Molecular identification of arthropods
Transcript of Molecular identification of arthropods
![Page 1: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/1.jpg)
Molecular identification of arthropods
Oleg MEDIANNIKOV
ESCMID Online Lecture Library
© by author
![Page 2: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/2.jpg)
Molecular identification?
• PCR followed by the sequencing of the amplicons
• Comparison of the obtained sequence with the records in the database
• Taking the decision on whose gene was amplified
ESCMID Online Lecture Library
© by author
![Page 3: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/3.jpg)
ESCMID Online Lecture Library
© by author
![Page 4: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/4.jpg)
… of arthropods? Why?
Arthropod
Infestation (parasitic diseases)
Vectors
Crustaceans
Pentastomida
Arachnidae Myriapoda
Insects
ESCMID Online Lecture Library
© by author
![Page 5: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/5.jpg)
Arthropod vectors
Acariens
Insects Ticks Mites
Soft Hard Lice: typhus, recurrent fever, trench fever Fleas: plague, murine typhus Mosquitoes: malaria, yellow fever, Saint-Louis encephalitis, dengue, West Nile, Chikungunya, Rift valley, Japanese encephalitis, dirofilariasis Biting midges (Cératopogonidae) Mansonella, veterinary pathogens Sandflies: leishmaniasis, pappataci fever Tsetse flies: sleeping sickness Triatoma: Chagas disease Lutzomia flies: bartonellose Bloodsucking Diptera: filariases Etc…
Scrub typhus
Spotted fevers, Lyme disease, Crimean-Congo hemorrhagic fever, tick-borne encephalitis, anaplasmose, ehrlichiose, babesiosis, tularemia, Q fever
Borreliosis (relapsing fever)
… of arthropod vectors?
ESCMID Online Lecture Library
© by author
![Page 6: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/6.jpg)
Other medically important arthropods (vectors or infesting)
• crayfish (paragonimiasis)
• ants (dicrocoeliasis)
• Cyclops (Guinea worm)
• Scorpions, spiders, Solifugae (wind scorpions)
• bedbugs
• House dust mites
ESCMID Online Lecture Library
© by author
![Page 7: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/7.jpg)
… but why?
1. Knowledge is the power 2. Clinical application
Presence of pathogenic agent, Capacity to transmit the pathogen
ESCMID Online Lecture Library
© by author
![Page 8: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/8.jpg)
… what to amplify/sequence?
Mitochondrial DNA: 1. Quantity (100-10,000 separate
copies of mtDNA per cell) 2. Quality (no recombination,
haploid) 3. House-keeping genes, but high
mutation rates.
ESCMID Online Lecture Library
© by author
![Page 9: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/9.jpg)
Human mitochondrion
ESCMID Online Lecture Library
© by author
![Page 10: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/10.jpg)
http://www.barcodeoflife.org/
ESCMID Online Lecture Library
© by author
![Page 11: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/11.jpg)
… what to amplify/sequence?
• COI so called « Folmer » primers (around 658 bps): – LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3‘ – HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'
• Redesigned primers: – Redesigned forward primer jgLCO1490 5’- TITCIACIAAYCAYAARGAYATTGG-3’ – Redesigned reverse primer jgHCO2198 5’-TAIACYTCIGGRTGICCRAARAAYCA-3’
• Ribosomal RNA – Mitochondrial (12S, 16S) – Cytoplasmic (28S, 5.8S, 18S and 5S). May have 300-400 repeats
• Cytochrome B
ESCMID Online Lectu
re Library
© by author
![Page 12: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/12.jpg)
Rools for successful identification
• Good quality specimen = good quality amplicon = good quality sequence
• Proper alignment. Do not forget to cut off the primers’ sequences
• Exhaustive search (BLAST)
• Decision taking
ESCMID Online Lecture Library
© by author
![Page 13: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/13.jpg)
blast.ncbi.nlm.nih.gov/
ESCMID Online Lecture Library
© by author
![Page 14: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/14.jpg)
BLAST results
ESCMID Online Lecture Library
© by author
![Page 15: Molecular identification of arthropods](https://reader035.fdocuments.in/reader035/viewer/2022062216/62a3b715e97b6866707787eb/html5/thumbnails/15.jpg)
BLAST problems
• Quality of submitted sequences: « garbage »
• Lack of sequences of all animals: Barcode of life is yet to do
• Delicate identification: no genetic criteria for most taxons. To be developped. ESCMID Online Lectu
re Library
© by author