| The monkeypox virus is the cause of the viral monkeypox disease. The monkeypox virus, a member of the Orthopoxvirus genus in the Poxviridae family, is closely related to the variola virus, which causes smallpox. It is important to note that monkeypox generally causes a milder illness. Monkeypox was first identified in laboratory monkeys in 1958, but the disease primarily affects rodents in the wild. | 
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First reported in humans in Africa in 1970 the disease can be transmitted to humans through direct contact with infected animals, such as rodents or primates, or through contact with bodily fluids, lesions, or contaminated materials. It can also spread from person to person through respiratory droplets or contact with contaminated materials.
Symptoms of monkeypox include fever, rash, and swollen lymph nodes. The rash often starts as flat, red spots (macules) on the face, trunk, and limbs, which then develop into raised bumps (papules), filled with clear fluid (vesicles), and then pus (pustules) before scabbing over. The illness usually lasts between 2 to 4 weeks.
Considered less severe than smallpox, but monkeypox it can still be serious, especially in young children, pregnant women, and individuals with weakened immune systems. Vaccination against smallpox has been shown to provide some protection against monkeypox, and there is also a specific vaccine for monkeypox that has been developed.
Monkeypox PCR Primers
Monkeypox PCR primers allow detection of the monkeypox virus. Specifically designed primers match sequences unique to the monkeypox virus genome.
These primers allow:
Detection and Diagnosis: PCR primers amplify viral DNA in patient samples and help confirm the presence of monkeypox infection.
Research: They are used in research settings to study the virus, track its spread, or evaluate the effectiveness of treatments and vaccines.
Surveillance: These primers help monitor outbreaks and understand epidemiological patterns.
Table 1: Primer Sets according to the CDC.
| | | | Temp | Time | Cycles |
| Assay | | 5’ - - - 3’ | ºC | sec | |
| Monkeypox Virus Generic | F | GGAAAATGTAAAGACAACGAATACAG | 95 | 20 | 1 |
| R | GCTATCACATAATCTGGAAGCGTA | 95 | 3 | 40 |
| P | FAM-AAGCCGTAATCTA{BHQ-1dT}GTTGTCTATCGTGT-SPACER C6 | 60 | 30 |
| Human DNA RNase P | F | AGATTTGGACCTGCGAGCG | | | |
| R | GAGCGGCTGTCTCCACAA | | | |
| P | FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1 | | | |
Table 2: Primer Sets developed by Maksyutov et al. 2016.
| Virus | | Oligonucleotide name | Oligonucleotide sequence |
| Variola Virus (smallpox) |
| VARV | P | VARV_B12R_probe | FAM-5′-CTGTCGGAGCCACAGTTTCGAGACG-3′-BHQ1 |
| B12R | F | VARV_B12R_upper | 5′-ATGTTCAAGCTGTTAATATCAATCTCG-3′ |
| | R | VARV_B12R_lower | 5′-TTTGCCACTGAACCATTCTATCAT-3′ |
| Monkeypox Virus |
| MPXV | P | MPXV_F3L_probe | JOE-5′-TGTAGGCCGTGTATCAGCATCCATT-3′-BHQ1 |
| F3L | F | MPXV_F3L_upper | 5′-CATCTATTATAGCATCAGCATCAGA-3′ |
| | R | MPXV_F3L_lower | 5′-GATACTCCTCCTCGTTGGTCTAC-3′ |
Table 3: Primer Sets developed by Khoo et al. 2022.
| # | Primer ID | Sequence (5′ → 3′) | Size (bp) | GC (%) | Tm (°C) |
| 1 | A28-F1 | CCTGGATAAACCACACATCTCC | 22 | 50.00 | 59.04 |
| | A28-R1 | ACTCATGCAGCATTCGAGTATT | 22 | 40.91 | 58.78 |
| | A28-Probe-F1 | ACTCTCCTATCTAATGCCGGTGTTCCA | 27 | 48.15 | 65.66 |
| 2 | A28-F2 | ACATTGTCGCATCGTGTTAAAT | 22 | 36.36 | 57.90 |
| | A28-R2 | AGTGGAGATGTGTGGTTTATCC | 22 | 45.45 | 58.37 |
| | A28-Probe-R2 | TGGATTTCAGGCAGAAGTTGGACCC | 25 | 52.00 | 65.62 |
| 3 | H3-F1 | CAGCGCCGTAGTAACTCTAATAA | 23 | 43.48 | 58.73 |
| | H3-R1 | ACCGAGCTTGTAATAGACAAAGA | 23 | 39.13 | 58.19 |
| | H3-Probe-R1 | CAGGAGGGTATGATGTTAGCTTATCCGC | 28 | 50.00 | 65.40 |
| 4 | H3-F2 | TTAGCAGCTACCGTTCCTATTC | 22 | 45.45 | 58.27 |
| | H3-R2 | ACACGATCCTCGTCTTGTTG | 20 | 50.00 | 58.50 |
| | H3-Probe-R2 | ACACCGCTTCGAAACCATGAAACC | 24 | 50.00 | 64.92 |
| 5 | H3-F3 | ACGTGTACATAACTCCTGGATAAC | 24 | 41.67 | 58.69 |
| | H3-R3 | CCGCTTCGAAACCATGAAAC | 20 | 50.00 | 58.60 |
| | H3-Probe-F3 | AGCAGCTACCGTTCCTATTCTAGACCA | 27 | 48.15 | 65.42 |
| 6 | DDRP-F1 | GGCAGACACGGACGATATTA | 20 | 50.00 | 57.71 |
| | DDRP-R1 | AGTGACTCTCCATCTTCTTCATC | 23 | 43.48 | 57.92 |
| | DDRP-Probe-F1 | TCCGATGATCTCACCGAATACGAGGA | 26 | 50.00 | 65.35 |
| 7 | DDRP-F2 | CTTCATGGTGGGAATATGCTCTA | 23 | 43.48 | 58.29 |
| | DDRP-R2 | AACCCGCATTGGCTACAT | 18 | 50.00 | 57.40 |
| | DDRP-Probe-R2 | AGCTGTCAATGAGGAATGGCTAACTGC | 27 | 48.15 | 66.02 |
| 8 | DDRP-F3 | CTCTACAGCAGTTAGCCATTCC | 22 | 50.00 | 59.05 |
| | DDRP-R3 | ATCGTCGTTGAACTCGAACC | 20 | 50.00 | 58.79 |
| | DDRP-Probe-F3 | ACTACTCCAATGTTTAACAAGGGCCA | 26 | 42.31 | 63.20 |
| 9 | DDRP-F4 | AGCCATTCCTCATTGACAGC | 20 | 50.00 | 58.74 |
| | DDRP-R4 | TCCACGGGAAGAGAATTCAATC | 22 | 45.45 | 58.64 |
| | DDRP-Probe-F4 | TAGCCAATGCGGGTTCGAGTTCAA | 24 | 50.00 | 65.49 |
| 10 | DDRP-F5 | CAACGTGTATCCTGGAGTATGG | 22 | 50.00 | 58.80 |
| | DDRP-R5 | GATCACAAGGCTGGTACAGATAA | 23 | 43.48 | 58.50 |
| | DDRP-Probe-F5 | TCGTGGATACTTGTGCGAAGCCAT | 24 | 50.00 | 65.28 |
| 11 | DDRP-F6 | TACCGGAACACTGGCTAGAA | 20 | 50.00 | 58.58 |
| | DDRP-R6 | CTGCGTACTTGATGAGCGTATTA | 23 | 43.48 | 59.22 |
| | DDRP-Probe-F6 | TATGGTGGTCGACGGATACGGACA | 24 | 54.17 | 65.59 |
Table 4: Primer and Probes Set according to Luciani et al. 2021.
| | Sequence; 5’ --- 3’ |
| F1 | CCDCAYCARYTVGCIACIBTIGAYT |
| R1 | GMDATIAYIGTYTTICCTGAICCCAT |
| R2 | GCCACGAATGTCTTACCACTTCCCAT |
| PA | FAM-WYRTGAAAYAWYADDRCDST-MGB |
| PE | FAM-TYATGAAAYADYADYAWNRCWYT-MGB |
| PC | FAM-ATRTGRAAHARYARHACRCTYYTRT-MGB |
| hGC | FAM-ATGTGRAASAGVARSAYRCT-MGB |
Reference
CDC Protocol: [CDC]
Khoo YW, Li S, Chong KP. In-silico primer designing and PCR for detection of monkeypox virus (MPXV). J Infect Public Health. 2022 Dec;15(12):1378-1380. [PMC]
Luciani, L., Inchauste, L., Ferraris, O. et al. A novel and sensitive real-time PCR system for universal detection of poxviruses. Sci Rep 11, 1798 (2021). [PMC]
Maksyutov RA, Gavrilova EV, Shchelkunov SN. Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay. J Virol Methods. 2016 Oct;236:215-220. [PMC]
Smallpox https://www.cdc.gov/smallpox/index.html
https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=10244
https://www.ncbi.nlm.nih.gov/datasets/taxonomy/10244/