scholarly journals Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2

2021 ◽  
Author(s):  
Sushama Telwatte ◽  
Nitasha Kumar ◽  
Albert Vallejo-Gracia ◽  
G. Renuka Kumar ◽  
Chuanyi M. Lu ◽  
...  
Keyword(s):  
Diagnostic Tools ◽  
Clinical Samples ◽  
List Type ◽  
Transcription Profile ◽  
Diagnostic Assays ◽  
Copy Numbers ◽  
Positive Sense ◽  
Viral Load Assay ◽  
Discontinuous Transcription ◽  
Negative Sense

AbstractThe exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription.Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.HighlightsWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2Our panel targets nongenic and genic regions in genomic and subgenomic RNAsAll assays detect 1-10 copies and are linear over 3-4 orders of magnitudeAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load AssayClinical samples showed higher copy numbers for targets at the 3’ end of the genome

The effect of rotation on the simpler modes of motion of a liquid in an elliptic paraboloid

1965 ◽  
Vol 22 (3) ◽  
pp. 529-545 ◽  
Author(s):  
F. K. Ball
Keyword(s):  
Linear Equations ◽  
The Other ◽  
List Type ◽  
Elliptic Paraboloid ◽  
The Earth ◽  
Positive Sense ◽  
Rotation Of The Earth ◽  
The Waves ◽  
Negative Sense ◽  
Non Linear Equations

The six simplest modes of motion are considered and three rotational effects investigated:The effect of the rotation of the earth.The effect of the rotation of the container.The effect of the rotation of the liquid within the container.The first two are shown to be equivalent for motion in a paraboloid, and the last two are also equivalent when the paraboloid iscircular. In the case of an elliptic paraboloid the last is rather more difficult and one must first derive a solution of the non-linear equations representing ‘elliptic rotation’ and then consider deviations from it.The changes in frequency consequent on the rotation are derived in all three cases for all six modes. In the case of the earth's rotation the disposition and character of the amphidromic (nodal) points and the amphidromic waves that rotate round these points are investigated in detail. One mode is particularly interesting because it has four amphidromic points, the waves rotate in a positive sense around two of these and in a negative sense round the other two.

scholarly journals Does the punishment fit the crime? Consequences and diagnosis of misspecified detection functions in Bayesian spatial capture-recapture modelling

2021 ◽  
Author(s):  
Soumen Dey ◽  
Richard Bischof ◽  
Pierre P. A. Dupont ◽  
Cyril Milleret
Keyword(s):  
Home Range ◽  
Space Use ◽  
Relative Bias ◽  
Diagnostic Tools ◽  
List Type ◽  
Detection Function ◽  
Density Estimates ◽  
Use Patterns ◽  
Wide Range ◽  
Capture Recapture

AbstractSpatial capture-recapture (SCR) is now used widely to estimate wildlife densities. At the core of SCR models lies the detection function, linking individual detection probability to the distance from its latent activity center. The most common function (half-normal) assumes a bivariate normal space use and consequently detection pattern. This is likely an oversimplification and misrepresentation of real-life animal space use patterns, but studies have reported that density estimates are relatively robust to misspecified detection functions. However, information about consequences of such misspecification on space use parameters (e.g. home range area), as well as diagnostic tools to reveal it are lacking.We simulated SCR data under six different detection functions, including the half-normal, to represent a wide range of space use patterns. We then fit three different SCR models, with the three simplest detection functions (half-normal, exponential and half-normal plateau) to each simulated data set. We evaluated the consequences of misspecification in terms of bias, precision and coverage probability of density and home range area estimates. We also calculated Bayesian p-values with respect to different discrepancy metrics to assess whether these can help identify misspecifications of the detection function.We corroborate previous findings that density estimates are robust to misspecifications of the detection function. However, estimates of home range area are prone to bias when the detection function is misspecified. When fitted with the half-normal model, average relative bias of 95% kernel home range area estimates ranged between −25% and 26% depending on the misspecification. In contrast, the half-normal plateau model (an extension of the half-normal) returned average relative bias that ranged between −26% and −4%. Additionally, we found useful heuristic patterns in Bayesian p-values to diagnose the misspecification in detection function.Our analytical framework and diagnostic tools may help users select a detection function when analyzing empirical data, especially when space use parameters (such as home range area) are of interest. We urge development of additional custom goodness of fit diagnostics for Bayesian SCR models to help practitioners identify a wider range of model misspecifications.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Tongues, a Sign for Unbelievers?: A Structural and Exegetical Study of I Corinthians XIV. 20–25

1979 ◽  
Vol 25 (2) ◽  
pp. 180-203 ◽  
Author(s):  
B. C. Johanson
Keyword(s):  
The Other ◽  
Logical Relation ◽  
Other Hand ◽  
Positive Sense ◽  
Negative Effect ◽  
Relationship Of ◽  
The Relationship ◽  
Negative Sense ◽  
Positive Effect

I Corinthians xiv. 20–25 has long posed severalcruces interpretationisfor commentators. The basic problems concern the relationship of the assertions made about tongues and prophecy in υ. 22 to the quotation of Isa. xxviii. 11–12 in υ. 21 and to the illustrations concerning tongues and prophecy in υυ. 23–5. As to the quotation, J. Ruef remarks that most commentators admit to the difficulty of seeing how it substantiates Paul's conclusion that tongues are meant as a sign for the unbeliever. Concerning the illustrations, both J. Héring and J. P. M. Sweet note that in the light of the assertions we would expect them to be the reverse of what they are. While tongues are asserted to be meant as a sign for unbelievers and prophecy for believers, the illustrations depict the negative effect of tongues upon unbelievers and the positive effect of prophecy not on believers but upon unbelievers. The second assertion (υ. 22b) in particular contradicts the second illustration (υυ. 24–5) in that it clearly states that ‘prophecy is meant as a signnot for unbelieversbut for believers’. This is so if σημεĩον is taken in a positive sense. If, on the other hand, it is taken in a negative sense, the logical relation of this second illustration to the second assertion becomes ambiguous.

DDX3 inhibitors show antiviral activity against positive-sense single-stranded RNA viruses but not against negative-sense single-stranded RNA viruses: The coxsackie B model

2020 ◽  
Vol 178 ◽  
pp. 104750 ◽  
Author(s):  
Paola Quaranta ◽  
Giulia Lottini ◽  
Giulia Chesi ◽  
Flavia Contrafatto ◽  
Roberta Russotto ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Viruses ◽  
Positive Sense ◽  
Negative Sense ◽  
Coxsackie B

scholarly journals Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

2018 ◽  
Vol 64 (12) ◽  
pp. 1704-1712 ◽  
Author(s):  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Malin Grabbe ◽  
Narayanan Madaboosi ◽  
Jan Albert ◽  
...  
Keyword(s):  
Seasonal Influenza ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Clinical Samples ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Diagnostic Assays ◽  
Padlock Probes ◽  
Probe Assay ◽  
Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

scholarly journals Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1164
Author(s):  
Shona C. Moore ◽  
Rebekah Penrice-Randal ◽  
Muhannad Alruwaili ◽  
Nadine Randle ◽  
Stuart Armstrong ◽  
...  
Keyword(s):  
Viral Genome ◽  
False Negative ◽  
Point Mutations ◽  
Read Length ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Number Of Patients ◽  
Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

scholarly journals Use of Monocyte-Derived Macrophage Culture Increases Zika Virus Isolation Rate from Human Plasma

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1058 ◽  
Author(s):  
Emilia Sippert ◽  
Bruno C. Rocha ◽  
Felipe L. Assis ◽  
Suzan Ok ◽  
Maria Rios
Keyword(s):  
Human Plasma ◽  
Cell Lines ◽  
Zika Virus ◽  
Basic Research ◽  
Host Cells ◽  
Clinical Samples ◽  
Viral Isolation ◽  
Diagnostic Assays ◽  
Primary Monocyte ◽  
Direct Inoculation

Viral isolation is desirable for many reasons, including development of diagnostic assays and reference materials, and for virology basic research. Zika virus (ZIKV) isolation from clinical samples is challenging, but isolates are known to infect various cell lines. Here, we evaluated suitability of Vero, C6/36 and JEG-3 as host cells, for direct isolation of ZIKV from human plasma. We also assessed the use of primary monocyte-derived macrophages (MDMs) culture to enhance ZIKV isolation from human plasma samples followed by virus expansion in Vero, C6/36 and JEG-3 cultures. Direct inoculation of cell lines with 42 ZIKV-RNA positive samples resulted in isolation rates of 9.52% (4/42) in Vero and C6/36, and of 7.14% (3/42) in JEG-3 cells. Inoculation of plasma in MDMs followed by supernatant testing by TaqMan RT-PCR, resulted in 33/42 (78.57%) ZIKV-RNA-positive supernatants, which expansion in cell lines increased isolation rates to 24.24% (8/33) in Vero and to 27.27% (9/33) in C6/36 and JEG-3 regardless of the presence of ZIKV-antibody. Isolates generated in JEG-3 cells were also produced in Vero and C6/36 with similar viral titers. These results suggest that efficiency of ZIKV isolation from human plasma can be enhanced when MDM culture is used before viral expansion in cell lines.

scholarly journals VIRTUS: a pipeline for comprehensive virus analysis from conventional RNA-seq data

2020 ◽  
Author(s):  
Yoshiaki Yasumizu ◽  
Atsushi Hara ◽  
Shimon Sakaguchi ◽  
Naganari Ohkura
Keyword(s):  
Sequence Data ◽  
Supplementary Information ◽  
Clinical Samples ◽  
Host Gene Expression ◽  
Rna Seq ◽  
Viral Transcript ◽  
Rna Transcripts ◽  
Copy Numbers ◽  
Infected Cells ◽  
New Treatments

Abstract Summary The possibility that RNA transcripts from clinical samples contain plenty of virus RNAs has not been pursued actively so far. We here developed a new tool for analyzing virus-transcribed mRNAs, not virus copy numbers, in the data of bulk and single-cell RNA-sequencing of human cells. Our pipeline, named VIRTUS (VIRal Transcript Usage Sensor), was able to detect 762 viruses including herpesviruses, retroviruses and even SARS-CoV-2 (COVID-19), and quantify their transcripts in the sequence data. This tool thus enabled simultaneously detecting infected cells, the composition of multiple viruses within the cell, and the endogenous host-gene expression profile of the cell. This bioinformatics method would be instrumental in addressing the possible effects of covertly infecting viruses on certain diseases and developing new treatments to target such viruses. Availability and implementation : VIRTUS is implemented using Common Workflow Language and Docker under a CC-NC license. VIRTUS is freely available at https://github.com/yyoshiaki/VIRTUS. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples

2020 ◽  
Vol 6 (4) ◽  
pp. 292
Author(s):  
Luis Pla ◽  
Anna Aviñó ◽  
Ramón Eritja ◽  
Alba Ruiz-Gaitán ◽  
Javier Pemán ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Pneumocystis Pneumonia ◽  
Rapid Screening ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Current Standard ◽  
Promising Alternative ◽  
Nanoporous Anodic Alumina ◽  
Dna Oligonucleotides ◽  
Risk Of Mortality

Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.

Sign in / Sign up

Export Citation Format

Share Document