Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Virus-Infected Samples
2.2. Clinical Samples
2.3. DNA Extraction
2.4. Multiplex PCR
2.5. Amplicon Library Preparation
2.6. Flongle Loading and Sequencing
2.7. Analysis
2.8. MPXV Real-Time PCR
3. Results and Discussion
3.1. Establishment of the Rapid Amplicon Nanopore Sequencing (RANS) Approach for the Differential Detection of MPXV and Other Vesicle-Forming Pathogens
3.2. Laboratory Samples
3.3. Evaluation of RANS in Clinical Samples
4. Summary and Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Subfamily | Chordopoxvirinae | Alphaherpesvirus | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Genus | Orthopoxvirus | Parapoxvirus | Moll. | Yata. | Simplexvirus | Varic. | |||||||||
Area | bp | VARV | MPXV | VACV | CPXV | CMLP | ECTV | ORF | BPSV | MOLL | TANA | HSV1 | HSV2 | HBV | VZV |
Chem | 597 | √ | √ | √ √ | √ | √ | |||||||||
R60 | 638 | √ √ | √ √ | √ √ | √ √ | √ √ | √ √ | ||||||||
R56 | 652 | √ √ | √ √ | √ √ | √ √ | √ | √ | ||||||||
Y71 | 785 | √ | √ | √ | √ √ | √ | √ √ | ||||||||
m8121L | 720 | √ √ | √ | √ | √ | √ √ | √ √ | √ √ | |||||||
Mine | 1017 | √ √ | √ | √ √ | √ | √ √ | √ √ | √ √ | √ √ | ||||||
B100 | 355 | √ √ | √ √ | √ √ | |||||||||||
ORF45 | 530 | √ √ | √ √ | √ √ | √ √ | ||||||||||
ORF27 | 460 | √ √ | √ √ | √ √ | √ √ |
Mix | Primer | Sequence |
---|---|---|
A | Mine-sF | CGATACGACGGGCGTACTAGCGAGGTGCTCwGCGAGAAGTTCAC |
Mine-sR1 | CGATACGACGGGCGTACTAGCGGGCAGCACCAGCATGAACTTG | |
Mine-sR2 | CGATACGACGGGCGTACTAGCGGGCAGCATGAGCATGAACTTG | |
Y71-F | CGATACGACGGGCGTACTAGCGCCCGTwTATGGATCwATTCAAGA | |
Y71-R | CGATACGACGGGCGTACTAGCGCCTCTTCCyTCyGGATCCTTAGA | |
R60-F | CGATACGACGGGCGTACTAGCGCAATATGAACAAGAAATAGAATCGTTAGAAG | |
R60-R | CGATACGACGGGCGTACTAGCGGCGCTTCTATATCTCTCATTAGCTAGAA | |
ORF45-F | CGATACGACGGGCGTACTAGCGTGCGAyGAyCACATGCCG | |
ORF45-R | CGATACGACGGGCGTACTAGCGTCCTGGCTGCTrTTkCCCTC | |
B | Chem-F | CGATACGACGGGCGTACTAGCGCAAmCATyATATGGGAATCGATGTTA |
Chem-R | CGATACGACGGGCGTACTAGCGTCGAyATACTTrAATCCATCCTTGAC | |
ORF27-F1 | CGATACGACGGGCGTACTAGCGCGTGGCCGACAACTGCCT | |
ORF27-F2 | CGATACGACGGGCGTACTAGCGACAGTGGCGGATAACTGCCT | |
ORF27-R1 | CGATACGACGGGCGTACTAGCGCGTGCTCTGCCACACGTG | |
ORF27-R2 | CGATACGACGGGCGTACTAGCGCGTGCTCTGCCACACGTG | |
B100-mF | CGATACGACGGGCGGACTAGCGGTACCACCCCAGCCAGTACG | |
B100-R | CGATACGACGGGCGTACTAGCGAyGCGCACCTCGTTCAT | |
R56-F | CGATACGACGGGCGTACTAGCGTTACCACGTCTGGATAGGAGATTGT | |
R56-R | CGATACGACGGGCGTACTAGCGCGTGTTCTTAGTTGCTTAGCTGAAAC | |
m8121-sF1 | CGATACGACGGGCGTACTAGCGTACTTGGAAAAGAATTTGGACCAA | |
m8121-sF2 | CGATACGACGGGCGTACTAGCGGAGAAATTCCATTTAATATGAAAGACATG | |
m8121-sR1 | CGATACGACGGGCGTACTAGCGCATGATAATATTAAAGATAAAGCGCTGAC | |
m8121-sR2 | CGATACGACGGGCGTACTAGCGGGTTCGCTTAAAAATATAGACTTGTTAAATG | |
Multiplex adapter | P* CGATACGACGGGCGTACTAGCG |
Sample # | Virus Strain Cultured | % Mapped Reads | Species Detected | Nearest Detected Strain (Accession Number) |
---|---|---|---|---|
1 VACV | Vaccinia Lister | 94.9 | Vaccinia virus | Vaccinia Lister (KX061501) |
2 VACV | Vaccinia Western Reserve | 93.1 | Vaccinia virus | Vaccinia Western Reserve (AY243312) |
3 ECTV | Ectromelia Moscow | 91.0 | Ectromelia virus | Ectromelia Moscow (AF012825) |
4 CMLP | Camelpox Negev 2016 | 98.4 | Camelpox virus | Camelpox Negev 2016 (MK901851) |
5 CPXV | Cowpox Brighton Red | 97.0 | Cowpox virus | Cowpox Brighton Red (AF482758) |
6 MPXV | Monkeypox virus Israel | 99.1 | Monkeypox virus | Monkeypox virus Israel 2018 (MN648051) |
7 VZV | Human herpesvirus 3 | 96.8 | Human herpesvirus 3 | Human herpesvirus 3 YCO1 (KU926318) |
8 HSV1 | Human herpesvirus 1 | 93.8 | Human herpesvirus 1 | Mckrae (MN136524) Human herpesvirus 1 |
Clinical Sample # | Source | MPXV Ct GE WA | % Mapped Reads | Species Detected | Nearest Strain (Accession Number) | |
---|---|---|---|---|---|---|
2022 | vesicle swab | 24.7 | 25.0 | 99.6 | Monkeypox virus | MPXV/Germany/2022/RKI015 (ON694331.1) |
2023 | throat swab | 32.2 | 32.7 | 99.6 | Monkeypox virus | MPXV/Germany/2022/RKI020 (ON694337.1) |
2043 | throat swab | 38.8 | 38.5 | 99.8 | Monkeypox virus | MPXV/Germany/2022/RKI03 (ON682263.2) |
2072 | semen | 34.1 | 33.7 | 99.5 | Monkeypox virus | MpxV/Spain/MD-HGUGM-6532064/2022 (ON720849) |
2012 | vesicle swab | undet. | undet. | 95.0 | Human herpesvirus 3 | YC02 (KU926319) |
2068 | vesicle swab | undet. | undet. | 99.9 | Human herpesvirus 3 | YC02 (KU926319) |
2089 | vesicle swab | undet. | undet. | 89.6 | Human herpesvirus 2 | HG52 (Z86099) |
2090 | vesicle swab | undet. | undet. | 97.9 | Molluscum contagiosum virus subtype 1 | MCV1_P05S02A (MN931749) |
2114 | vesicle swab | undet. | undet. | 0.0 | unknown | unknown |
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Israeli, O.; Guedj-Dana, Y.; Shifman, O.; Lazar, S.; Cohen-Gihon, I.; Amit, S.; Ben-Ami, R.; Paran, N.; Schuster, O.; Weiss, S.; et al. Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens. Viruses 2022, 14, 1817. https://doi.org/10.3390/v14081817
Israeli O, Guedj-Dana Y, Shifman O, Lazar S, Cohen-Gihon I, Amit S, Ben-Ami R, Paran N, Schuster O, Weiss S, et al. Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens. Viruses. 2022; 14(8):1817. https://doi.org/10.3390/v14081817
Chicago/Turabian StyleIsraeli, Ofir, Yehoudit Guedj-Dana, Ohad Shifman, Shirley Lazar, Inbar Cohen-Gihon, Sharon Amit, Ronen Ben-Ami, Nir Paran, Ofir Schuster, Shay Weiss, and et al. 2022. "Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens" Viruses 14, no. 8: 1817. https://doi.org/10.3390/v14081817