Honey Bees and Honey Collected in Apiaries across the Campania Region.

Coronaviruses (CoVs), a subfamily of Orthocoronavirinae, are viruses that sometimes present a zoonotic character. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the recent outbreak of COVID-19, which, since its outbreak in 2019, has caused about 774,593,066 confirmed cases and 7,028,881 deaths. Aereosols are the main route of transmission among people; however, viral droplets can contaminate surfaces and fomites as well as particulate matter (PM) in suspensions of natural and human origin. Honey bees are well known bioindicators of the presence of pollutants and PMs in the environment as they can collect a great variety of substances during their foraging activities. The aim of this study was to evaluate the possible role of honey bees as bioindicators of the prevalence SARS-CoV-2. In this regard, 91 samples of honey bees and 6 of honey were collected from different apiaries of Campania region (Southern Italy) in four time periods from September 2020 to June 2022 and were analyzed with Droplet Digital RT-PCR for SARS-CoV-2 target genes Orf1b and N. The screening revealed the presence of SARS-CoV-2 in 12/91 in honey bee samples and in 2/6 honey samples. These results suggest that honey bees could also be used as indicators of outbreaks of airborne pathogens such as SARS-CoV-2.

Assessing the efficacy of different bead-based assays in capturing hepatitis E virus

Hepatitis E virus (HEV) generally causes acute liver infection in humans and its transmission could be waterborne, foodborne, bloodborne, or zoonotic. To date, there is no standard method for the detection of HEV from food and environmental samples. Herein, we explored the possibility of using magnetic beads for the capture and detection of HEV. For this purpose, we employed Dynabeads M-270 Epoxy magnetic beads, coated with different monoclonal antibodies (mAbs) against HEV capsid protein, and the Nanotrap Microbiome A Particle magnetic beads, which are coated with chemical affinity baits, to capture HEV-3 particles in suspension. Viral RNA was extracted by heat-shock or QIAamp viral RNA kit and subjected to quantification using digital-droplet RT-PCR (ddRT-PCR). We demonstrated that the mAb-coupled Dynabeads and the Nanotrap particles, both were able to successfully capture HEV-3. The latter, however had lower limit of detection (<140gc compared with <1400 gc) and significantly higher extraction efficiency in comparison to the mAb-coupled Dynabeads (41.1% vs 8.8%). We have also observed that viral RNA extraction by heat-shock is less efficient compared to using highly denaturing reagents in QIAmp viral RNA extraction kit. As such, magnetic beads have the potential to be used to capture HEV virions for research and surveillance purposes.

Selective Globin Gene Regulation By the Non-Canonical Baf Chromatin Remodeling Complex

The regulation of the beta-type globin genes has served as a powerful model for developmental transcriptional control, with humans expressing embryonic (HBE), two fetal (HBG1/2) and two adult (HBB and HBD) versions at the corresponding developmental stages. Reactivation of HBG in adult erythroid cells is of benefit to patients with sickle cell disease and β-thalassemia. While past efforts focused on the identification of repressors of the HBG genes, including BCL11A, ZBTB7A, and NFI-A/X, to date no transcriptional activators have been identified with selectivity for any of the globin genes. Using a CRISPR-Cas9 screen in the HUDEP2 erythroid cell line, we recently identified BRG1/SMARCA4 as a potential activator of HBG production. BRG1 is a core ATPase component of the multi-subunit BAF (BRG1/BRM-associated factor) complex; different subtypes of this large chromatin remodeler family are specific to particular cell types or developmental stages. BRG1 was previously implicated in globin gene regulation but its molecular composition and whether it has selectivity for any of the genes in the cluster is unknown. Notably, we found that loss of BRG1 in both HUDEP2 and primary erythroid cells impairs transcription of HBG to a much greater extent than that of HBB. Here we functionally dissected BAF subunits to decipher composition of the relevant BAF complex and the mechanism for this unexpected HBG gene selectivity. We used CRISPR-Cas9 in HUDEP2 cells and human primary erythroid cultures to test the requirement for 14 different BAF subunits for selective HBG transcription. Among these, the BAF60A component emerged as critically required for the normal production of HBG but not HBB in HUDEP2 cells. Depletion of BAF60A in human primary erythroid cultures reduced HBG by up to 80% with no reduction in HBB as assessed by western blot, flow cytometry, HPLC, RT-PCR, and droplet digital RT-PCR. We also observed a reduction in HBD expression, suggesting that BAF60A functions in a gene selective manner but not necessarily favoring the fetal over the adult-type globin genes. α-type globin genes were unaffected by BAF60A loss, further demonstrating the gene selectivity of BAF60A action. RNA-seq of BAF60A-targeted cells showed that the most downregulated transcripts were from the two HBG genes and the BGLT3 long non-coding RNA, which typically parallels HBG in expression. These results revealed an unexpected gene selectivity of the BAF complex, conveyed at least in part by BAF60A. To assess global chromatin accessibility upon BAF60A loss in primary erythroid cells we utilized ATAC-seq. Among ~132,000 accessibility peaks, ~10,000 were reduced in BAF60A targeted cells. Interestingly, we saw a strong decrease in chromatin accessibility at the HBG and HBD genes with minimal to no effect on the HBB or α-globin genes, in line with gene expression data, again reflecting the gene specificity of BAF60A. BAF60A can be found in different classes of BAF complexes, but is absolutely required for the non-canonical BAF (ncBAF) subtype. ncBAF is a relatively recently described BAF complex that is distinguished from other classes of BAF complexes by the presence of the bromodomain protein BRD9. Therefore, we hypothesized that ncBAF is the relevant BAF complex required for selective HBG transcription. Genetic or pharmacologic disruption of BRD9 in HUDEP2 cells recapitulated the BAF60A phenotype, with decreased HBG and HBD expression but sparing of the HBB and α-globin genes. This is the first study of the role of the non-canonical BAF complex in hemoglobin gene regulation. Our results demonstrate remarkable selectivity of this subtype of a broadly active chromatin remodeling complex for specific hemoglobin genes in adult erythroid cells. Further work on the ncBAF complex will focus on rerouting the BAF complex to promote the transcription of HBG and HBD in hemoglobinopathies.

Human norovirus (HuNoV) GII RNA in wastewater solids at 145 United States wastewater treatment plants: comparison to positivity rates of clinical specimens and modeled estimates of HuNoV GII shedders

BackgroundHuman norovirus (HuNoV) is a leading cause of disease globally, yet actual incidence is unknown. HuNoV infections are not reportable in the United States, and surveillance is limited to tracking severe illnesses or outbreaks. Wastewater monitoring for HuNoV has been done previously and results indicate it is present in wastewater influent and concentrations are associated with HuNoV infections in the communities contributing to wastewater. However, work has mostly been limited to monthly samples of liquid wastewater at one or a few wastewater treatment plants (WWTPs).ObjectiveThe objectives of this study are to investigate whether HuNoV GII preferentially adsorbs to wastewater solids, investigate concentrations of HuNoV GII in wastewater solids in wastewater treatment plants across the county, and explore how those relate to clinical measures of disease occurrence. In addition, we aim to develop and apply a mass-balance model that predicts the fraction of individuals shedding HuNoV in their stool based on measured concentrations in wastewater solids.MethodsWe measured HuNoV GII RNA in matched wastewater solids and liquid influent in 7 samples from a WWTP. We also applied the HuNoV GII assay to measure viral RNA in over 6000 wastewater solids samples from 145 WWTPs from across the United States daily to three times per week for up to five months. Measurements were made using digital droplet RT-PCR.ResultsHuNoV GII RNA preferentially adsorbs to wastewater solids where it is present at 1000 times the concentration in influent. Concentrations of HuNoV GII RNA correlate positively with clinical HuNoV positivity rates. Model output of the fraction of individuals shedding HuNoV is variable and uncertain, but consistent with indirect estimates of symptomatic HuNoV infections in the United States.Impact statementIllness caused by HuNoV is not reportable in the United States so there is limited data on disease occurrence. Wastewater monitoring can provide information about the community spread of HuNoV. Data from wastewater can be available within 24 h of sample receipt at a laboratory. Wastewater is agnostic to whether individuals seek medical care, are symptomatic, and the severity of illness. Knowledge gleaned from wastewater may be used by public health professionals to make recommendations on hand washing, surface disinfection, or other behaviors to reduce transmission of HuNoV, or medical doctors to inform clinical decision making.

Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study

Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence. A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1-4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets. We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0-9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000-56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32-0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses. Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications. CDC Foundation.

Rise and fall of SARS-CoV-2 variants in Rotterdam: Comparison of wastewater and clinical surveillance

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity.

Quantification of in vitro replication kinetics of Alagoas vesiculovirus isolates by digital droplet RT-PCR.

Vesicular stomatitis caused by Alagoas vesiculovirus (VSAV) has generated disease outbreaks in Brazil, mainly in the northeast region. Phylogenetic studies divide the isolates into three distinct genotypes (A, B, and C). However, there is no description of how this genetic divergence reflects on the phenotype of VSAV isolates such as in vitro replication fitness. Therefore, the objective of this work was to evaluate the ability of three distinct genotypes of Brazilian isolates of VSAV to grow in different cell-culture lines (BHK-21, Vero, and NCI-H1299). Quantification of viral RNA was performed using RT-PCR digital droplet from supernatant of cell culture collected every 4 h for a period of 24 h of viral growth in three different cell lines (BHK-21, Vero, and NCI-H1299). It was observed that the genotype C isolate has the lowest replication efficiency among the three analyzed viruses, without major changes in the copies of viral RNA over the entire time of the study.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Visualizing Cathepsin K-Cre Expression at the Single-Cell Level with GFP Reporters.

The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so-called nonspecific/off-target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk-Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk-Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk-Cre expression at the single-cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT-nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT-PCR. The results demonstrate that Ctsk-Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk-Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk-Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effect of SARS-CoV-2 digital droplet RT-PCR assay sensitivity on COVID-19 wastewater based epidemiology

We developed and implemented a framework for examining how molecular assay sensitivity for a viral RNA genome target affects its utility for wastewater-based epidemiology. We applied this framework to digital droplet RT-PCR measurements of SARS-CoV-2 and Pepper Mild Mottle Virus genes in wastewater. Measurements were made using 10 replicate wells which allowed for high assay sensitivity, and therefore enabled detection of SARS-CoV-2 RNA even when COVID-19 incidence rates were relatively low (~10−5). We then used a computational downsampling approach to determine how using fewer replicate wells to measure the wastewater concentration reduced assay sensitivity and how the resultant reduction affected the ability to detect SARS-CoV-2 RNA at various COVID-19 incidence rates. When percent of positive droplets was between 0.024% and 0.5% (as was the case for SARS-CoV-2 genes during the Delta surge), measurements obtained with 3 or more wells were similar to those obtained using 10. When percent of positive droplets was less than 0.024% (as was the case prior to the Delta surge), then 6 or more wells were needed to obtain similar results as those obtained using 10 wells. When COVID-19 incidence rate is low (~ 10−5), as it was before the Delta surge and SARS-CoV-2 gene concentrations are &lt;104 cp/g, using 6 wells will yield a detectable concentration 90% of the time. Overall, results support an adaptive approach where assay sensitivity is increased by running 6 or more wells during periods of low SARS-CoV-2 gene concentrations, and 3 or more wells during periods of high SARS-CoV-2 gene concentrations.

Avian metapneumovirus: A five-plex digital droplet RT-PCR method for identification of subgroups A, B, C, and D.

End-point and real-time avian metapneumovirus (AMPV) RT-PCRs have been developed to detect one or two of the four recognized subgroups (A,B,C, and D) simultaneously or for broad range AMPV detection. Current subgroup specific tests target variable areas of the genome which makes these PCRs sensitive to specificity defects as recently documented. In the current study, a single five-plex digital droplet RT-PCR targeting the conserved viral polymerase gene of AMPV, which is less prone to genetic drift, has been designed. This digital droplet RT-PCR was capable of identifying each of the four AMPV subgroups. Each subgroup was identified according to a specifically assigned fluorescent amplitude. Specificity, which was tested including 31 AMPV strains, non-AMPV avian viruses and closely related human respiratory viruses, was 100%. The specific limit of detection for extracted viral RNA was estimated between 1 and 3 copies/μl. This tool simplifies the number of tests required for AMPV genotype diagnostics and should be theoretically less effected by viral genome evolution due to its target region. Ultimately, application of this test will contribute to an improved understanding of the global geographic distribution and subgroup host range of field strains.

Sponge Whirl-Pak Sampling Method and Droplet Digital RT-PCR Assay for Monitoring of SARS-CoV-2 on Surfaces in Public and Working Environments.

The SARS-CoV-2 can spread directly via saliva, respiratory aerosols and droplets, and indirectly by contact through contaminated objects and/or surfaces and by air. In the context of COVID-19 fomites can be an important vehicle of virus transmission and contribute to infection risk in public environments. The aim of the study was to analyze through surface sampling (sponge method) the presence of SARS-CoV-2 in public and working environments, in order to evaluate the risk for virus transmission. Seventy-seven environmental samples were taken using sterile sponges in 17 animal farms, 4 public transport buses, 1 supermarket and 1 hotel receptive structure. Furthermore, 246 and 93 swab samples were taken in the farms from animals and from workers, respectively. SARS-CoV-2 detection was conducted by real-time RT-PCR and by digital droplet RT-PCR (dd RT-PCR) using RdRp, gene E and gene N as targets. None of the human and animal swab samples were positive for SARS-CoV-2, while detection was achieved in 20 of the 77 sponge samples (26%) using dd RT-PCR. Traces of the RdRp gene, gene E and gene N were found in 17/77 samples (22%, average concentration 31.2 g.c./cm2, range 5.6 to 132 g.c./cm2), 8/77 samples (10%, average concentration 15.1 g.c./cm2, range 6 to 36 g.c./cm2), and in 1/77 (1%, concentration 7.2 g.c./cm2). Higher detection rates were associated with sampling in animal farms and on public transport buses (32% and 30%) compared to the supermarket (21%) and the hotel (no detection). The result of the study suggests that the risk of contamination of surfaces with SARS-CoV-2 increases in environments in which sanitation strategies are not suitable and/or in highly frequented locations, such as public transportation. Considering the analytical methods, the dd RT-PCR was the only approach achieving detection of SARS-CoV-2 traces in environmental samples. Thus, dd RT-PCR emerges as a reliable tool for sensitive SARS-CoV-2 detection.

Polycyclic aromatic hydrocarbons, long non-coding RNA expression, and DNA damage in coke oven workers.

Exposure to polycyclic aromatic hydrocarbons (PAHs) was associated with DNA damage, while the roles of long non-coding RNAs (lncRNAs) in the associations were unclear. We aimed to assess the association of lncRNA NR_024564 with urinary monohydroxy PAHs (OH-PAHs) and DNA damage among 332 coke oven workers. We determined 12 OH-PAHs by gas chromatography-mass spectrometry, and the expression level of NR_024564 by droplet digital RT-PCR and DNA damage by the comet assay. In total participants, we found that NR_024564 was not significantly associated with OH-PAHs or comet parameters. However, among workers with ≥ 20 working years, multiple OH-PAHs including urinary 1-hydroxyphenanthrene (1-OHPh), 2-OHPh, 3-OHPh, 9-OHPh, 1‑hydroxypyrene, and total PAH metabolites were related to increased comet parameters. Moreover, NR_024564 was significantly associated with 2-OHPh and four comet parameters. Each 1% increase in 2-OHPh was associated with 0.35% reduction (95% CI: 0.16%, 0.55%) in NR_024564 (P-FDR = 0.005), and 2-OHPh was marginally interacted with working years in relation to NR_024564 decrease. Also, each 1% increment of NR_024564 was related to 0.04-0.13% decrease of Olive tail moment, percent DNA in the comet tail, tail length, and tail moment (all P-FDR < 0.05). Furthermore, low NR_024564 level combined with high levels of 1-OHPh and 2-OHPh or ≥ 20 working years was positively associated with the comet parameters among the total participants. Our results indicated that NR_024564 might be linked to the adverse associations of PAHs with the DNA damage of coke oven workers who worked for ≥ 20years.

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14)

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs.A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region.Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4–100.0), 98.7 % (92.1–100.0), and 99.6 % (97.5–100.0) for the rt RT-qPCR; 92.4 % (87.4–95.6), 100.0 % (94.2–100.0), and 94.7 % (91.1–97.0) for the dd RT-PCR.The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR.

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater

Wastewater surveillance has shown to be a valuable and efficient tool to obtain information about the trends of COVID-19 in the community. Since the recent emergence of new variants, associated with increased transmissibility and/or antibody escape (variants of concern), there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. In this study, we demonstrate the use of RT-ddPCR on wastewater samples for specific detection of mutation N501Y. This assay enabled simultaneous enumeration of lineage B.1.351 (containing the 501Y mutation) and Wild Type (WT, containing 501N) SARS-CoV-2 RNA. Detection of N501Y was possible in samples with mixtures of WT with low proportions of B.1.351 (0.5%) and could accurately determine the proportion of N501Y and WT in mixtures of SARS-CoV-2 RNA. The application to raw sewage samples from the cities of Amsterdam and Utrecht demonstrated that this method can be applied to wastewater samples. The emergence of N501Y in Amsterdam and Utrecht wastewater aligned with the emergence of B.1.1.7 as causative agent of COVID-19 in the Netherlands, indicating that RT-ddPCR of wastewater samples can be used to monitor the emergence of the N501Y mutation in the community. It also indicates that RT-ddPCR could be used for sensitive and accurate monitoring of current (like K417N, K417T, E484K, L452R) or future mutations present in SARS-CoV-2 variants of concern. Monitoring these mutations can be used to obtain insight in the introduction and spread of VOC and support public health decision-making regarding measures to limit viral spread or allocation of testing or vaccination.

SARS-CoV-2 RNA Quantification Using Droplet Digital RT-PCR

Quantitative viral load assays have transformed our understanding of viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time RT-PCR, yield semiquantitative results only. Droplet digital RT-PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification. Eight primer/probe sets originally developed for real-time RT-PCR–based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR; three were identified as the most efficient, precise, and sensitive for RT-ddPCR–based SARS-CoV-2 RNA quantification. For example, the analytical efficiency for the E-Sarbeco primer/probe set was approximately 83%, whereas assay precision, measured as the coefficient of variation, was approximately 2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19–positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. RT-ddPCR–derived SARS-CoV-2 E gene copy numbers were further calibrated against cycle threshold values from a commercial real-time RT-PCR diagnostic platform. This log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from cycle threshold values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

Polycyclic aromatic hydrocarbons associated long non-coding RNAs and heart rate variability in coke oven workers.

Epidemiological studies have showed that polycyclic aromatic hydrocarbons (PAHs) were associated with heart rate variability (HRV), but the role of long non-coding RNAs (lncRNAs) in the association is unknown. We aimed to identify PAHs-related lncRNAs and assess their associations with HRV among coke oven workers. Differential lncRNAs expression between 12 exposed workers and 12 controls was tested by Human 8X60k LncRNA Arrays in discovery stage, then selected NR_024564 was validated in 353 workers using droplet digital RT-PCR. Microarray results showed that 1234 lncRNAs were downregulated with 805 lncRNAs upregulated in exposed group (≥ 2-fold change). In validation stage, no significant association was observed between NR_024564 and PAH exposure or HRV in total subjects, while urinary 2-hydroxyfluorene (2-OHFlu) was inversely related to root mean square successive difference (RMSSD). However, in current smokers, NR_024564 was inversely related to urinary 2-OHFlu, 2-hydroxyphenanthrene, 1-hydroxypyrene (1-OHP), and total PAHs metabolites (ΣOH-PAHs), of which 1-OHP accounted for the strongest estimation for interaction with smoking status (Pinteraction = 0.011). Also, the positive associations of NR_024564 with RMSSD and high frequency power showed an interaction with smoking status (Pinteraction = 0.034 and 0.023, respectively). Also, urinary 2-OHFlu and ΣOH-PAHs were inversely associated with RMSSD in current smokers. In addition, elevated NR_024564 was dose-responsive related to increased RMSSD in above high-PAHs groups among smokers (all Ptrend < 0.05). Our results revealed that NR_024564 and its interactions with smoking status might act as novel mechanisms regulating the adverse effects of PAHs on HRV.