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Mansonella Diagnostics

A. Staining Microfilariae after Blood Filtration (Qualitative)

Materials

  • Glass slides
  • Giemsa stain (Modified Solution, Fluka Analytical 48900)
  • Methanol (Fisher BP1105 4)

Note: Follow the mf Filtration protocol to filter microfilariae from blood.

Protocol

  1. After filtration, place the filter on a glass slide.

  2. Fix dried mf on the filter in 100% methanol for 30-60 sec.

  3. Wash lightly with H2O and let air dry.

  4. Stain with diluted Giemsa (1:20) for 20 min.

    • Store at RT.

  5. 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

  1. 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.

  2. Check a few samples for purity and concentration using the NanoDrop.

  3. 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

  • Primers

    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.

LAMP Protocol

  1. 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

  2. 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.

    • Gently mix.

    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.

  3. 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.

  4. Image plates after the reaction using the Cytiva ImageQuant 800 and record results.