TITLE: Schistosome DNA Isolation and Colorimetric LAMP protocol for the detection of Schistosomiasis
AUTHORS: Isaac Owusu-Frimpong, Linda B. Debrah, samuel.k.armoo, Edward J. Tettevi, Yvonne A. Ashong, Naa A. Kuma, Frank Twum FTA Aboagye, Bright K. Idun, Mike Y. Osei-Atweneboana

[DESCRIPTION]
Schistosomiasis keeps devastating the world despite many measures put in place by control programs. Molecular diagnostics has played a significant role in the detection and monitoring of the treatment of the disease. Loop-medicated isothermal amplification has been developed with increased sensitivity and specificity to help the diagnosis of schistosomiasis. This protocol aids the successful isolation of schistosome DNA from urine and stool using Quick-DNA Miniprep kits (ZymoResearch, USA) with slight modification. Progressively, specific-specific Schisto-LAMP assay has been developed from the cytochrome c oxidase subunit 1 (COX1) gene of the schistosome mitochondrial genome. In addition, to effectively detect successful amplification, a laboratory-based colorimetric buffer which is pH dependent, was used. The neutral red dye changes from pale yellow to pink when positive and to a deep yellow color if negative. This protocol spells out the performance of two LAMP assays to detect S. haematobium and S. mansoni in urine and stool, respectively. 

[BEFORE_START]
Make sure urine and stool specimens are well stored since storage can affect the integrity of the DNA. 
Please make to sterilize all consumables to be used for this experiment. 
Please work in a sterile environment to prevent carryover contamination. 
pH adjustment of the colorimetric buffer must be made with a well-cleaned pH meter to prevent contamination.

[GUIDELINES]
Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
Preparation of 2X colorimetric solution mix protocol
Preparation of 50 mM Neutral Red Dye Stock protocol.
Preparation of 25X Dye Stocks of Neutral red protocol.
Preparation of 10X SCH-LAMP Primer mix protocol
Preparation of Colourimetric LAMP reaction protocol
Agarose Gel Electrophoresis Protocol

[STEPS]
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
1. 0.12-gram stool is suspended in 300µL of PBS (1X phosphate-buffered saline) with 2% PVPP (polyvinylpolypyrrolidone) in a 2ml microcentrifuge tube containing glass beads and vortex.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
2. Freeze the sample for 1 hour at -50 ºC or overnight at -20 ºC.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
3. Defrost and bead beat in MagNA lyser for 5 minutes at 3000 rpm.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
4. Add 400μL Genomic Lysis Buffer and 10μL Proteinase K to the mixture and mix completely by vortexing for 15-30 seconds, then incubate at 56ºC overnight (14-16 hours).
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
5. Mix by vortexing at 3000rpm for 15-30 seconds and centrifuge at 10,000 x g for one minute.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
6. Transfer the supernatant to a Zymo-Spin™ IIC Column in a Collection Tube. Centrifuge at 10,000 x g for one minute. Discard the Collection Tube with the flow-through.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
7. Transfer the Zymo-Spin™ IIC Column to a new Collection Tube. Add 200μl of DNA Pre-Wash Buffer to the spin column. Centrifuge at 10,000 x g for one minute.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
8. Add 500μl of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for one minute. Repeat the centrifugation to dry the spin column.
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
9. Transfer the spin column to a clean microcentrifuge tube. Add 70μl DNA Elution Buffer to the spin column. Incubate for 15-30 minutes at room temperature and then centrifuge at top speed for 30 seconds to elute the DNA. 
SECTION: Schistosoma-DNA Extraction from Stool (Zymo Kits) Protocol
10. Repeat the elution step into a different microcentrifuge tube as the second elution. The eluted DNA can be used immediately for molecular-based applications or stored ≤-20ºC for future use.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
11. Mix urine sample by pipetting up and down, and aliquot 300µL in a sterile 1.5ml microcentrifuge. 
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
12. Add 200µL of PBS (1X phosphate-buffered saline) with 2% PVPP (polyvinylpolypyrrolidone) to the sample and vortex.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
13. Add 300μL BioFluid & Cell Buffer and 10μL Proteinase K to the mixture and mix completely by vortexing for 15-30 seconds, then incubate at 56ºC overnight (14-16 hours).
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
14. Mix by vortexing at 3000rpm for 15-30 seconds and centrifuge at 10,000 x g for one minute.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
15. Add 300μL Genomic Binding Buffer to the digested sample. Mix thoroughly or vortex for 10-15 seconds.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
16. Transfer the supernatant to a Zymo-Spin™ IIC Column in a Collection Tube. Centrifuge at 10,000 x g for one minute. Discard the Collection Tube with the flow-through.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
17. Transfer the Zymo-Spin™ IIC Column to a new Collection Tube. Add 200μl of DNA Pre-Wash Buffer to the spin column. Centrifuge at 10,000 x g for one minute.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
18. Add 500μl of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for one minute. Repeat the centrifugation to dry the spin column.
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
19. Transfer the spin column to a clean microcentrifuge tube. Add 70μl DNA Elution Buffer to the spin column. Incubate for 15-30 minutes at room temperature and then centrifuge at top speed for 30 seconds to elute the DNA. 
SECTION: Schistosoma-DNA Extraction from Urine (Zymo Kit Plus) Protocol
20. Repeat the elution step again in a different microcentrifuge tube as the second elution. The eluted DNA can be used immediately for molecular-based applications or stored ≤-20ºC for future use.
SECTION: Preparation of 2X colorimetric solution mix protocol
21. 
 

SECTION: Preparation of 50 mM Neutral Red Dye Stock protocol.
22. Weigh 0.7222g of Neutral red powder in a beaker and dissolve in 40 mL ddH2O.
SECTION: Preparation of 50 mM Neutral Red Dye Stock protocol.
23. Mix thoroughly with a magnetic stirrer, and adjust the pH to 8.6 – 8.8 with 1 M KOH.
SECTION: Preparation of 50 mM Neutral Red Dye Stock protocol.
24. Top up the solution to the 50 mL mark with ddH2O.
SECTION: Preparation of 25X Dye Stocks of Neutral red protocol.
25. 


 

SECTION: Preparation of 10X SCH-LAMP Primer mix protocol
26. 
 

 

SECTION: Preparation of Colourimetric LAMP reaction protocol
27. 


 

SECTION: Agarose Gel Electrophoresis Protocol
28. The successful LAMP amplification can be confirmed with agarose gel electrophoresis and visualized under UV light. The steps for the gel preparation is as follows;