A. Staining Microfilariae after Blood Filtration (Qualitative)
- Glass slides
- Giemsa stain (Modified Solution, Fluka Analytical 48900)
- Methanol (Fisher BP1105 4)
Note: Follow the mf Filtration protocol to filter microfilariae from blood.
After filtration, place the filter on a glass slide.
Fix dried mf on the filter in 100% methanol for 30-60 sec.
Wash lightly with H2O and let air dry.
Stain with diluted Giemsa (1:20) for 20 min.
- Store at RT.
Wash with H2O and let air dry.
Note: Protocol adapted from the CDC Bench aid for Brugia spp. and from Medeiros et al 2018.
B. Loop-Mediated Isothermal Amplification (LAMP) Diagnostic
Materials for DNA Extraction from Whole Blood
- Quick-DNA 96 plus kit (Zymo D4071)
- Disposable lab coats
- Sterile H2O
- Plate foil cover (zymo C2007-4)
DNA Extraction Protocol
Follow the kit protocol for Solid Tissue with the following modifications:
- Step 1: Add 50 μL of blood instead of water.
- After step 3 (Add 2 volumes of Genomic Binding Buffer): spin the Deep Well Block for 2 min at 3,500g.
- Add the supernatant to the Zymo-Spin 96 plate.
Check a few samples for purity and concentration using the NanoDrop.
Cover the plates with plate foil covers and store DNA at 4 °C overnight or -20 °C for long term storage.
Materials for LAMP Reaction
- DNA from blood extractions
- 100 µM stock primers
- WarmStart Colorimetric LAMP 2x Master Mix (NEB M1804L)
- 10X GuHCL
- Sterile H2O
- 96 well plates
M. perstans M. ozzardi FIP (F1c+F2): TGTGAGCACATTTCAGTAAGT-GATGAATCCACTAAATTCWC FIP (F1c-F2): CGCAAACAGAAGCCCGAAAC-GCTCGCAATTTCATAGTGG BIP (B1+B2c): GGATTCTTTCTAAAAGTTGAG-GATCGATTTCGTTAAAAACAGY BIP(B1c-B2): CTTGCGCGTAGCATTAGATCC-TCCGAAATGTATACGACAGAT F3: ACAGTTGATTATTTGAAGGTGCTR F3: GCACGAAATGTTTTTGTACG B3: AYAATGATTATTTYTAAAGAATC B3: CGTATCACCGTTGATGACG LF: AGACTTGATTACTGTTTGG LF: AAGCCTAAGCCTAAGCCTGA LB: ACAATTTGGTAATCGCTTAAACTG LB: GCACATCTTCAATCTCCTCTTGC
Note * W= A or T, Y= C or T, R = A or G * Higher Sensitivity can be achieved with HPLC purified primers.
Make a 10x Primer Mix by following the chart below, be cognizant of species:
Primers µL of 100 µM stock FIP 16 µL F3 2 µL BIP 16 µL B3 2 µL LF 4 µL LB 4 µL H2O 56 µL Final Volume 100 µL
Assemble 20 µL reactions in 96-well plates.
Add 2 µL of DNA to each well of a 96-well plate.
Create a master mix outlined in table below, pipette gently to mix.
Add 18 µL of the master mix to the 96-well plate containing the DNA.
Reagent One Reaction 100 Reactions WarmStart Colorimetric LAMP 2x Master Mix 10.0 µL 1 mL 10x Primer Mix 2.0 µL 200 µL 10X GuHCL 2.0 µL 200 µL H2O 4 µL 400 µL Final Volume 18 µL 1.8 mL
Note: H2O can be used instead of DNA as a non-template control.
Incubate at 63° for either 30 minutes (M. ozzardi ) or 60 minutes (M. perstans)
- Pink = Negative
- Yellow = Positive
Note: Samples can be removed periodically and checked for color change without affecting the reaction.
Image plates after the reaction using the Cytiva ImageQuant 800 and record results.