scholarly journals Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ramesh Yelagandula ◽  
◽  
Aleksandr Bykov ◽  
Alexander Vogt ◽  
Robert Heinen ◽  
...  
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
High Sensitivity ◽  
Cost Effective ◽  
Massively Parallel ◽  
Rt Pcr ◽  
Viral Spread ◽  
Saliva Analysis ◽  
Double Blinded ◽  
Generation Sequencing

AbstractThe COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

scholarly journals SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections

2020 ◽  
Author(s):  
Ramesh Yelagandula ◽  
Aleksandr Bykov ◽  
Alexander Vogt ◽  
Robert Heinen ◽  
Ezgi Özkan ◽  
...  
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
False Positive Rate ◽  
Cost Effective ◽  
Rt Pcr ◽  
Robust Performance ◽  
Saliva Analysis ◽  
Positive Rate ◽  
Double Blinded ◽  
Generation Sequencing

During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massively parallel, yet cost-effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total, for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to a few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.

scholarly journals Surveillance of Respiratory Viruses in Long Term Care Facilities

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Checivich ◽  
Shari Barlow ◽  
Peter Shult ◽  
Erik Residorf ◽  
Jonathan L. Temte
Keyword(s):  
Influenza A ◽  
Nasal Swab ◽  
Long Term Care ◽  
Respiratory Infections ◽  
Respiratory Viruses ◽  
Influenza B ◽  
Rt Pcr ◽  
Term Care ◽  
Care Facilities

ObjectiveTo assess the feasibility of conducting respiratory virus surveillance for residents of long term care facilities (LTCF) using simple nasal swab specimens and to describe the virology of acute respiratory infections (ARI) in LCTFs.IntroductionAlthough residents of LTCFs have high morbidity and mortality associated with ARIs, there is very limited information on the virology of ARI in LTCFs.[1,2] Moreover, most virological testing of LCTF residents is reactive and is triggered by a resident meeting selected surveillance criteria. We report on incidental findings from a prospective trial of introducing rapid influenza diagnostic testing (RIDT) in ten Wisconsin LTCFs over a two-year period with an approach of testing any resident with ARI.MethodsAny resident with new onset of respiratory symptoms consistent with ARI had a nasal swab specimen collected for RIDT by nursing staff. Following processing for RIDT (Quidel Sofia Influenza A+B FIA), the residual swab was placed into viral transport medium and forwarded to the Wisconsin State Laboratory of Hygiene and tested for influenza using RT-PCR (IVD CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel), and for 17 viruses (Luminex NxTAG Respiratory Pathogen Panel [RPP]). The numbers of viruses in each of 7 categories [influenza A (FluA ), influenza B (FluB), coronaviruses (COR), human metapneumovirus (hMPV), parainfluenza (PARA), respiratory syncytial virus (RSV) and rhinovirus/enterovirus (R/E)], across the two years were compared using chi-square.ResultsTotals of 164 and 190 specimens were submitted during 2016-2017 and 2017-2018, respectively. RPP identified viruses in 56.2% of specimens, with no difference in capture rate between years (55.5% vs. 56.8%). Influenza A (21.5%), influenza B (16.5%), RSV (19.0%) and hMPV (16.5%) accounted for 73.5% of all detections, while coronaviruses (15.5%), rhino/enteroviruses (8.5%) and parainfluenza (2.5%) were less common. Specific distribution of viruses varied significantly across the two years (Table: X2=48.1, df=6; p<0.001).ConclusionsSurveillance in LTCFs using nasal swabs collected for RIDT is highly feasible and yields virus identification rates similar to those obtained in clinical surveillance of ARI with collection of nasopharyngeal specimens by clinicians and those obtained in a school-based surveillance project of ARI with collection of combined nasal and oropharyngeal specimens collected by trained research assistants. Significant differences in virus composition occurred across the two study years. RSV varied little between years while hMPV demonstrated wide variation. Simple approaches to surveillance may provide a more comprehensive assessment of respiratory viruses in LTCF settings.References(1) Uršič T, Gorišek Miksić N, Lusa L, Strle F, Petrovec M. Viral respiratory infections in a nursing home: a six-month prospective study. BMC Infect Dis. 2016; 16: 637. Published online 2016 Nov 4. doi: 10.1186/s12879-016-1962-8(2) Masse S, Capai L, Falchi A. Epidemiology of Respiratory Pathogens among Elderly Nursing Home Residents with Acute Respiratory Infections in Corsica, France, 2013–2017. Biomed Res Int. 2017; 2017: 1423718. Published online 2017 Dec 17. doi: 10.1155/2017/1423718

scholarly journals Evaluation of RNA extraction free method for detection of SARS-COV-2 in salivary samples for mass screening for COVID-19

2021 ◽  
Author(s):  
Sally A. Mahmoud ◽  
Esra Ibrahim ◽  
Subhashini Ganesan ◽  
Bhagyashree Thakre ◽  
Juliet G Teddy ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Gold Standard ◽  
Rna Extraction ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Good Percentage

AbstractIn this current COVID - 19 pandemic, there is a dire need for cost effective and less time-consuming alternatives for SARS-COV-2 testing. The RNA extraction free method for detecting SARS-COV-2 in saliva is a promising option, this study found that it has high sensitivity (85.34%), specificity (95.04%) and was comparable to the gold standard nasopharyngeal swab. The method showed good percentage of agreement (kappa coefficient) 0.797 between salivary and NPS samples. However, there are variations in the sensitivity and specificity based on the RT-PCR kit used. The Thermo Fischer-Applied biosystems showed high sensitivity, PPV and NPV but also showed higher percentage of invalid reports. Whereas the BGI kit showed high specificity, better agreement (kappa coefficient) between the results of saliva and NPS samples and higher correlation between the Ct values of saliva and NPS samples. Thus, the RNA extraction free method for salivary sample serves as an effective alternative for SARS-CoV 2-testing.

scholarly journals Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

2020 ◽  
Author(s):  
Chenyu Li ◽  
David N. Debruyne ◽  
Julia Spencer ◽  
Vidushi Kapoor ◽  
Lily Y. Liu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Multiplex Pcr ◽  
High Sensitivity ◽  
Detection Methods ◽  
Next Generation ◽  
Rt Pcr ◽  
Copy Numbers ◽  
Virus Rna ◽  
Highly Sensitive ◽  
Generation Sequencing

AbstractMany detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

scholarly journals A new Syber Green real time PCR to detect SARS-CoV-2

2020 ◽  
Author(s):  
D.R. Marinowic ◽  
G. Zanirati ◽  
F.V.F. Rodrigues ◽  
M.V.C. Grahl ◽  
A.M. Alcará ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Large Scale ◽  
Low Cost ◽  
Phylogenetic Analyses ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Etiologic Agent ◽  
Rt Pcr ◽  
Human Respiratory Tract

Abstract Phylogenetic analyses demonstrated that etiologic agent of pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by WHO is the RT-PCR. To detect low viral load and large-scale screening a low-cost diagnostic test becomes necessary. Here we develop a cost-effective test capable of to detect the new coronavirues. We validated an auxiliary protocol for molecular diagnosis with RT-PCR SYBR Green methodology to successfully screen negative cases of SARS-CoV-2. Our results demonstrated that a set of primers with high specificity, and no homology with other viruses from Coronovideae family or human respiratory tract pathogenic viruses. Optimization of annealing temperature and polymerization time led to an high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on large scale populational to soften panic and economic burden through guidance for isolation strategies.

scholarly journals Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Masateru Taniguchi ◽  
Shohei Minami ◽  
Chikako Ono ◽  
Rina Hamajima ◽  
Ayumi Morimura ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Machine Learning ◽  
High Speed ◽  
Simple Procedure ◽  
Rna Extraction ◽  
Disease Outbreaks ◽  
High Sensitivity ◽  
Cost Effective ◽  
Rt Pcr ◽  
Emerging Viruses

AbstractHigh-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.

scholarly journals A new SYBR Green real-time PCR to detect SARS-CoV-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
D. R. Marinowic ◽  
G. Zanirati ◽  
F. V. F. Rodrigues ◽  
M. V. C. Grahl ◽  
A. M. Alcará ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Large Scale ◽  
Low Cost ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Etiologic Agent ◽  
Sybr Green ◽  
Rt Pcr ◽  
Human Respiratory Tract

AbstractPhylogenetic analysis has demonstrated that the etiologic agent of the 2020 pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by the Word Health Organization (WHO) is RT-PCR. To detect low viral loads and perform large-scale screening, a low-cost diagnostic test is necessary. Here, we developed a cost-effective test capable of detecting SARS-CoV-2. We validated an auxiliary protocol for molecular diagnosis with the SYBR Green RT-PCR methodology to successfully screen negative cases of SARS-CoV-2. Our results revealed a set of primers with high specificity and no homology with other viruses from the Coronovideae family or human respiratory tract pathogenic viruses, presenting with complementarity only for rhinoviruses/enteroviruses and Legionella spp. Optimization of the annealing temperature and polymerization time led to a high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on a large scale to soften panic and economic burden through guidance for isolation strategies.

scholarly journals A nanofluidic device for rapid and multiplexed SARS-CoV-2 serological antibody detection

2021 ◽  
Author(s):  
Thomas Mortelmans ◽  
Dimitrios Kazazis ◽  
Celestino Padeste ◽  
Philipp Berger ◽  
Xiaodan Li ◽  
...  
Keyword(s):  
Influenza A ◽  
Point Of Care ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Modern Society ◽  
Antibody Detection ◽  
Multiplexed Detection ◽  
Viral Spread ◽  
Point Of Care Diagnostics ◽  
Nanofluidic Device

Abstract The outbreak of COVID-19 has led to a substantial death toll and has hindered the functioning of modern society, sending the world into a medical and economic crisis1,2. This underlined the importance of point-of-care diagnostics, as well as accurate, cost-effective serological antibody tests as well as point-of-care diagnostics to monitor the viral spread and contain pandemics and endemics. Here, we present a three-dimensional (3D) nanofluidic device for rapid and multiplexed detection of viral antibodies. The device is designed to size-dependently immobilize particles from a multi-particle mixture at predefined positions in nanochannels through capillary forces only, resulting in distinct trapping lines. We show that individual lines can be used as an on-chip fluorescence-linked immunosorbent assay for multiplexed detection of serological immunoglobulin antibodies against viral proteins with high sensitivity. Further device versatility is exhibited by on-bead color multiplexing for simultaneous detection of IgG and IgM antibodies in convalescent human serum and by concurrent detection of anti-spike (SARS-CoV-2) and anti-hemagglutinin (Influenza A) antibodies. The device’s applications can be further extended to detect a plethora of diseases simultaneously in a reliable and straightforward manner.

scholarly journals Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

2014 ◽  
Vol 53 (3) ◽  
pp. 1002-1004 ◽  
Author(s):  
Sophie Edouard ◽  
Elsa Prudent ◽  
Philippe Gautret ◽  
Ziad A. Memish ◽  
Didier Raoult
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Scale Detection ◽  
The Cost

We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.

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