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Freeze-drying additive, fluorescent PCR (Polymerase Chain Reaction) mixture dry powder, and preparation method

A technology of reaction mixtures and additives, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of increased reagent cost, high cost of freeze-drying, limited use, etc., to save freeze-drying time and increase performance Good, improve the effect of eutectic point

Pending Publication Date: 2020-02-04
NANJING LIMING BIO PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reagents need cold chain transportation during sales and transportation, which increases the cost of reagents and greatly limits the use of remote or even small urban hospitals
[0003] More and more people are beginning to study freeze-dried PCR products. The main problem is that the freeze-drying time is long and the cost of freeze-drying is high; Not ideal; reduced long-term storage performance

Method used

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  • Freeze-drying additive, fluorescent PCR (Polymerase Chain Reaction) mixture dry powder, and preparation method
  • Freeze-drying additive, fluorescent PCR (Polymerase Chain Reaction) mixture dry powder, and preparation method
  • Freeze-drying additive, fluorescent PCR (Polymerase Chain Reaction) mixture dry powder, and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Additive component screening

[0034] Additives (excipients, protective agents) need not inhibit PCR amplification. In order to test the impact of various additive additions on PCR, different concentrations were added to the reaction system to test the impact on amplification.

[0035] 1. Preparation of each additive mother liquor

[0036] Dithiothreitol (DTT) was formulated to be 10mmol / L, gelatin was formulated to be 10% (w / v), trehalose was formulated to be 2mol / L, bovine serum albumin (BSA) was formulated to be 10mg / ml, all in water Dissolve; Tween-20 (tween-20) and tert-butanol are diluted with water to 10% (v / v). DTT, gelatin, trehalose, BSA and tween-20 were purchased from Shanghai Sangong, and tert-butanol was purchased from Shanghai Aladdin Reagent.

[0037] 2. Add 0.2μl, 0.4μl, 1μl, 2μl, 4μl, 6μl, and 8μl of each additive to the following amplification systems separately, and compare with the one without additives:

[0038] Table 1 Amplification system

[...

Embodiment 2

[0049] Eutectic point detection

[0050] 1. DTT is prepared at 1mol / L, gelatin is prepared at 10% (w / v), trehalose is prepared at 2mol / L, BSA is prepared at 100mg / ml, all of which are dissolved in water.

[0051] 2. Preparation of amplification reagents for eutectic point detection, the detection machine is LGJ-20F gland type freeze dryer (Beijing Songyuan Huaxing). The prepared reagent is loaded into the vial, and the eutectic point detection program of the machine is started for detection. The sample addition ratio is as follows:

[0052] Table 3 Amplification system

[0053]

[0054]

[0055] The concentration of additives in the system and the corresponding detected eutectic point are as follows:

[0056] Table 4 The concentration of additives in the system and the eutectic point corresponding to the detection

[0057]

[0058] Among the five additives, it can be seen from the comparison between additive group 1 and additive group 7 that only the content of Tw...

Embodiment 3

[0063] Comparison of freeze-drying time and amplification efficiency of different components

[0064] According to Example 2, as shown in Table 4: the combination and concentration of additive components 1-7 are added to the amplification system, and other components are as follows:

[0065] Table 6 Amplification system

[0066]

[0067] Put it into LGJ-20F gland-type freeze dryer and freeze-dry. The freeze-drying program is as follows: pre-freezing, -30°C, 2 hours; sublimation, -15°C, 3 hours; analytical drying, 10°C, 3 hours, 25°C, 0.5 hours . Freeze-dried finished products are all uniform white and will not disperse when shaken.

[0068] After freeze-drying, each group of products was put into an oven at 56°C for acceleration test, respectively accelerated for 1 day, 3 days, 5 days, 7 days, 8 days, 9 days, and 10 days. control.

[0069] Add 16.5 μl of deionized purified water to the lyophilized single-serving reagent, add 2 μl of template (containing 1000 copies of N...

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Abstract

The invention provides a freeze-drying additive, a fluorescent PCR (Polymerase Chain Reaction) mixture dry powder, and a preparation method, and particularly relates to the biotechnology field. The freeze-drying additive comprises a component A and a component B, wherein the component A consists of dithiothreitol, Tween-20, trehalose, gelatin and BSA (Bull Serum Albumin), and the component B consists of tertiary butanol. The component A is added into other ingredients of a fluorescent PCR mixture stock solution to be stored for standby, the component B is added into the fluorescent PCR mixturestock solution before freeze drying, and finally, freeze drying is carried out to obtain the fluorescent PCR mixture dry powder. While the fluorescent PCR mixture dry powder prepared by the inventionguarantees augmentation performance, freeze-drying time is shortened, freeze-drying cost is reduced, and a freeze-drying finished product is firm, is unlikely to be broken, and can be saved for a long time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a freeze-dried additive and fluorescent PCR reaction mixture dry powder and a preparation method. Background technique [0002] In vitro diagnostic reagents for nucleic acid detection by fluorescent PCR method generally include DNA polymerase, dNTPs, primers, PCR buffer, etc. These components polymerase, primers need to be stored at low temperature to maintain biological activity. Reagents need cold chain transportation during sales and transportation, which increases the cost of reagents and greatly limits the use of remote or even small urban hospitals. [0003] More and more people are beginning to study freeze-dried PCR products. The main problem is that the freeze-drying time is long and the cost of freeze-drying is high; Not ideal; reduced long-term storage performance. [0004] The basic process of freeze-drying includes pre-freezing and freezing of products; th...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2521/101C12Q2563/107
Inventor 张树文葛斌文刘光明
Owner NANJING LIMING BIO PROD CO LTD
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