TITLE: LAMP Protocol for Entomopathogenic Serratia spp. in Insect Frass
AUTHORS: Nicholas P Doidge

[DESCRIPTION]
A protocol for the detection of insect-associated Serratia spp. in the frass of live insects, using a LAMP assay targeting the ureD gene of urease-positive species from the Serratia marcescens complex. In order to improve the DNA extraction from folivorous insects a bacterial separation and concentration protocol has been developed prior to the DNA extraction, but this part of the protocol is not required for carnivorous and omnivorous insects. To improve accuracy the LAMP assay is conducted in triplicate, and a pH-linked calorimetric assay changes from pink to yellow for positive results. This protocol is potentially suitable for field applications with minimal equipment required.

[STEPS]
SECTION: Bacterial Separation and Concentration
3. Collect 300 mg fresh   per DNA extraction and store at -20 °C until processing.
 

 

SECTION: DNA Extraction
12. Briefly spin the PowerBead Pro tube to ensure the beads have settled at the bottom of the tube.
 

SECTION: LAMP Assay
29. Make up stock solutions of the LAMP primers using MG H20. Add the same amount of MG H20 to the dried primers as the primer amount in moles to make 1000 micromolar (µM) stock solutions. Dillute the F3, B3, LF and LB primers 1:10 to make 100 micromolar (µM) working stocks.
SECTION: Bacterial Separation and Concentration
5. Add 1.0 mL sterile phosphate buffered saline (PBS) to each of the five microcentrifuge tubes containing  .
SECTION: Equipment and Materials Required
1. Equipment Required:
-Pipettors and pipette tips in a range of sizes (1000 µL 100 µL 10 µL )
-Qiagen Tissue Lyser II, alternatively a high-speed horizontal vortex
-Low speed swing bucket centrifuge (500 x g) (ideally but can be done without)
-High speed microcentrifuge (16000 x g)
-Equipment for measuring DNA concentration and quality (e.g. Thermo Fisher Nanodrop or Implen Nanophotometer)
-Heating element fitted to the tube size used for LAMP (PCR block, heating block, hot water bath)
-Access to a fridge (4 °C) and freezer (-20 °C) or ice to achieve these temperatures
SECTION: Equipment and Materials Required
2. Materials Required (per extraction):
-5 x 1.5 mL microcentrifuge tubes
-5 mL sterile phospate buffered saline
-Sterile surface for handling frass (e.g. parafilm or petri dish)
-1 x sterile forceps (or reusable forceps sterilised between each pooled sample)
-1 x sterile pestle (Axygen Cat. AX-PES-15-B-SI-1)
-1 x 10 mL graduated centrifuge tube (ideally screw-top)
-1 x 2.0 mL round-bottomed microcentrifuge tube
-DNeasy PowerSoil Pro kit (Qiagen Cat. 47014/6)
-Molegular Grade water (MG H20)
-WarmStart® Colorimetric LAMP 2X Master Mix with UDG (NEB Cat. M1804S/L)
-LAMP Primers (IDT):
    Serr_ureD_F3: ATCGCCTGACGCTCGA
    Serr_ureD_B3: TAGAGCAGCGTCGCCGT
    Serr_ureD_FIP: CTGCATCTGCGCCGCGTA-GCCTGCGGGCATGTCA
    Serr_ureD_BIP: TCTGGAGATGATGCCCGATCCG-CGTAATGCGGGTATCGGTAA
    Serr_ureD_LF: TGGCGTGGATCTTGGTCG
    Serr_ureD_LB: CTGCGGTTCGCGCTTCA
-3 x 0.2 mL PCR tubes
SECTION: Bacterial Separation and Concentration
4. Tip out   onto a sterile surface (e.g. parafilm or petri dish) and using sterile forceps add approximately 60 mg frass (for D.australis approximately 2 adult, 8 subadult or 17 juvenile frass pieces) to each of five 1.5 mL microcentrifuge tubes (300 mg total).
 

 

SECTION: Bacterial Separation and Concentration
6. Using one sterile pestle for each pooled frass sample crush the   in each tube until well homogenised in the PBS. A 100 µL pipette tip can be used to seperate crushed frass from the bottom of the tube and assist with homogenization.
SECTION: Bacterial Separation and Concentration
7. Transfer the contents of all five microcentrifuge tubes into one 10 mL graduated centrifuge tube.
SECTION: Bacterial Separation and Concentration
8. Mix the 10 mL tube well by shaking. Using a large swing bucket centrifuge spin down the graduated centrifuge tube at 600 x g for 5 mins.
 

 

SECTION: Bacterial Separation and Concentration
9. Using a 1000 µL pipette tip transfer up to 1.8 mL (2 x 900 µL) of supernatant to a clean 2.0 mL round-bottom microcentrifuge tube. To maximise bacterial yield, aspirate close to the pellet without disturbing it.
SECTION: Bacterial Separation and Concentration
10. Pellet the supernatant in the 2.0 mL tube using a standard microcentrifuge at 16000 x g, 5 Room temperature for 5 mins. Carefully discard most of the supernatant from the 2.0 mL tube, taking care not to disturb the bacterial pellet.

SECTION: Bacterial Separation and Concentration
11. Repeat Steps 8 -10   , adding to the same 2.0 mL tube. If significant supernatant is still present in the 10 mL tube can repeat a third time to maximise bacterial cell yield.

SECTION: DNA Extraction
13. Using a 1000 µL pippette tip re-homogenise the bacterial cell pellet in 800 µL of Solution CD1, then transfer all the contents of the 2.0 mL tube to a PowerBead tube. Shake briefly to mix.
SECTION: DNA Extraction
14. Conduct cell disruption and lysis by shaking the PowerBead tube in the Tissue Lyser II at 25 Hz for 10 mins.
 

SECTION: DNA Extraction
15. Centrifuge the PowerBead tube at 15000 x g for 1 min. Transfer the supernatant to a clean 2.0 mL microcentrifuge tube. Expect 500 µL to 700 µL supernatant, which may contain some frass particles.
SECTION: DNA Extraction
16. Add 200 µL Solution CD2 and mix well by gentle pipetting.
SECTION: DNA Extraction
17. Centrifuge the 2.0 mL tube at 15000 x g for 1 min. Avoiding the pellet, transfer up to 700 µL supernatant to a clean 2.0 mL microcentrifuge tube.
SECTION: DNA Extraction
18. Add 600 µL of Solution CD3 and mix well by gentle pipetting.
SECTION: DNA Extraction
19. Load 650 µL of lysate into an MB Spin Column. Centrifuge at 15000 x g for 1 min.
SECTION: DNA Extraction
20. Discard the flow-through and repeat the step above   to ensure all of the lysate has passed through the spin column.
SECTION: DNA Extraction
21. Carefully place the spin column into a clean 2.0 mL collection tube. Take care not to splash any of the flow-through onto the spin column.
SECTION: DNA Extraction
22. Add 500 µL of Solution EA to the spin column. Centrifuge at 15000 x g for 1 min.
SECTION: DNA Extraction
23. Discard the flow-through and place the spin column back into the same 2.0 mL collection tube.
SECTION: DNA Extraction
24. Add 500 µL of Solution C5 to the spin column. Centrifuge at 15000 x g for 1 mins.
SECTION: DNA Extraction
25. Discard the flow-through and place the spin column into a clean 2.0 mL collection tube. Centrifuge at 16000 x g for 2 min.
SECTION: DNA Extraction
26. Carefully place the spin column into a clean 1.5 mL elution tube, without allowing any flow through to be transferred to the new tube on the spin column. Carefully add 100 µL of MG H20 to the centre of the white filter membrane in the spin column. Centrifuge at 15000 x g for 1 min.
SECTION: DNA Extraction
27. Discard the spin column. The DNA is now contained in the flow-though and ready to be used. If not using immediately store at -20 °C. Measure the concentration and quality of extracted DNA using spectrophotometry (e.g. Thermo Fisher Nanodrop or Implen Nanophotometer).
SECTION: LAMP Assay
30. Prepare a 25X LAMP Primer Mix using the LAMP worksheet. The Primer Mix can be stored at -20 °C for use in multiple assays.   

SECTION: LAMP Assay
28. Prepare a positive control of Serratia ureilytica AM923 DNA at a concentration of app. 50 ng/µL. Can be stored at 4 °C for up to 7 days.

SECTION: LAMP Assay
31. Prepare the working Master Mix using the LAMP worksheet, with MG H2O, 1 X LAMP Master Mix and 1 X LAMP Primer Mix. Use 2 or 3 LAMP assays per extraction (duplicate or triplicate) to ensure confidence in the results.   

SECTION: LAMP Assay
32. Vortex the working Master Mix thoroughly and spin briefly. Transfer 24 µL to each of PCR tube. Add 1 uL MG H2O to the negative control tubes and close caps on negative controls.
SECTION: LAMP Assay
33. Add 1 uL DNA sample to each of the tubes, including the positive controls loaded last to minimise false positives. Close all caps snugly and spin down PCR tubes to ensure DNA sample is in contact with the Master Mix.
 

SECTION: LAMP Assay
34. Incubate the LAMP assays for 30 mins at 65 °C.
 

SECTION: LAMP Assay
35. At the end of the 30 min incubation, the results of the LAMP assays can be interpreted and photographed.
 

SECTION: LAMP Assay
36. The LAMP products can be stored at 4 °C for several months, but over time the amplification products will break down, affecting the pH and changing the colour. If left at Room temperature the products will break down and cause colour change much faster.