Rapid Detection of Measles Virus Using Reverse Transcriptase/Recombinase Polymerase Amplification Coupled with CRISPR/Cas12a and a Lateral Flow Detection: A Proof-of-Concept Study
Abstract
:1. Introduction
2. Material and Methods
2.1. Biological Materials
2.2. Genotyping
2.3. Design of Measles Specific Primers, Exo Probe and crRNAs
2.4. Real-Time RT-RPA Reaction Conditions
2.5. RT-RPA CRISPR/Cas12a Reaction and Lateral Flow Detection
2.6. Statistical Analysis
3. Results
3.1. RPA Primer Pair Validation
3.2. Analytical Performances of RT-RPA-CRISPR-LFD Assay on Reference Standard
3.3. Diagnostic Performances of RT-RPA-CRISPR-LFD Assay on Biological Samples
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Oligo Name | Sequence | Position |
---|---|---|
Meas_NS1 | CAA-AGG-CGG-TTA-CGG-CYC-CAG-ACA-CRG-CAG-CTG-AT | 643-677 |
Meas_NR1 | ACC-ACA-TCC-AAC-CAT-TTT-CTC-TCC-AAT-CTA-AAT-TC | 763-729 |
Meas_Probe | AAT-CTA-AAT-TCA-CCA-ACT-ACC-CTT-CTT-TGT-(dT-FAM)G(THF)-G(dT-BHQ)G-TAY-TTT-ATC-CAC-C-(C3) | 735-691 |
crRNA1 | UAA-UUU-CUA-CUA-AGU-GUA-GAU-UUG-GGU-GUA-CUU-UAU-CCA-CCU-UC | 710-688 |
crRNA2 | UAA-UUU-CUA-CUA-AGU-GUA-GAU-UUG-GGU-GUA-UUU-GAU-CCA-CCU-UC | 710-688 |
Reporter | 6-FAM-TTATT-Biotin |
Sample Type | Samples Correctly Detected | Analytical Specificity † % | Diagnostic Sensitivity * % (95% CI) | Diagnostic Specificity § % | Accuracy ‡ % (95% CI) |
---|---|---|---|---|---|
Measles (crRNA1) | 24/27 | 89 (71–96) | 94 (87–100) | ||
Measles (crRNA1 and 2) | 26/27 | 96 (82–99) | 98 (93–100) | ||
Healthy | 29/29 | 100 | |||
Other viruses § | 20/20 | 100 |
RNA Extract | Genotype | RT-PCR | RT-RPA | |||
---|---|---|---|---|---|---|
Fluorescence | Agarose | Fluorescence | crRNA1 | crRNA2 | ||
(Ct) | Electrophoresis | (TwistAmp Exo) | ||||
C1 | D8 | 19.8 | + | + | + | |
C2 | D8 | 19.9 | + | + | + | |
C3 | ND | 20.1 | + | + | + | |
C4 | D8 | 20.5 | + | + | + | |
C5 | D8 | 20.7 | + | + | + | |
C6 | D8 | 21.1 | + | + | + | |
C7 | D8 | 21.9 | + | + | + | |
C8 | D8 | 22.1 | + | + | + | |
C9 | D8 | 23.6 | + | + | + | |
C10 | B3 | 23.8 | + | + | − | − |
C11 | D8 | 23.8 | + | + | + | |
C12 | D8 | 24.5 | + | + | + | |
C13 | D8 | 25 | + | + | + | |
C14 | D8 | 26 | + | + | + | |
C15 | D8 | 26.1 | + | + | + | |
C16 | ND | 26.2 | + | + | + | |
C17 | D8 | 26.5 | + | + | + | |
C18 | D8 | 26.8 | + | + | + | |
C19 | D8 | 27 | + | + | + | |
C20 | A | 27.2 | + | + | + | |
C21 | B3 | 28.5 | + | + | − | + |
C22 | B3 | 28.9 | + | + | − | + |
C23 | D8 | 29.5 | + | + | + | |
C24 | ND | 31 | − | + | + | |
C25 | ND | 31 | − | − | + | |
C26 | ND | 35.5 | − | − | + | |
C27 | ND | 36.1 | − | − | + |
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Pinchon, E.; Henry, S.; Leon, F.; Fournier-Wirth, C.; Foulongne, V.; Cantaloube, J.-F. Rapid Detection of Measles Virus Using Reverse Transcriptase/Recombinase Polymerase Amplification Coupled with CRISPR/Cas12a and a Lateral Flow Detection: A Proof-of-Concept Study. Diagnostics 2024, 14, 517. https://doi.org/10.3390/diagnostics14050517
Pinchon E, Henry S, Leon F, Fournier-Wirth C, Foulongne V, Cantaloube J-F. Rapid Detection of Measles Virus Using Reverse Transcriptase/Recombinase Polymerase Amplification Coupled with CRISPR/Cas12a and a Lateral Flow Detection: A Proof-of-Concept Study. Diagnostics. 2024; 14(5):517. https://doi.org/10.3390/diagnostics14050517
Chicago/Turabian StylePinchon, Elena, Steven Henry, Fanny Leon, Chantal Fournier-Wirth, Vincent Foulongne, and Jean-François Cantaloube. 2024. "Rapid Detection of Measles Virus Using Reverse Transcriptase/Recombinase Polymerase Amplification Coupled with CRISPR/Cas12a and a Lateral Flow Detection: A Proof-of-Concept Study" Diagnostics 14, no. 5: 517. https://doi.org/10.3390/diagnostics14050517