A Multiplex, Fluorescent, and Isothermal Method for Detecting Genetically Modified Maize

Bektaş, Ali

doi:10.1007/s12161-017-1041-9

Cited by 11 publications

(9 citation statements)

References 37 publications

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“…ALERT’s central reaction is RT-LAMP with QUASR reporting for detection of viral RNA, reducing the possibility of false positives and allowing for multiplexing applications. We demonstrate, following other QUASR-LAMP publications [34], that this mode of signal generation is compatible with at least two targets in the same closed tube (BPIFA1 and SARS-CoV-2 or SARS-CoV-2 and Influenza B). A simple LED excitation source and a colored plastic gel filter allows easy discrimination between positive and negative QUASR signals.…”

Section: Resultssupporting

confidence: 80%

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Preprint

Self Cite

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The COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests, that bind and detect the surface proteins of a virus, are rapid and inexpensive but suffer from high false negative rates. To address this problem, we have created an inexpensive, simple, and robust 60-minute Do-It-Yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μl) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, 4) Quality control with a nasopharyngeal-specific mRNA target, and 5) Co-detection of other respiratory viruses, such as Influenza B, by duplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for centralized labs and hospitals. With minimal effort, SARS-CoV-2-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms or nucleic acid-based markers.

show abstract

Section: Resultssupporting

confidence: 80%

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…ALERT’s central reaction is RT-LAMP with QUASR reporting for detection of viral RNA, reducing the possibility of false positives and allowing for multiplexing applications. We demonstrate, following other QUASR-LAMP publications [ 39 ], that this mode of signal generation is compatible with at least two targets in the same closed tube (BPIFA1 and SARS-CoV-2 or SARS-CoV-2 and influenza B). A simple light-emitting diode (LED) excitation source and a colored plastic gel filter allows easy discrimination between positive and negative QUASR signals.…”

Section: Discussionsupporting

confidence: 80%

Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2

Bektaş¹,

Covington²,

Aidelberg

et al. 2021

Viruses

Self Cite

View full text Add to dashboard Cite

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

show abstract

“…The insertion of biotin into amplicons by biotin-labelled primers is unproblematic regarding the efficiency of amplication due to Aedes aegypti (Wolbachia infection) 85,86 Ammonia-oxidizing enzyme in environmental bacteria 64 Avian reovirus 70 Bacteriophage MS2 73 Bemisia tabaci 60 BRAF allele (V600E) 83 Brucella 72 Caenorhabditis elegans 68 Chikungunya virus 73 Chlamydia trachomatis 61 Dengue virus 87,112,156 Diarrheal disease 82 Escherichia coli 68 Fomitiporia torreyae 59 Fulviformes umbrinellus 59 Fusarium oxysporum f. sp. lycopersici (point mutations in xylem 3 (SIX3) gene) 66 Genetically modied maize [77][78][79][80][81] Haemophilus ducreyi 101 Haemophilus inuenzae 105 hBRCA1 68 HeLa 68 Hepatitis B and C virus 62,87,107 Herpes simplex virus 1 (HSV1) US4 83 Human immunodeciency virus 87,101 HLA-B*15:02 allele 163 Human papillomavirus 153,171 Human T-lymphotropic virus 1 101 Inuenza virus 63,114 Lambda DNA 68 Leptospira 148 Leishmania 155 Listeria ivanovii 103 Listeria monocytogenes 103 Methicillin-resistant Staphylococcus aureus (MRSA) 71,166 Middle east respiratory sy...…”

Section: Heterogeneous Methodsmentioning

confidence: 99%

“…Similar approaches allowing visual endpoint detection through the addition of quencher oligonucleotides aer amplication were demonstrated for the human immunodeciency virus (HIV) and genetically modied maize. [77][78][79][80][81] Detection of diarrheal disease by QUASR was performed on a centrifugal microuidic platform. 82 QUASR is limited to biplex LAMP if visual detection is performed due to overlapping of the colours.…”

Section: Alternately Binding Quenching Probe Competitive Lamp (Abc-lamp)mentioning

confidence: 99%