Jun 26, 2023

Public workspaceGenetic Characterization of Schistosomes V.1

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3nobv4o/v1
  • 1Biomedical and Public Health Research Unit, CSIR - Water Research Institute
Yvonne A. Ashong
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo3nobv4o/v1
Protocol Citation: Frank Twum Aboagye, Isaac Owusu-Frimpong, Naa A. Kuma, Samuel .K. Armoo, Mike Y. Osei-Atweneboana, Yvonne A. Ashong 2023. Genetic Characterization of Schistosomes. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3nobv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 20, 2023
Last Modified: July 04, 2023
Protocol Integer ID: 83740
Keywords: Schistosomes, Cercariae, PCR, Vector, Snails, Xenomonitoring
Funders Acknowledgement:
COUNTDOWN (UKAID)
Grant ID: PO6407 to LSTM
Abstract
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.
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
Materials
Consumables
PCR tubes (0.2 ml)
1.5 ml microcentrifuge tube
Filtered tips 100 - 1000μL
Filtered tips 20 - 200μL
Filtered tips 2 - 20μL
Filtered tips 0.5 - 10μL
Autoclavable glass bottles (500 mL)

Reagents
OneTaq® 2X Master Mix
Nuclease-free water
Polyvinylpolypyrrolidone (PVPP)
Proteinase K (20 ng/mL)
Agarose Powder
TBE Buffer (1 X)
TE Buffer (1X)
6X Loading Dye
Ethidium Bromide
Molecular Weight Marker (50 bp and 100 bp)
Zymo Quick DNA Miniprep Kit (Zymo Research, USA)

Equipment and Materials
Automatic micropipettes
Thermocycler (Eppendorff)
Microcentrifuge (Eppendorff)
MagNa Lyser
Vortex Mixer
Microwave
Gel Electrophoresis tank and power supply system
Ultraviolet Light Transilluminator



Safety warnings
1. β-mercaptoethanol has a pungent smell, it should be opened in a well-aerated space.
2. Handle all solutions as potential biohazard materials
3. Care should be taken during the electrophoresis process as it involves the use of electricity.
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
Cercariae Suspension Pre-Treatment
Cercariae Suspension Pre-Treatment
Centrifuge the cercariae suspension at Centrifigation1600 rpm for Duration00:05:00 to concentrate the cercaria

5m
Pipette off the supernatant leaving Amount200 µL of cercariae suspension for nucleic acid isolation.

Schistosome Vector (Snail) Pre-Treatment
Schistosome Vector (Snail) Pre-Treatment
Crush each snail in Amount200 µL of 1XTE Buffer using a pestle in a Amount1.5 mL microcentrifuge tube. Ensure the end mixture is as fine as possible.

Nucleic Acid Isolation
Nucleic Acid Isolation
36m
36m
Transfer Amount200 µL of the pre-treated sample (cercariae suspension or snail) into a sterile Amount1.5 mL microcentrifuge tube.

Add Amount200 µL of Concentration2 Mass / % volume PVPP in 1XPBS and vortex the sample with glass beads at Shaker3000 rpm for Duration00:01:00

1m
Add Amount400 µL of Genomic Lysis Buffer and Amount10 µL of Proteinase K (Concentration20 ng/mL ) to the sample.

Vortex briefly and incubate the sample at Temperature56 °C for 3 - 5 hours or DurationOvernight

20m
Vortex the sample at Shaker3000 rpm for Duration00:00:30 and centrifuge at Centrifigation10000 rpm for Duration00:01:00

1m 30s
Transfer the supernatant into a Zymo-Spin IIC column in a new collection tube.
Centrifuge at Centrifigation10000 rpm for Duration00:01:00 . Discard the flow-through liquid.

1m
Transfer the Zymo-Spin IIC column into a new collection tube
Add Amount200 µL of DNA Pre-Wash Buffer to the spin column and centrifuge at Centrifigation10000 rpm for Duration00:01:00 .

1m
Add Amount500 µL of gDNA Wash Buffer to the spin column and centrifuge at Centrifigation10000 rpm for Duration00:01:00

1m
Transfer the Zymo-Spin IIC column into the sterile Amount1.5 mL microcentrifuge tube.

Add Amount100 µL of DNA Elution Bufferto the spin column and incubate for Duration00:30:00

30m
Centrifuge at Centrifigation13000 rpm for Duration00:00:30 to elute the DNA.

30s
Store the DNA at Temperature-20 °C pending further analysis.

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

ABC
ConcentrationReagent1X Volume (µL)
2XOne Taq Mastermix5.0
10 µMForward Primer2.0
10 µMReverse Primer2.0
NANuclease-Free Water1.6
Template DNA3.0
Total Reaction Volume10.0
ABCDE
Schistosome speciesForward PrimerReverse PrimerFragment SizePrimer Tm
ITS - Schistosome sp.TCT TGA CCG GGG TAC CTAATT AAG CCA CGA CTC GAG CAC691 bp60.1°C
S. mansoniGAG GGG TCT GGT TTT GGT GTGCA GAT AAA GCC ACC CCT GT659 bp58.7°C
S. haematobiumTTG AGC CTAT GGG TGG TGG TACC AGT AAC ACC ACC TAT CGT410 bp58.7°C
S. bovisTGG GCA TCC TGA GGT GTA TCAC AGG ATC AGA CAA ACG AGT ACC301 bp55.6°C
S. haematobium/S. bovis hybridCCT CCA TTA TCT ATA TCT GAG AAT TCTCGA AGT CTT AAA ATC CAC ACA ACT141 bp55.6°C

Thermal Cycling Conditions
Thermal Cycling Conditions

ABCD
StepTemperatureTimeCycles
Initial Denaturation95°CNA
Denaturation95°C45 seconds40
AnnealingTm45 seconds
Extension72°C45 seconds
Final Extension72°C5 minutesNA
Tm: Refer to the primer list for the individual annealing temperatures of the primer pairs
NA: Not applicable

Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
20m
20m
To prepare Concentration1.5 Mass / % volume agarose solution, weigh Amount1.5 g of agarose into a glass beaker containing Amount100 mL 1XTBE Buffer

Microwave the solution until the agarose completely dissolves and forms a clear solution.
Allow the solution to cool at TemperatureRoom temperature to about Temperature50 °C .

Add Amount5 µL of Concentration10 mg/mL Ethidium Bromide to the agarose solution. Swirl to ensure complete mixing of the stain with the agarose solution.

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 Duration00:20:00 at TemperatureRoom temperature

20m
Gently remove the combs from the solidified agarose gel and move the casting tray into the electrophoresis tank.
Note
Ensure the agarose gel is submerged in the 1XTBE Buffer in the electrophoresis tank

Load Amount5 µL to Amount12 µL the SampleSample into each well (mix 1µL of 6X Loading with 5µL of amplicon).

Load Amount3 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.

Electrophorese the amplicons at 100 volts until the molecular weight marker has travelled two-thirds of the length of the agarose gel.
Visualize the agarose gel under UV light using the transilluminator.
Expected Results
Expected Results
Mk: 100 bp molecular weight marker
P1 – P3: S. mansoni (659 bp);
P4 – P6: S. haematobium (410 bp);
P7 – P9: S. bovis (301 bp);
P10 – P12: S. haematobium/S. bovis (141 bp)

Protocol references
Lockyer AE, Olson PD, Ostergaard P, Rollinson D, Johnston DA, Attwood SW, et al. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology. 2003;126(Pt 3):203-24.
WHO. Scistosomiasis 2022 [Available from: https://www.who.int/news-room/fact-sheets/detail/schistosomiasis.
Chitsulo L, Engels D, Montresor A, Savioli L. The global status of schistosomiasis and its control. Acta Trop. 2000;77(1):41-51.
Tchuem Tchuente LA, Rollinson D, Stothard JR, Molyneux D. Moving from control to elimination of schistosomiasis in sub-Saharan Africa: time to change and adapt strategies. Infect Dis Poverty. 2017;6(1):42.
Hailegebriel T, Nibret E, Munshea A. Prevalence of Schistosoma mansoni and S. haematobium in Snail Intermediate Hosts in Africa: A Systematic Review and Meta-analysis. J Trop Med-Us. 2020;2020.