describes isolation of DNA from adult helminths, larval stages, protozoa, faecal sample and arthropods

2. Endoparasites
Helminths
• Trematodes
• Cestodes
• Nematodes
Protozoa
Ectoparasites
(Arthropods)
Insects
Ticks and mites
Classification of Parasites

3. 1
• Collection of helminths from sample
2 • Trituration with pestle and morter in 200 μl PBS
3
• Addition of 10 ml of extraction buffer (10 mMTrisHCl, 0.1 mM EDTA and
0.5% SDS) for each 1 ml of suspension and incubation for 1 hr at 37 ºC.
4
• Addition of Proteinase K to a final concentration of 200 μg/ml and the
suspension is incubated at 55ºC for 3 hr.
5
• Phenol and chloroform isoamyl alcohol extraction procedures
Isolation of DNA from Helminths
Sambrook and Russell (2001)

4. • .
Isolation of DNA from larvae
Coles et al. (2006)
1
• Collection of larvae from coproculture
2 • Exsheathment of larva
3
Addition of 5 μl digestion buffer (1mMTris-HCl, 0.1mM EDTA and 5mg/ml
proteinase K).
4
These larvae were incubated overnight at 41ºC.
5
• Proteinase K was inactivated by incubation at 95ºC for 20 minutes.

5. • Stool is chemically complex and the extraction of DNA from stool samples is
extremely difficult.
• Haemoglobin breakdown products, such as bilirubin, bile acids and mineral
ions, that are present in the stool samples, can inhibit DNA amplification and
cause molecular assays to produce false-negative results.
• The stool samples are washed three times in sterile distilled water prior to
DNA isolation.
Isolation of DNA from faecal sample
Kuk et al ., 2012

6.  From whole Blood eg: Trypanosoma, Theileria, Babesia,
Anaplasma etc.
 From lymphocytes eg: Leishmania spp., Theileria spp.
 From uterine secretions eg: Tritrichomonas foetus
 From oocysts eg: Eimeria spp., Cryptosporidium spp.
 From culture on artificial media:
 NNN media: Trypanosoma, Leishmania spp
 Diamonds medium: Tritrichomonas foetus
 Balmuth’s medium: Entamoeba histolytica
Isolation of DNA from Protozoa

7. Isolation of DNA from blood protozoa

8. Isolation of DNA from blood protozoa
For Leishmania spp.:
Promastigotes obtained for
culture to peripheral blood from
healthy donors.
Isolation of white blood cells:
Ficoll separation method

9. • By glass-bead grinding:
 The suspended oocysts in TE buffer are put in a sterile, 2-ml round bottom microfuge
tube, 1–2 sterile, 4-mm glass beads are added, and the tube was vortexed vigorously.
 Breakage is monitored using a compound microscope 40x at 2-min intervals until all
the oocysts and their sporocysts appear to be ruptured approximately 10 min.
 The suspension containing freed sporozoites is added to 1 ml of CTAB buffer
containing proteinase K (100 µg/ml) , and incubated at 65ºC for 1 h.
 DNA is then purified by phenolrchloroform extraction followed by ethanol
precipitation. The air-dried DNA pellet was resuspended in MiliQ water or TE buffer.
 By freeze-thaw method: Cryptosporidium oocysts.
 15 cycles of freezing in LN2 followed by thawing at 65ºC in lysis buffer containing
SDS
Isolation of DNA from oocysts
Zhao et al., 2009
Harith et al., 2014

10. Isolation of DNA from arthropods
Zhao et al., 2009
Asghar et al., 2015

11. References
Kuk, S., & Cetinkaya, U. (2012). Stool sample storage conditions for the preservation
of Giardia intestinalis DNA. Memórias do Instituto Oswaldo Cruz, 107(8), 965-968.
Zhao, X., Duszynski, D. W., & Loker, E. S. (2001). A simple method of DNA extraction
for Eimeria species. Journal of Microbiological Methods, 44(2), 131-137.
Al-Warid, H. S. the effect of freezing-thawing on DNA extraction from
Cryptosporidium oocysts in fecal samples.
Coles, G.C., Jackson, F., Pomroy, W.E., Prichard, R.K., von Samson-Himmelstjerna,
G., Silvestre, A., Taylor, M.A. and Vercruysse, J. 2006. The detection of anthelmintic
resistance in nematodes of veterinary importance. Vet. Parasitol. 136: 167–185.
Sambrook, J. and Russell, D. W.2001. Molecular cloning: a laboratory manual.New
York Cold Spring Harbor Laboratory.
Asghar, U., Malik, M. F., Anwar, F., Javed, A., & Raza, A. (2015). DNA extraction
from insects by using different techniques: a review. Advances in Entomology, 3(04),
132.