scholarly journals Surveillance and correlation of SARS-CoV-2 viral RNA, antigen, virus isolation, and self-reported symptoms in a longitudinal study with daily sampling

2021 ◽  
Author(s):  
Gaston Bonenfant ◽  
Jessica Deyoe ◽  
Terianne Wong ◽  
Carlos G. Grijalva ◽  
H. Keipp Talbot ◽  
...  
Keyword(s):  
Positive Culture ◽  
Viral Rna ◽  
Rt Pcr ◽  
Predictive Values ◽  
Qrt Pcr ◽  
Household Transmission ◽  
Daily Sampling ◽  
Diagnostic Panel ◽  
Pcr Assays ◽  
Novel Coronavirus

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of FDA emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, i.e., the presence of replicable virus. As the number of tests conducted increased, persistent SARS-CoV-2 RNA positivity by RT-PCR in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR (qRT-PCR) assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike (S), nucleocapsid (N), membrane (M), envelope (E), and ORF8. The absolute copy number of each RNA target was determined in longitudinal specimens from a household transmission study. Calculated viral RNA levels over the 14-day follow up period were compared with antigen testing and self-reported symptoms to characterize the clinical and molecular dynamics of infection and infer predictive values of these qRT-PCR assays relative to culture isolation. When detection of sgS RNA was added to the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, we found a qRT-PCR positive result was 98% predictive of a positive culture (negative predictive value was 94%). Our findings suggest sgRNA presence correlates with active infection, may help identify individuals shedding culturable virus, and that similar multiplex assays can be adapted to current and future variants.

scholarly journals Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

2019 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Fereshteh Amiri ◽  
Zohreh Fadajan ◽  
Azadeh Rasooli ◽  
Iman Salahshourifar ◽  
Rouzbeh Bashar ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Rt Pcr ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

scholarly journals Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

2005 ◽  
Vol 17 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Susan K. Schommer ◽  
Sang-Myeong Lee ◽  
Sandy Watkins ◽  
Wayne Chittick ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
North American ◽  
Simultaneous Detection ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Field Isolates ◽  
Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

scholarly journals One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1560 ◽  
Author(s):  
Rashi Gautam ◽  
Slavica Mijatovic-Rustempasic ◽  
Mathew D. Esona ◽  
Ka Ian Tam ◽  
Osbourne Quaye ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Group A ◽  
One Step ◽  
Stool Samples ◽  
Pcr Assays ◽  
Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

scholarly journals A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2

2020 ◽  
Author(s):  
Zhen Zhao ◽  
Haodong Cui ◽  
Wenxing Song ◽  
Xiaoling Ru ◽  
Wenhua Zhou ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Clinical Diagnosis ◽  
Molecular Diagnosis ◽  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Novel Coronavirus ◽  
Rna Complexes

1AbstractThe ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 105 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.

Circulating tumor cell detection: A direct comparison between the CellSearch System, the AdnaTest, and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22117-e22117
Author(s):  
L. Y. Dirix ◽  
H. Elst ◽  
I. Benoy ◽  
I. Van der Auwera ◽  
A. Prové ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Tumor Cells ◽  
Metastatic Breast ◽  
Rt Pcr ◽  
Cellsearch System ◽  
Blood Samples ◽  
Detection Techniques ◽  
Qrt Pcr ◽  
Pcr Assays

e22117 Background: The detection, enumeration and isolation of circulating tumor cells (CTC) has considerable potential to influence the clinical management of patients with breast cancer. There is however a substantial variability in the rates of positive samples using existing detection techniques. Methods: This study was designed to compare three techniques for detecting CTC in blood of 80 patients with metastatic breast cancer (MBC) and 20 healthy controls: the CellSearch System, which is an automated, standardized and regulatory-approved system for the immunocytochemical detection and quantification of CTC in blood; ii) the AdnaTest Breast Cancer Select/Detect, which involves the detection of tumor-associated transcripts by RT-PCR after an immunomagnetically enrichment of tumor cells; iii) an in-house developed multimarker qRT-PCR assay, which involves the quantification of tumor-associated transcripts (CK-19 and MAM) by qRT- PCR. Results: As a result, 23% of patients with MBC were positive by the CellSearch System (≥5 CTC), 22% by the AdnaTest (>0.30 ng/μl for any of the amplicons), 31% by qRT-PCR for CK-19 and 49% by qRT-PCR for MAM. Samples were more likely to be positive by qRT-PCR for at least one mRNA marker than by the CellSearch System (P<0.001) or the AdnaTest (P <0.001). The concordance between samples analyzed by the CellSearch System and the AdnaTest was substantial (κ = 0.667, P <0.001). Agreement between both detection techniques was observed in 88% of blood samples. When the CellSearch System was compared with the qRT-PCR assays for CK-19 and MAM, we observed agreement percentages of 78% and 58%, respectively (κ = 0.462, P <0.001 and κ = 0.159, P = 0.09). Agreement between the AdnaTest and the qRT-PCR assays for CK-19 and MAM was observed in 78% and 53% of blood samples, respectively (κ = 0.443, P <0.001 and κ = 0.05, P = 0.607). Conclusions: We observed a substantial variation in the detection rates of CTC in blood from MBC patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and MAM, which suggests that this is currently the most sensitive technique for detecting CTC. No significant financial relationships to disclose.

scholarly journals Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

2020 ◽  
Vol 21 (12) ◽  
pp. 4396 ◽  
Author(s):  
Tuna Toptan ◽  
Sebastian Hoehl ◽  
Sandra Westhaus ◽  
Denisa Bojkova ◽  
Annemarie Berger ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
World Health ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
M Gene ◽  
Control And Prevention ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Health Organization

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

scholarly journals Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260487
Author(s):  
Justin S. Lee ◽  
Jason M. Goldstein ◽  
Jonathan L. Moon ◽  
Owen Herzegh ◽  
Dennis A. Bagarozzi ◽  
...  
Keyword(s):  
Real Time ◽  
False Positive ◽  
Bulk Material ◽  
Virus Detection ◽  
Rt Pcr ◽  
Nucleocapsid Gene ◽  
Positive Reactivity ◽  
Control And Prevention ◽  
Diagnostic Panel ◽  
Novel Coronavirus

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the “bulk” manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3’ end of the N3 probe and the 3’ end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the “bulk” material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

scholarly journals Ocular manifestations of a hospitalised patient with confirmed 2019 novel coronavirus disease

2020 ◽  
Vol 104 (6) ◽  
pp. 748-751 ◽  
Author(s):  
Lu Chen ◽  
Meizhou Liu ◽  
Zheng Zhang ◽  
Kun Qiao ◽  
Ting Huang ◽  
...  
Keyword(s):  
Threshold Value ◽  
Viral Rna ◽  
Rt Pcr ◽  
Illness Onset ◽  
Reverse Transcription Pcr ◽  
Ocular Manifestations ◽  
Conjunctival Swab ◽  
Precautionary Measures ◽  
Novel Coronavirus ◽  
Hospitalised Patient

PurposeTo report the ocular characteristics and the presence of viral RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in conjunctival swab specimens in a patient with confirmed 2019 novel coronavirus disease (COVID-19).Participant and methodsA 30-year-old man with confirmed COVID-19 and bilateral acute conjunctivitis which occurred 13 days after illness onset. Based on detailed ophthalmic examination, reverse transcription PCR (RT-PCR) was performed to detect SARS-CoV-2 virus in conjunctival swabs. The ocular characteristics, presence of viral RNA and viral dynamics of SARS-CoV-2 in the conjunctival specimens were evaluated.ResultsSlit lamp examination showed bilateral acute follicular conjunctivitis. RT-PCR assay demonstrated the presence of viral RNA in conjunctival specimen 13 days after onset (cycle threshold value: 31). The conjunctival swab specimens remained positive for SARS-CoV-2 on 14 and 17 days after onset. On day 19, RT-PCR result was negative for SARS-CoV-2.ConclusionSARS-CoV-2 is capable of causing ocular complications such as viral conjunctivitis in the middle phase of illness. Precautionary measures are recommended when examining infected patients throughout the clinical course of the infection. However, conjunctival sampling might not be useful for early diagnosis because the virus may not appear initially in the conjunctiva.

scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Comparative Study ◽  
Clinical Diagnosis ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Predictive Values ◽  
Health Crisis ◽  
Pcr Assays ◽  
Sensitivity Specificity

Abstract SARS-CoV-2 is a newly emerged coronavirus that was isolated from human infections in recent months. Since drugs and vaccines of Covid-19 are still being developed, accurate pathogen detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been reliably used for the detection and confirmation of SARS-CoV-2 since the beginning of outbreak, whereas isothermal nucleic acid amplification based point of care automated assays has also been considered as a simpler and rapid alternative. However, since these assays have only been developed and applied for clinical use within a short timeframe, their analytical performance has not been adequately compared to-date. We describe a comparative study between a newly developed cross primer isothermal amplification (CPA) assay (Kit A) and five RT-PCR assays (Kits B to F), using clinical diagnosis as the reference standard to evaluate their sensitivity, specificity, predictive values and accuracy analysis. Clinical samples used (n=52) included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprised of positive (n=26) and cleared cases (n=26) by clinical diagnosis. For the CPA assay (Kit A), the sensitivity, specificity, positive and negative predictive values and accuracy were 100%. Among the five RT-PCR kits, Kits B, C and F had good agreement with clinical diagnosis (Kappa≥0.75), Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all assays were statistically significant (P<0.001). The reproducibility of RT-PCR assays was determined using a positive sample that was verified by all assays, with standard deviations (SD) between 0.35 and 0.87, and coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. To further evaluate the CPA assay (Kit A) compared to Kits B and F, throat swabs from close contacts of cases (n=200) were analyzed. All three kits identified the same positive samples and showed total agreement. This is the first comparative study to evaluate a CPA assay and RT-PCR assays for SARS-CoV-2 detection, which could serve as a reference for clinical laboratories and inform testing protocols amid the rapidly evolving COVID-19 pandemic.

scholarly journals Simple Strategy for Rapid and Sensitive Detection of 2019 novel coronavirus Based on Antibody

2020 ◽  
Author(s):  
Xinjun Hu ◽  
Huiyang Deng ◽  
Yibing Shang ◽  
Mengnan Fan ◽  
Feng Yue
Keyword(s):  
Colloidal Gold ◽  
Detection Rate ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Immunochromatographic Strip ◽  
Simple Strategy ◽  
Immunochromatographic Strip Test ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Strip Test

Abstract Objectives: Since December 2019, acute respiratory disease due to 2019 novel coronavirus emerged in Wuhan city and rapidly spread throughout China. Real-time RT-PCR is widely deployed in diagnostic virology. However, the positive detection rates of RT-PCR are only 30% to 50%. Therefore, we propose a simple strategy for rapidly and sensitively detecting the IgM/IgG antibody against 2019-nCoV using a colloidal gold-based immunochromatographic strip test.Methods A total of 41 clinically 2019-nCoV suspected cases (23 males and 18 females) were enrolled. The sensitivity of colloidal gold-based immunochromatographic strip test and of RT-PCR were compared and evaluated. McNemar’s test was used to compare the detection rate of both assays (P<0.05).Results: The Antibody was detected in 63.4% (26/41) of blood specimens using the assay. In contrast, the 2019-nCoV was detected in 46.3% (19/41) of nasal and pharyngeal swab specimens using the RT-PCR assays. The detection rate obtained by this assay was markedly higher than that obtained by the RT-PCR assays (P=0.039)Conclusion: This detection assay exhibits a higher detection sensitivity than RT-PCR. More important, the assay shows the benefits of easy operation and setup. We believe that the sensitive and time-saving approach may be used as an auxiliary diagnostic tool for 2019-nCoV detection and virus screening and confirmation.

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