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 pe

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

Example Embodiment

[0032] Example one

[0033] Additive component screening

[0034] Additives (excipients, protective agents) need not inhibit PCR amplification. To test the effect of various additives on PCR, different concentrations are added to the reaction system to test the effect on amplification.

[0035] 1. Preparation of mother liquor of each additive

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

[0037] 2. Each additive is added in seven kinds of dosages, including 0.2μl, 0.4μl, 1μl, 2μl, 4μl, 6μl, 8μl, etc., and each is added separately to the following amplification system, and compared with the without a...

Example Embodiment

[0048] Example two

[0049] Eutectic point detection

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

[0051] 2. Prepare amplification reagents for eutectic point detection. The detection machine is LGJ-20F gland type freeze dryer (Beijing Songyuan Huaxing). The prepared reagents are 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 points are as follows:

[0056] Table 4 Concentrations of additives in the system and corresponding eutectic points detected

[0057]

[0058] Among the five additives, it can be seen from the comparison between the additive group 1 and the additive group 7 that the two are only different in the content of Tween 20, so it is ...

Example Embodiment

[0062] Example 3

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

[0064] According to Example 2, Table 4: The combination and concentration of additive components 1-7 are added to the amplification system, and the 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 procedure is pre-freezing, -30°C, 2 hours; sublimation, -15°C 3 hours; analytical drying 10°C, 3 hours, 25°C, 0.5 hours . The freeze-dried finished products are all uniform white, and will not shake.

[0068] Each group of products after lyophilization were put into a 56℃ oven for acceleration test, respectively accelerated for 1 day, 3 days, 5 days, 7 days, 8 days, 9 days, 10 days, and put them in a 4℃ refrigerator as 0 days Contrast.

[0069] After freeze-drying, add 16.5 μl of deionized purified water to the single reagent reagent, add 2 μl of template (contain...

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PUM

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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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