LAMP Enzyme Mix
EXAMPLE 1
The functionality of the dry format containing the reagents necessary for LAMP were compared. The differences in the format are shown in Table 1.
TABLE 1
Standard LAMP kit Dry Format Lamp Kit
2 x Reaction Mix (1 tube) 1.5 ml Reaction Tube (1 tube)
2M betaine 32U Bst enzyme
40 mM Tris-Cl pH 8.8 [and 3U AMV reverse
20 mM KCl transcriptase (if RNA target)]
20 mM (NH4)2SO4 Primers (kit dependent)
16 mM MgSO4 dNTPs
dinucleotide triphosphates (dNTPs)
0.2% Tween 20
Primer Mix (1 tube) Aqueous Buffer (1 tube)
Primers (target specific) 25 mM Tris-HCl pH 8.8
12.5 mM KCl
10 mM MgSO4
12.5 mM (NH4)2SO4
0.125% Tween 20
1.25 M betaine
Enzyme Mix (1 tube)
8 U/μl Bst polymerase
[and 1 U/μl AMV reverse
transcriptase (if RNA target)]
50% Glycerol
Distilled Water Negative Control of
(1 tube) DNAse/RNAse free water
(1 tube)
Positive Control Positive Control
(1 tube) (1 tube)
Wet Format LAMP. In the standard LAMP kit, the kit components must be stored at −20° C. The recommended protocol is as follows: Remove reagents from −20° C. and thaw at room temperature. Once thawed, keep on ice. Prepare Master Mix (prepare on ice) either in 0.5 ml or 1.5 ml tubes. Briefly, the Master Mix is prepared by adding 12.5 μl 2× Reaction Mix; 2.5 μl Primer Mix; and 4.0 μl distilled water into a reaction tube. Reagents are mixed by tapping or inverting tube or vortex ˜1 second×3 times followed by a brief centrifugation. The tube was heated @ 95° C. for 5 minutes. Then, the tube was cooled on ice. After cooling, 1 μl Enzyme Mix was added to the tube, followed by vortexing and/or centrifuging. Once the Master Mix preparation was complete, 20 μl of Master Mix was dispensed into each sample and control tube (0.2 ml PCR tubes). 5 μl of DNA or RNA sample were added to the tube and mix by pipetting or taping, and then centrifuged briefly. The tubes were heated at ˜60° C. for 1 hour, followed by inactivation of the enzyme at 80° C. for 5 minutes. Turbidity was determined by visual inspection.
Dry Format LAMP. The dry format LAMP reagent preparation greatly reduces the number of steps, thereby reducing errors and increasing sensitivity. The components in the dry format LAMP reagent preparation can be stored at −20° C. to 30° C.
Preparation of dry format. Enzyme-containing solution was dialyzed against enzyme storage buffer that was glycerol-free using a tangential flow microdialyser. Typically, dialysis occurred in less than 2 hours. The dialyzed enzyme solution as well as undialyzed enzyme solution was dried using a lyophilizer. The undialyzed solution was unable to be dried after 24 hours. The tubes containing dried, dialyzed enzyme were stored in a sealed foil pouch containing desiccant.
Protocol. The reaction tube containing the dry reagent preparation was removed the from the foil pouch. 80 μl of the reaction buffer and 20 μl of the sample were added to each reaction tube. The contents were mixed by gently vortexing, and then heat at ˜60° C. for 1 hour. Turbidity was determined visually.
EXAMPLE 2
The purpose of this experiment was to determine if reverse transcriptase LAMP (RT-LAMP) would function if Bst polymerase and AMV reverse transcriptase were lyophilized in the same tube.
Materials included dNTPS (25 mM) (New England Biolabs); Eiken Norovirus GI primer mix set; dialyzed Bst DNA polymerase (˜37 u/μl, no glycerol); AMV reverse transcriptase (20 u/μl) (Stratagene); AMV dialysis buffer (200 mM KH2PO4, 2 mM dithiothreitol (DTT) and 0.2% Triton X-100), pH 7.2; reconstitution buffer (2×): 40 mM Tris-HCl pH 8.8, mM KCl, 16 mM MgSO4, 20 mM (NH4)2SO4, and 0.2% Tween 20; and betaine.
Procedure—Lyophilization of Enzyme Mix
1. Prepared enzyme dilutions
a. Bst 8 u/μl: 2.4 μl dialyzed enzyme +
7.6 μl dH2O
b. AMV 0.5 u/μl: 0.7 μl dialyzed enzyme +
9.3 μl dH2O
2. Prepare enzyme mix in three 0.2 ml tubes
a. Norovirus GI primer mix: 2.5 μl per tube
b. Diluted Bst 1.0 μl per tube
c. Diluted AMV 1.0 μl per tube
d. 25 mM dNTPs 1.4 μl per tube
3. Enzyme mix lyophilized 30 minutes.
4. Added reconstitution buffer components to reaction tube
a. 2X reaction buffer 12.5 μl/tube
b. Betaine 4.0 μl/tube
c. dH2O 3.5 μl/tube
d. Norovirus GI RNA or dH2O 5.0 μl/tube (1 positive/
2 negative)
5. Incubated at 63° C. for 60 minutes
6. Results interpreted visually.
Results—Lamp Reaction with Lyophilized Reagents:
Norovirus GI positive control: (+)
Water (negative control): (−), (−)
Conclusions: Reverse transcriptase LAMP can successfully be performed with AMV reverse transcriptase and Bst enzyme lyophilized in the same tube.
EXAMPLE 3
Purpose: The purpose of this experiment was to confirm the requirement to remove glycerol from the enzyme storage buffer prior to lyophilization.
Materials included dNTPS (25 mM) (New England Biolabs); Clostridium difficile TcdB (Toxin B) Loopamp primer set; Bst DNA polymerase (120 u/μl) (New England Biolabs); Bst DNA polymerase (8 u/μl) (New England Biolabs); and Bst DNA dialysis buffer (50 mM KCl, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol (DTT) and 0.1% Triton X-100), pH 7.5.
Procedure—Lyophilization of Enzyme Mix: 10 reactions tubes each were prepared for the undialyzed and dialyzed enzyme by preparing a 10.5 reaction volume for each enzyme condition in one tube and aliquoting single reaction volume into 10 tubes as follows:
10.5 volume per reaction tube
Undialyzed:
dNTP 58.8 μl 5.6 μl
Primer mix 42 μl 4.0 μl
Bst (8 u/μl) 84 μl 8.0 μl
Dialyzed:
dNTP 58.8 μl 5.6 μl
Primer mix 42 μl 4.0 μl
Bst (37 u/μl) 21 μl 2.0 μl
Lyophilization monitored through glass at 8 minutes, 30 minutes, 45 minutes, 60 minutes, 120 minutes and 27.5 hours (for undialyzed enzyme reagent tubes only). Reaction tubes containing dialyzed enzyme were removed after 2 hours of lyophilization. Reaction tubes containing undialyzed enzyme were removed after 27.5 hours of lyophilization.
Results—Lyophilization of Enzyme Mix. All ten reagent tubes containing dialyzed enzyme appeared to be visually dry at 8 minutes. Reagent confirmed to be dry after 2 hours of lyophilization. (“Dry” is defined as material that has transitioned from a clear liquid to a white “fluffy” solid). All tubes containing undialyzed enzyme did not appear visually dry at any time during lyophilization however at 45 minutes, a visually noticeable decrease in volume was observed. All tubes containing undialyzed enzyme still appeared wet and clear after 27.5 hours of lyophilization.
Conclusion: The glycerol supplied with the Bst enzyme must be removed prior to lyophilization for the product to form a dry reagent.
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