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Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process

A technology of freeze-drying protective agent and detection kit, applied in the field of freeze-drying, can solve the problems of large workload, high cost, long cycle, etc., achieve good protection effect, strong versatility, improve freeze-drying activity and storage stability Effect

Active Publication Date: 2022-02-08
无锡百泰克生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to differences in sources, components, proportions and preparation processes of fluorescent PCR reagents produced by various manufacturers, it is necessary to screen suitable lyoprotectant components and optimize the reaction system for fluorescent PCR reagents produced by different manufacturers. The process cycle is long, the workload is heavy, and the cost is high

Method used

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  • Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process
  • Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process
  • Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 is to the freeze-drying protective effect of different manufacturer's enzyme raw material

[0058] (1) Preparation of 2× lyoprotectant: Weigh 8 g of trehalose and 8 g of hydroxyethyl starch, add 100 μL of Tween 20, add sterile purified water to dissolve, dilute to 100 mL, and set aside.

[0059] (2) Preparation of fluorescent PCR reagents: purchase three enzyme raw materials of lyophilized Taq enzyme, reverse transcriptase, and RNase inhibitor from various reagent manufacturers without glycerol or with low glycerol content (<1%) for RT-PCR system construction, Then add primers, probes, etc. for influenza virus detection.

[0060] Among them, each reagent manufacturer is a common fluorescent PCR reagent manufacturer and enzyme raw material manufacturer on the market, including Roche Diagnostics (Shanghai) Co., Ltd., Takara Bio Inc., Nanjing Novizan Biotechnology Co., Ltd., MeridianBioscience, Yisheng Biotechnology (Shanghai ) Co., Ltd., Zhuhai Baorui Biotec...

Embodiment 2

[0094] Embodiment 2 uses vials as the freeze-drying experiment of carrier

[0095] (1) Preparation of 5× lyoprotectant: weigh 40 g of trehalose and 10 g of hydroxyethyl starch, add 1.5 mL of Tween 20, add sterile purified water to dissolve, dilute to 100 mL, and set aside.

[0096] (2) Preparation of fluorescent PCR reagents: Three enzyme raw materials, glycerol-free freeze-drying Taq enzyme, reverse transcriptase, and RNase inhibitor from Nanjing Nuoweizan Biotechnology Co., Ltd. were selected for PCR system construction, and then primers for influenza virus detection were added. , probes, etc.

[0097] (3) Preparation of the test mixture: the above 5×lyoprotectant was mixed with the fluorescent PCR reagent to obtain a PCR reaction system. Wherein, the final concentration of each component of the lyoprotectant is shown in Table 8, and other components are shown in Table 9.

[0098] The final concentration of each component of table 8 lyoprotectant

[0099]

[0100] Conc...

Embodiment 3

[0120] Example 3 Freeze-drying experiments using a microfluidic chip as a carrier

[0121] (1) Preparation of 2.5× lyoprotectant: Weigh 5 g of trehalose and 10 g of hydroxyethyl starch, add 125 mL of Tween 20, add sterile purified water to dissolve, dilute to 100 mL, and set aside.

[0122] (2) Preparation of fluorescent PCR reagents: select glycerol-free lyophilized Taq enzyme, reverse transcriptase, and RNase inhibitor from Zhuhai Baorui Biotechnology Co., Ltd. to construct a PCR system, and then add primers for influenza virus detection, Probes etc. The following 8 systems were prepared, among which, systems 1 to 6 are fluorescent RT-PCR systems; systems 7 and 8 are DNA amplification systems without adding reverse transcriptase and RNase inhibitors.

[0123] (3) Preparation of the test mixture: the above-mentioned 2.5×lyoprotectant was mixed with the fluorescent PCR reagent to obtain a PCR reaction system. Wherein, the final concentration of each component of the lyoprote...

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Abstract

The invention provides a freeze-drying protective additive, a fluorescent PCR detection kit and a freeze-drying process, wherein the freeze-drying protective additive is used for freeze-drying a fluorescent PCR reagent, and the freeze-drying protective additive comprises the following components: trehalose, hydroxyethyl starch and Tween 20. According to the freeze-drying protective agent, the trehalose, the hydroxyethyl starch and the Tween 20 are adopted, so that the active conformation of protein is maintained in the freeze-drying process, the freeze-drying protective agent has a proper glass transition temperature, the protein is well protected in the freezing and dehydration processes, and the inhibition effect on PCR is avoided. The freeze-drying protective agent can be applied to freeze-drying of fluorescent PCR reagents of different manufacturers, and the product is high in universality and has no inhibition effect on the performance of the fluorescent PCR reagents.

Description

technical field [0001] The invention relates to the technical field of freeze-drying, in particular to a freeze-drying protective agent, a fluorescent PCR detection kit and a freeze-drying process thereof. Background technique [0002] During the freeze-drying process of fluorescent PCR reagents, a lyoprotectant must be added to protect the inherent structure of the protein and prevent protein active substances from being damaged during the freezing and drying process. However, due to differences in sources, components, proportions and preparation processes of fluorescent PCR reagents produced by various manufacturers, it is necessary to screen suitable lyoprotectant components and optimize the reaction system for fluorescent PCR reagents produced by different manufacturers. The process cycle is long, the workload is heavy, and the cost is high. Contents of the invention [0003] The main purpose of the present invention is to provide a lyoprotectant, a fluorescent PCR de...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2563/107C12Q2527/125C12Q2565/629
Inventor 蔡禹希李锦锦周志图
Owner 无锡百泰克生物技术有限公司
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