Jan 15, 2025
Version 1
LAMP Meat identification V.1
This protocol is a draft, published without a DOI.
- 1OBS
Protocol Citation: openbioscience Adrian 2025. LAMP Meat identification. protocols.io https://protocols.io/view/lamp-meat-identification-dsa46agw
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: In development
We are still developing and optimizing this protocol
Created: October 29, 2024
Last Modified: January 15, 2025
Protocol Integer ID: 112700
Abstract
Identifying species by identifying unique DNA sequences is common practice.
In this case we will identify the presence or absence of pork DNA in a meat sample.
We will use the alkaline method for DNA extraction and LAMP method for DNA amplification and detection.
This is not a new protocol but rather the detailing of a previously published protocol listed in the manuscript citation.
Materials
Labware: minimum 2 1.5 mL PCR tubes, 2 x 0.1 mL PCR tubes with flat caps, commonly known as low-profile PCR tubes that match the device well size.
Devices:, LAMP/PCR/Water bath and thermometer, scale, 1- 10 μl micropipette, 100- 1000 μl micropipette and tips, centrifuge, Optional: vortex, scale and boat.
Reagents: Primers, LAMP master-mix, DNase free water, aliquot of meat to be tested in a prepared form
Others: crushed ice and tray, nitrile gloves, pipette rack , tube rack, labeling pen.
Grand labware total for all the protocols required:
TRIS prep
50 ml falcon tube labeled Tris Buffer
Meat prep:
15 ml falcon for NaOH
100 ul Prepared sample
1.5 mL Final prepared sample centrifuge tube
LAMP
2 x1.5 uL centrifuge tubes for 10x primers pairs
2 x 100 ul Target and NTC tube for LAMP
1 x 1.5 ul centrifuge tube for 10x stain
Total:
1 x 50 mL
1 x 15 mL
4 x 1.5 ml3 100 uL
Safety warnings
Use appropriate PPE, follow Safe Work Practices and follow local regulation.
Prepare primers 100 uM concentrated stock for each of the 4 primers F3, B3, FIP and BIP
Prepare primers 100 uM concentrated stock for each of the 4 primers F3, B3, FIP and BIP
centrifuge the 4 lyophilized primer tubes 1 minute at 1000 G to move the powder to bottom as per IDT guide
Add 264 μL DNase free water to F3 tube ,
242 μl to B3 tube
277 μl to FIP tube
271 μl to BIP tube
Note: water can be substituted with 10 mM Tris pH8.0 (25 Celsius).
Note: the quantities for 100 μM were specified by IDP in the "oligonucleotide specification sheet" shipped with the IDT product.
Oligonucleotide specification sheet excerpt by IDT
Incubate the primer tubes at room temperature for at least 20 minutes following the addition of the water to allow for complete hydration of the DNA.
Relabel the original tubes to 100 μM F3, 100 μM B3, 100 μM FIP and 100 μM BIP.
Label several (for instance 2) new 1.5 mL centrifuge tubes 10 ul-50 μM F3B3 and add in each 5 μl F3 and 5 ul B3 aliquots.
We do this to minimize freeze thaw cycle impact.
Label several (for instance 2) new 1.5 mL centrifuge tubes 10 ul-50 μM FIP-BIP and add in each 5 μl FIP and 5 ul BIP aliquots.
We do this to minimize freeze thaw cycle impact.
Store all the 100 uM and all the 10 uL- 50 uM combined primer tubes at -15 Celsius
Prepare 10X stock of primers
Prepare 10X stock of primers
Make 10x of combined F3 and B3 primer stock.
The final reaction requires 0.2μM F3 so a 10x stock should be 2 μM. The same for B3.
For F3 the initial concentration(ic) is 50uM and the initial volume(iv) of the aliquot is 10 uL. ic*iv=fc*fv so fv=(50 uM*10uL)/2 uM=250 uL. Same for B3.
Add 250-10=240 μl of DNase free water to the 10 ul-50 μM F3B3 and re-label the tube to 10x F3B3 primer tube.
Note: some source recommends premixing the all primers in one 10x stock but we do only 2 pairs.
Note: Additional explanation of similar calculations at https://www.optigene.co.uk/wp-content/uploads/2012/06/OptiGene-LAMP-User-Guide-Assay-Design-Primers-1.pdf
Make 10x of combined FIP and BIP primer stock.
The final concentration for FIP is 1.6 μM so a 10x stock should be 16 μM. Same for BIP.
The initial concentration is 50 uM and the initial volume of the aliquot is 10uL. ic*iv=fc*fv so fv=(50uM*10uL)/16 uM=31.25 uL.
Add 31.25-10=21.25 μl of DNase free water to the 10ul@50 μM FIP tube and relabel the tube to 10x FIP-BIP primer tube.
vortex briefly to mix all
LAMP
LAMP
Take a 100 μL tube and label it and label it NTC (no template negative control).
Take a 100 μL tube and label it Target.
Prepare ice tray for the reaction. On ice, thaw all components ( dye, Superscript LAMP master mix and template) to be used.
Use the 10X F3-B3 stock to make the final 0.2μM μM F3-B3 concentration at 25uL.
2uM*iv=0.2 uM*25uL iv=0.2*25/2=2.5 uL
so add 2.5 ul of 10x F3-B3 10x stock to each target and NTC tube.
Use the 10X FIP-BIP stock to make the final 1.6 μM FIP-BIP concentration at 25uL.
16uM*iv=1.6 uM*25uL iv=1.6*25/16=2.5 uL
so add 2.5 ul of 10x FIP-BIP 10x stock to each target and NTC tube.
On ice tray we have 1X RT-LAMP Master Mix SuperScript IV (https://www.fishersci.ca/shop/products/superscript-iv-rt-lamp-master-mix-3/p-9568005- 1250 uL) .
The final volume should be 25μl and initial concentration of Master Mix stock is 2X.
RT-LAMP Master Mix SuperScript IV 2x*iv=1x*25ul iv=1*25/2=12.5 uL
so add 25uL*(1X/2X)=12.5 μl from RT-LAMP Master Mix SuperScript IV stock to each target and NTC tube.
On ice tray prepare SYTO 9 stain to 5 μM final concentration.
Note:
Superscript4 Invitrogen kit came with 30 μL of 5000 μM SYTO 9 stain as stated on the tube
ic*iv=fc*fv 5000uM*iv uL=5 uM*25 uL iv=5*25/5000=25/1000=0.025 uL. That is too little to accurately pipette so we need to dilute the initial stock to 10X concentration stock.
Prepare SYTO 9 stain 10x stock @ 50uM
Centrifuge the SYTO 9 tube for 1 min at 1000G to gather the stain at the bottom.
Label a new 1.5 mL centrifuge tube to 10X SYTO 9 stock.
Aliquot 2 ul SYTO 9 in a new 1.5 mL centrifuge tube.
ic*iv=fc*fv 5000uM*2uL=50uM*fv so fv=5000*2/50=200 μL. Since we start with 2 ul we just need to add 198 ul water.
Add 198 ul DNase free water to the 10X SYTO 9 stock to a total of 200 ul
Store at -15 and protect from light
To add SYTO 9 stain to a final concentration of 5 μM we add 5uM/500uM*25 μL=2.5 μL from the newly prepared 10x SYTO9 stock
Add 2.5 μL 10X SYTO 9 stock to each target and NTC tube.
Add 1 μL template DNA to target tube and 1 μL DNase free water to NTC tube.
Add DNase free water to 25 μL to each target and NTC tube. That is 4ul for Target tube and 4ul to NTC tube.
Load target and negative control in the LAMP device and perform the LAMP protocol as specified by manufacturer as in https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/superscript-iv-rt-lamp-master-mix-detect-dna-app-note.pdf
Heat at 65 Celsius for 15-30 minutes. Note: that would be for fast reaction and this is a normal reaction so maybe 1 minute.
Inactivate at 95 Celsius for 2 minutes.
An orange color indicates no amplification, whereas a change from orange to
yellow-green demonstrates the presence of LAMP amplicons.
Protocol references
Girish et al: Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique. Published, journal pre-proof can be downloaded from Academia https://www.academia.edu/114013841/Rapid_detection_of_pork_using_alkaline_lysis_Loop_Mediated_Isothermal_Amplification_AL_LAMP_technique
Superscript4 RT LAMP master-mix
https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/superscript-iv-rt-lamp-master-mix-detect-dna-app-note.pdf. Please note on Table 2 the numbers were incorrect at the time of writing this protocol. The company support line was contacted and replied that they will be fixed.
For examples of calculations and some steps
IDT oligo resuspension guide https://www.idtdna.com/pages/education/decoded/article/tips-for-resuspending-and-diluting-your-oligonucleotides
IDT dilution calculator https://www.idtdna.com/Calc/Dilution/