Freeze-drying protection system required for nucleic acid amplification reagent and preparation method of freeze-drying protection system
A technology of freeze-drying protection and freeze-drying protection agent, which is applied in the field of pharmaceutical preparation, can solve the problems of high temperature and inactivation of reagents, and achieve the effects of less energy consumption, convenient storage, and avoiding repeated freezing and thawing.
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Embodiment 1
[0056] A kind of LAMP amplification reagent lyophilization protection system and lyophilization treatment steps are as follows:
[0057] (1). Freeze-drying of the whole system: Mix all the reagents required for the Lamp reaction together. The specific components of the reagents are as follows:
[0058] components
volume
content
Bst enzyme (8U / μL)
80μL
640U
UNG enzyme (1U / μL)
0.5μL
0.5U
dNTPS (100mM)
20 μL
2μM
MgSO4(100mM)
40μL
4μM
Buffer(10X)
100μL
/
SYBR GREEN I(250X)
15μL
3.75X
Betaine (5M)
2000μL
15mM
44.5μL
/
[0059] The lyoprotectant is one of the following formulations:
[0060] components
volume
content
Mannitol (18%, w / v)
1110μL
5%
BSA (10%, w / v)
200μL
0.5%
Glycine (100mM)
200μL
5mM
290μL
/
[0061] (2). Fractional freeze-dry...
Embodiment 2
[0069] The formula screening of embodiment 2 freeze-drying protection reagent
[0070] The formula of the freeze-drying protective agent is as follows, and the balance is purified water:
[0071] Formula 1: dextran 5%, polyethylene glycol 2%, sucrose 2%;
[0072] Formula 2: mannitol 8%, polyethylene glycol 1%, threonine 1%;
[0073] Formula 3, Mannitol 5%, Bovine Serum Albumin 2%, Glycine 1%;
[0074] Formula 4, polyvinylpyrrolidone 3%, bovine serum albumin 2%, trehalose 1%.
[0075] The above four groups of formulas were mixed with the LAMP reagent (the A solution part in the sub-system) and then lyophilized. The lyophilized reagents were subjected to sensitivity experiments and morphological comparisons, and a control group was set up. The control group was the reaction reagent without lyophilization ( Does not contain lyoprotectant).
[0076] serial number
[0077] After lyophilization, the PCR tube was taken out of the lyophilizer, and the tube was fastened a...
Embodiment 3
[0078] Embodiment 3 thermal stability test
[0079] After screening the lyophilized reagent formula, formula 2 and formula 3 were selected for reagent thermal stability test (37°C constant temperature), and the stability of the reagent was initially evaluated. At the same time, a control group (without adding lyoprotectant reaction system), the test results refer to Figure 2-5 as shown,
[0080] sensitivity
[0081] In the present invention, the response curve Ct≤48 in the sensitivity test can determine the effectiveness of the reagent. From the above results, it can be seen that the freeze-dried protection reagent of the present invention, especially reagent 2, can be stored at 37°C for 4 weeks or even longer, which is greatly improved. The storage stability of the LAMP amplification reagent is improved.
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