scholarly journals Optical Biosensor Platforms Display Varying Sensitivity for the Direct Detection of Influenza RNA

Biosensors ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 367
Author(s):  
Samantha Courtney ◽  
Zachary Stromberg ◽  
Adán Myers y Gutiérrez ◽  
Daniel Jacobsen ◽  
Loreen Stromberg ◽  
...  
Keyword(s):  
Influenza A ◽  
Optical Biosensor ◽  
Molecular Beacons ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Probe Design ◽  
Sequence Coverage ◽  
High Coverage ◽  
Fast Evaluation ◽  
High Background

Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention’s (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC’s probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.

Development of an antigen-ELISA and a colloidal gold–based immunochromatographic strip based on monoclonal antibodies for detection of avian influenza A(H5) viruses

2021 ◽  
pp. 104063872110275
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Linfang Cheng ◽  
Hangping Yao ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Disease Virus ◽  
Colloidal Gold ◽  
Rapid Detection ◽  
Influenza A ◽  
Field Investigation ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Immunochromatographic Strip ◽  
Antigen Elisa

Avian influenza A(H5) viruses (avian IAVs) pose a major threat to the economy and public health. We developed an antigen-ELISA (ag-ELISA) and a colloidal gold–based immunochromatographic strip for the rapid detection of avian A(H5) viruses. Both detection methods displayed no cross-reactivity with other viruses (e.g., other avian IAVs, infectious bursal disease virus, Newcastle disease virus, infectious bronchitis virus, avian paramyxovirus). The ag-ELISA was sensitive down to 0.5 hemagglutinin (HA) units/100 µL of avian A(H5) viruses and 7.5 ng/mL of purified H5 HA proteins. The immunochromatographic strip was sensitive down to 1 HA unit/100 µL of avian A(H5) viruses. Both detection methods exhibited good reproducibility with CVs < 10%. For 200 random poultry samples, the sensitivity and specificity of the ag-ELISA were 92.6% and 98.8%, respectively, and for test strips were 88.9% and 98.3%, respectively. Both detection methods displayed high specificity, sensitivity, and stability, making them suitable for rapid detection and field investigation of avian A(H5) viruses.

scholarly journals A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 479
Author(s):  
Soumi Sukla ◽  
Prasenjit Mondal ◽  
Subhajit Biswas ◽  
Surajit Ghosh
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Magnetic Beads ◽  
Molecular Beacon ◽  
Detection System ◽  
Denv Infection ◽  
Molecular Beacons ◽  
Nucleic Acid Amplification ◽  
Complementary Sequence ◽  
Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

scholarly journals Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

2021 ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Gregory D. Van Duyne ◽  
Frederic D. Bushman
Keyword(s):  
Genetic Variants ◽  
Molecular Beacons ◽  
Spike Protein ◽  
Detection Methods ◽  
Gene Encoding ◽  
Detection And Identification ◽  
Rapid Spread ◽  
The World ◽  
Primer Sets ◽  
Current Detection

AbstractOver the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

scholarly journals Detection of Influenza A and B Viruses and Respiratory Syncytial Virus by Use of Clinical Laboratory Improvement Amendments of 1988 (CLIA)-Waived Point-of-Care Assays: a Paradigm Shift to Molecular Tests

2018 ◽  
Vol 56 (7) ◽  
Author(s):  
Marwan M. Azar ◽  
Marie L. Landry
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Therapy ◽  
Influenza A ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Molecular Tests ◽  
Syncytial Virus ◽  
Clinical Laboratory Improvement Amendments

ABSTRACT An accurate laboratory diagnosis of influenza, respiratory syncytial virus (RSV), and other respiratory viruses can help to guide patient management, antiviral therapy, infection prevention strategies, and epidemiologic monitoring. Influenza has been the primary driver of rapid laboratory testing due to its morbidity and mortality across all ages, the availability of antiviral therapy, which must be given early to have an effect, and the constant threat of new pandemic strains. Over the past 30 years, there has been an evolution in viral diagnostic testing, from viral culture to rapid antigen detection, and more recently, to highly sensitive nucleic acid amplification tests (NAAT), as well as a trend to testing at the point of care (POC). Simple rapid antigen immunoassays have long been the mainstay for POC testing for influenza A and B viruses and respiratory syncytial virus (RSV) but have been faulted for low sensitivity. In 2015, the first POC NAAT for the detection of influenza was approved by the Food and Drug Administration (FDA), ushering in a new era. In 2017, the FDA reclassified rapid influenza diagnostic tests (RIDTs) from class I to class II devices with new minimum performance standards and a requirement for annual reactivity testing. Consequently, many previously available RIDTs can no longer be purchased in the United States. In this review, recent developments in Clinical Laboratory Improvement Amendments of 1988 (CLIA)-waived testing for respiratory virus infections will be presented, with the focus on currently available FDA-cleared rapid antigen and molecular tests primarily for influenza A and B viruses and RSV.

Impact of molecular testing on reported Clostridoides difficile infection rates

2019 ◽  
Vol 41 (3) ◽  
pp. 306-312 ◽  
Author(s):  
Iulian Ilieş ◽  
James C. Benneyan ◽  
Tiago Barbieri Couto Jabur ◽  
Arthur W. Baker ◽  
Deverick J. Anderson
Keyword(s):  
Change Point Detection ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Testing ◽  
Infection Rates ◽  
Hospital Level ◽  
The Mean ◽  
Point Detection ◽  
The Impact ◽  
Individual Hospital

AbstractBackground:The reported incidence of Clostridoides difficile infection (CDI) has increased in recent years, partly due to broadening adoption of nucleic acid amplification tests (NAATs) replacing enzyme immunoassay (EIA) methods. Our aim was to quantify the impact of this switch on reported CDI rates using a large, multihospital, empirical dataset.Methods:We analyzed 9 years of retrospective CDI data (2009–2017) from 47 hospitals in the southeastern United States; 37 hospitals switched to NAAT during this period, including 24 with sufficient pre- and post-switch data for statistical analyses. Poisson regression was used to quantify the NAAT-over-EIA incidence rate ratio (IRR) at hospital and network levels while controlling for longitudinal trends, the proportion of intensive care unit patient days, changes in surveillance methodology, and previously detected infection cluster periods. We additionally used change-point detection methods to identify shifts in the mean and/or slope of hospital-level CDI rates, and we compared results to recorded switch dates.Results:For hospitals that transitioned to NAAT, average unadjusted CDI rates increased substantially after the test switch from 10.9 to 23.9 per 10,000 patient days. Individual hospital IRRs ranged from 0.75 to 5.47, with a network-wide IRR of 1.75 (95% confidence interval, 1.62–1.89). Reported CDI rates significantly changed 1.6 months on average after switching to NAAT testing (standard deviation, 1.9 months).Conclusion:Hospitals that switched from EIA to NAAT testing experienced an average postswitch increase of 75% in reported CDI rates after adjusting for other factors, and this increase was often gradual or delayed.

scholarly journals The Panther Fusion System with Open Access Functionality for Laboratory-Developed Tests for Influenza A Virus Subtyping

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Kathleen A. Stellrecht ◽  
Jesse L. Cimino ◽  
Vincente P. Maceira
Keyword(s):  
Open Access ◽  
Influenza A ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Influenza B ◽  
Fusion System ◽  
Valuable Complement ◽  
Syncytial Virus

ABSTRACT Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion (Fusion; Hologic) system has an array of highly sensitive in vitro diagnostic (IVD) real-time PCR assays for respiratory viruses, including an assay for influenza A (FluA) virus, influenza B (FluB) virus, and respiratory syncytial virus (RSV) (FFABR assay). The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays. We developed two LDTs for FluA virus strain typing on the Panther Fusion instrument, enabling side-by-side testing with the FFABR assay. The LDT-FAST assay uses proprietary primers and probes designed by Hologic for the Prodesse ProFAST+ (PFAST) assay. The exWHO-FAST assay is an expanded redesign of the WHO-recommended reverse transcriptase PCRs (RT-PCRs). To evaluate the performance of these two LDTs, 110 FluA virus-positive samples were tested. Of these, 104 had been subtyped previously; 54 were H3, 46 were 09H1, and 4 were fsH1. All were appropriately subtyped by both LDTs. Of the untyped FluA virus samples, three were subtyped as H3 by both LDTs and two were subtyped as H3 by the LDT-FAST assay only. The sample not subtyped by either LDT was retested with the FFABR assay and was now negative. Limit-of-detection (LOD) analyses were performed with five FluA virus strains. The LDT-FAST LODs were similar to the FFABR assay LODs, while the exWHO-FAST LODs were higher for two H3N2 strains, findings that were explained by analysis of primer/probe homology. In conclusion, either FluA virus typing assay would be a valuable complement to the Panther Fusion respiratory menu given the performance of these LDTs, the system’s full automation, and the ability to split eluates for both IVD and LDT testing.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

Integrating nucleic acid sequence-based amplification and microlensing for high-sensitivity self-reporting detection

The Analyst ◽  
2020 ◽  
Vol 145 (23) ◽  
pp. 7528-7533
Author(s):  
Feiyue Teng ◽  
Xinpei Wu ◽  
Tao Hong ◽  
Gary B. Munk ◽  
Matthew Libera
Keyword(s):  
Electron Beam ◽  
Nucleic Acid ◽  
Solid Phase ◽  
High Sensitivity ◽  
Molecular Beacons ◽  
Solution Phase ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Sequence ◽  
Viral Detection

We use electron-beam patterned functional microgels to integrate self-reporting molecular beacons, dielectric microlenses, and solid-phase and/or solution-phase nucleic acid amplification in a viral-detection microarray model.

Nucleic acid amplification-based methods for microRNA detection

2015 ◽  
Vol 7 (6) ◽  
pp. 2258-2263 ◽  
Author(s):  
Hui-Ling Chen ◽  
Meng-Meng Guo ◽  
Hao Tang ◽  
Zhan Wu ◽  
Li-Juan Tang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Basic Principles ◽  
Microrna Detection

This review traces the basic principles of several nucleic acid amplification-based microRNA detection methods that have been developed in recent three years.

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