TITLE: Genetic Characterization of Schistosomes
AUTHORS: Frank Twum Aboagye, Isaac Owusu-Frimpong, Naa A. Kuma, Samuel .K. Armoo, Mike Y. Osei-Atweneboana, Yvonne A. Ashong

[DESCRIPTION]
Schistosomiasis is a parasitic disease transmitted through water by blood-fluke trematodes of the genus Schistosoma. After malaria and soil-transmitted helminthiasis, it is the third most important parasitic tropical disease. An estimated 236 million people are infected, with another 700 million at risk of infection. It is estimated that 90% of all schistosomiasis cases occur in Africa. The disease is regarded as a public health threat, with numerous debilitating effects on growth, well-being, and overall health. The importance of snail-intermediate hosts in schistosomiasis transmission cannot be overstated, and thus monitoring the prevalence and distribution of Schistosoma cercariae is critical for indirect estimation of schistosomiasis in human or animal populations. As a result, developing sensitive tools to aid in the characterization of specific schistosomes is critical, and this protocol outlines procedures ranging from DNA isolation to PCR and the expected outcomes from newly developed oligonucleotides.

[BEFORE_START]
Aliquot the required volume of reagents for extraction into a sterile tube for the assay. 
Allow the frozen reagents to thaw completely at 4°C before use and avoid centrifuging to thaw.

Reagent Preparation for Nucleic Acid Isolation
Genomic Lysis Buffer
Add 500µL of β-mercaptoethanol to 100 mL of Genomic Lysis Buffer. Shake slightly to mix

2% PVPP in 1 X TE Buffer
Dissolve 2 g of PVPP powder in 100 mL of 1X TE Buffer

[GUIDELINES]
Cercaria Suspension Pre-Treatment
Schistosome Vector (Snail) Pre-Treatment
Nucleic Acid Isolation
Preparation of PCR Master Mix and Reaction
Thermal Cycling Conditions
Agarose Gel Electrophoresis
Expected Results

[STEPS]
SECTION: Nucleic Acid Isolation
4. Transfer 200 µL of the pre-treated sample (cercariae suspension or snail) into a sterile 1.5 mL microcentrifuge tube.

SECTION: Nucleic Acid Isolation
5. Add 200 µL of 2 Mass / % volume PVPP in 1XPBS and vortex the sample with glass beads at   for 1 min

SECTION: Nucleic Acid Isolation
6. Add 400 µL of Genomic Lysis Buffer and 10 µL of Proteinase K (20 ng/mL) to the sample.

SECTION: Nucleic Acid Isolation
7. Vortex briefly and incubate the sample at 56 °C  for 3 - 5 hours or 20 min

SECTION: Nucleic Acid Isolation
8. Vortex the sample at   for 30 s and centrifuge at 10000 rpm for 1 min

SECTION: Nucleic Acid Isolation
9. Transfer the supernatant into a Zymo-Spin IIC column in a new collection tube.
SECTION: Nucleic Acid Isolation
10. Centrifuge at 10000 rpm for 1 min. Discard the flow-through liquid.

SECTION: Nucleic Acid Isolation
11. Transfer the Zymo-Spin IIC column into a new collection tube
SECTION: Nucleic Acid Isolation
12. Add 200 µL of DNA Pre-Wash Buffer to the spin column and centrifuge at 10000 rpm for 1 min.

SECTION: Nucleic Acid Isolation
13. Add 500 µL of gDNA Wash Buffer to the spin column and centrifuge at 10000 rpm for 1 min

SECTION: Nucleic Acid Isolation
14. Transfer the Zymo-Spin IIC column into the sterile 1.5 mL microcentrifuge tube.

SECTION: Nucleic Acid Isolation
15. Add 100 µL of DNA Elution Bufferto the spin column and incubate for 30 min

SECTION: Nucleic Acid Isolation
16. Centrifuge at 13000 rpm for 30 s to elute the DNA.

SECTION: Nucleic Acid Isolation
17. Store the DNA at -20 °C pending further analysis.

SECTION: Preparation of PCR Master Mix and Reaction
18. Prepare the master mix for each schistosome species separately following the protocol:

 
 
 

2. Pipette off the supernatant leaving 200 µL of cercariae suspension for nucleic acid isolation.

SECTION: Cercariae Suspension Pre-Treatment
1. Centrifuge the cercariae suspension at 1600 rpm for 5 min to concentrate the cercaria

SECTION: Schistosome Vector (Snail) Pre-Treatment
3. Crush each snail in 200 µL of 1XTE Buffer using a pestle in a 1.5 mL microcentrifuge tube. Ensure the end mixture is as fine as possible.

SECTION: Thermal Cycling Conditions
19. 
 

SECTION: Agarose Gel Electrophoresis
20. To prepare 1.5 Mass / % volume agarose solution, weigh 1.5 g of agarose into a glass beaker containing 100 mL 1XTBE Buffer

SECTION: Agarose Gel Electrophoresis
21. Microwave the solution until the agarose completely dissolves and forms a clear solution.
SECTION: Agarose Gel Electrophoresis
22. Allow the solution to cool at Room temperature to about 50 °C. 

SECTION: Agarose Gel Electrophoresis
23. Add 5 µL of 10 mg/mL Ethidium Bromide to the agarose solution. Swirl to ensure complete mixing of the stain with the agarose solution. 

SECTION: Agarose Gel Electrophoresis
24. Pour the stained agarose molten solution into a casting tray (5mm deep) fitted with combs of the desired size for a well. Allow the molten solution to solidify for about 20 min at Room temperature

SECTION: Agarose Gel Electrophoresis
25. Gently remove the combs from the solidified agarose gel and move the casting tray into the electrophoresis tank.
 

SECTION: Agarose Gel Electrophoresis
26. Load 5 µL to 12 µL the   into each well (mix 1µL of 6X Loading with 5µL of amplicon). 

SECTION: Agarose Gel Electrophoresis
27. Load 3 µL of 100 or 50 bp (depending on the fragment size of the amplicon of interest) Molecular Weight Marker into the first well which will serve as a reference.

SECTION: Agarose Gel Electrophoresis
28. Electrophorese the amplicons at 100 volts until the molecular weight marker has travelled two-thirds of the length of the agarose gel.
SECTION: Agarose Gel Electrophoresis
29. Visualize the agarose gel under UV light using the transilluminator.
SECTION: Expected Results
30.