CN114015758B - Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process - Google Patents
Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process Download PDFInfo
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- CN114015758B CN114015758B CN202111207256.6A CN202111207256A CN114015758B CN 114015758 B CN114015758 B CN 114015758B CN 202111207256 A CN202111207256 A CN 202111207256A CN 114015758 B CN114015758 B CN 114015758B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention provides a freeze-drying protective agent, a fluorescent PCR detection kit and a freeze-drying process thereof, wherein the freeze-drying protective agent is used for freeze-drying a fluorescent PCR reagent, and comprises the following components: trehalose, hydroxyethyl starch and tween 20. The freeze-drying protective agent disclosed by the invention maintains the active conformation of the protein and has a proper vitrification temperature in the freeze-drying process by adopting the trehalose, the hydroxyethyl starch and the Tween 20, plays a good protection role on the protein in the freezing and dehydration processes, and does not inhibit the PCR reaction. The freeze-drying protective agent can be applied to freeze-drying of fluorescent PCR reagents of different manufacturers, has strong universality of products, and has no inhibiting effect on the performance of the fluorescent PCR reagents.
Description
Technical Field
The invention relates to the technical field of freeze-drying, and particularly relates to a freeze-drying protective agent, a fluorescence PCR detection kit and a freeze-drying process thereof.
Background
In the freeze drying process of the fluorescent PCR reagent, a freeze-drying protective agent is added to protect the inherent structure of the protein and prevent the protein active substances from being damaged in the freezing and drying processes. However, because the fluorescent PCR reagents produced by various manufacturers have differences in sources, components, proportions and preparation processes, the fluorescent PCR reagents produced by different manufacturers need to be screened for suitable components of the freeze-drying protective agent, a reaction system is optimized, and the whole research process is long in period, large in workload and high in cost.
Disclosure of Invention
The invention mainly aims to provide a freeze-drying protective agent, a fluorescence PCR detection kit and a freeze-drying process thereof, and aims to provide a universal freeze-drying protective agent.
In order to achieve the purpose, the invention provides a lyoprotectant, which is used for freeze-drying of a fluorescent PCR reagent and comprises the following components: trehalose, hydroxyethyl starch and tween 20.
In one embodiment, the lyoprotectant further includes sterile purified water.
The invention also provides a fluorescent PCR detection kit, which comprises the freeze-drying protective agent, wherein the freeze-drying protective agent is used for freeze-drying the fluorescent PCR reagent and comprises the following components: trehalose, hydroxyethyl starch and tween 20.
In one embodiment, the fluorescence PCR detection kit comprises a mixed solution, the mixed solution comprises a fluorescence PCR reagent and the freeze-drying protective agent, the final concentration of trehalose in the mixed solution is 1-12% (W/V), the final concentration of hydroxyethyl starch is 1-10% (W/V), and the final concentration of Tween 20 is 0.01-1.00%.
In one embodiment, the final concentration of trehalose in the mixture is 1.5-9.0% (W/V), the final concentration of hydroxyethyl starch is 1.2-9.0% (W/V), and the final concentration of tween 20 is 0.03-0.80%.
The invention also provides a freeze-drying process for freeze-drying the fluorescent PCR reagent by adopting the freeze-drying protective agent, which is characterized by comprising the following steps of:
uniformly mixing a freeze-drying protective agent and a fluorescent PCR reagent to obtain a mixed solution;
and (3) filling the mixed solution into a bearing object, and performing freeze drying according to a preset freeze-drying program to obtain a freeze-dried finished product.
In one embodiment, the preset lyophilization procedure is:
a pre-freezing stage: the temperature of the clapboard is-45 ℃ to-55 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, and the temperature is kept for 2.5 to 4 hours;
a sublimation drying stage: the temperature of the clapboard is-40 ℃ to-30 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, the vacuum is started, the vacuum degree is less than or equal to 10Pa, the temperature is started to rise to-18 ℃ to-12 ℃ after the vacuum is maintained for 6 to 8 hours, and the holding time is 1 to 2 hours;
a re-sublimation drying stage: continuously keeping the vacuum degree to be less than or equal to 10Pa, keeping the temperature of the cold trap at-65 ℃ to-75 ℃, increasing the temperature of the plate layer to 0-10 ℃, keeping the temperature for 1-2 hours, increasing the temperature of the plate layer to 15-25 ℃, and keeping the temperature for 3-6 hours.
In one embodiment, the carrier includes a PCR tube, a vial, or a microfluidic chip.
According to the technical scheme, trehalose is adopted to effectively protect protein in the fluorescent PCR reagent, the trehalose and hydroxyethyl starch are combined for use, so that the defects can be mutually compensated, the phenomenon that the glass transition temperature is too low or too high is avoided, the natural configuration of the protein can be maintained, and the freeze-drying activity and the storage stability of the protein are improved. In addition, tween 20 reduced protein freezing and dehydration denaturation caused by ice water interfacial tension during lyophilization, and served as a lubricant to protect proteins during reconstitution.
The freeze-drying protective agent disclosed by the invention adopts the trehalose, the hydroxyethyl starch and the Tween 20, so that the active conformation of the protein is maintained and the proper vitrification temperature is realized in the freeze-drying process, the protein is well protected in the freezing and dehydration processes, and the PCR reaction cannot be inhibited. The freeze-drying protective agent can be applied to freeze-drying of fluorescent PCR reagents of different manufacturers, has strong universality and has no inhibiting effect on the performance of the fluorescent PCR reagents.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is an appearance diagram of lyophilized products of enzymes of different manufacturers in example 1 of the present invention;
FIG. 2 is an appearance diagram of a lyophilized product of the enzyme of different manufacturers of comparative example 1;
FIG. 3 is an appearance diagram of a lyophilized product of the enzyme of different manufacturers of comparative example 2;
FIG. 4 is an appearance diagram of a lyophilized product of the enzyme of different manufacturers of comparative example 3;
FIG. 5 is an appearance diagram of lyophilized product of sample in penicillin bottle in example 2 of the present invention;
FIG. 6 is an appearance diagram of a freeze-dried finished product of sample 4 on a microfluidic chip in example 3 of the present invention;
fig. 7 is an appearance diagram of the freeze-dried products of the comparative examples 5 to 7 on the microfluidic chip according to example 3 of the present invention (the comparative examples 5 to 7 are shown from left to right).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Due to the characteristics of rapidness, sensitivity and accuracy of the fluorescent PCR technology, the fluorescent PCR technology is widely applied to the fields of pathogen diagnosis, accurate cancer treatment, Single Nucleotide Polymorphism (SNP) detection, forensic identification, food safety and the like, and for example, the nucleic acid detection of novel coronaviruses becomes one of indispensable important technologies in modern medicine. At present, the fluorescent PCR reagent can be stored at the temperature below-20 ℃ to keep the activity, but the detection performance of the reagent is reduced by the temperature rise and repeated freeze thawing in the processes of storage, transportation and use, particularly, the transportation is hindered, the risk of reagent failure and the cold chain management cost are greatly increased during epidemic situations, and the fluorescent PCR reagent cannot be applied to primary medical institutions with poor cold chain conditions and international long-distance transportation.
The freeze-drying technique is a drying method in which a solvent is frozen at a low temperature and then its solid state is directly sublimated into a gaseous state. Its advantage does: drying at low temperature can reduce the denaturation of heat-sensitive substances (such as protein); the product has unchanged property after freeze-drying, and still retains good activity after redissolution. The freeze drying technology is applied to the fluorescent PCR reagent, so that the fluorescent PCR reagent can be stored at normal temperature, the requirement of long-distance transportation is met, unfreezing is not needed before use, and the influence of repeated freeze thawing on the activity of the reagent is completely avoided.
However, the fluorescent PCR reagent must be added with a lyoprotectant to protect the inherent structure of the protein active substance and prevent the protein active substance from being damaged during the freezing and drying processes. The existing fluorescent PCR reagent has differences in sources, components, proportions and preparation processes of fluorescent PCR reagents produced by various manufacturers, so that the fluorescent PCR reagents produced by different manufacturers are matched with corresponding freeze-drying protective agents, and the universal performance of the freeze-drying protective agents is poor.
The invention provides a freeze-drying protective agent.
In the embodiment of the invention, the freeze-drying protective agent is used for freeze-drying of a fluorescent PCR reagent and comprises the following components: trehalose, hydroxyethyl starch and tween 20.
According to the technical scheme, trehalose is adopted to effectively protect protein in the fluorescent PCR reagent, the trehalose and hydroxyethyl starch are combined for use, so that the defects can be mutually compensated, the phenomenon that the glass transition temperature is too low or too high is avoided, the natural configuration of the protein can be maintained, and the freeze-drying activity and the storage stability of the protein are improved. In addition, tween 20 reduced protein freezing and dehydration denaturation caused by ice water interfacial tension during lyophilization, and served as a lubricant to protect proteins during reconstitution.
The freeze-drying protective agent disclosed by the invention adopts the trehalose, the hydroxyethyl starch and the Tween 20, so that the active conformation of the protein is maintained and the proper vitrification temperature is realized in the freeze-drying process, the protein is well protected in the freezing and dehydration processes, and the PCR reaction cannot be inhibited. The freeze-drying protective agent can be applied to freeze-drying of fluorescent PCR reagents of different manufacturers, has strong universality and has no inhibiting effect on the performance of the fluorescent PCR reagents.
Specifically, trehalose can increase the free energy of protein in the freezing process so as to prevent protein denaturation, and can replace hydrogen bonds between protein and water molecules to stabilize protein in the drying and dehydration process so as to prevent activity reduction caused by the dehydration effect of the protein, so that the protein can maintain a natural configuration and has a protective effect on the protein.
Trehalose is formed by condensing two glucose molecules through hemiacetal hydroxyl, and under the conditions of high temperature, high cold, high osmotic pressure, drying and water loss and the like, trehalose can form a unique protective film on the surface, so that the protein molecules are effectively protected from invariance and inactivation, on one hand, the damage of low temperature to proteins is avoided, and on the other hand, the protein molecules are supported and wrapped, so that the trehalose is not easy to deform. The trehalose has high glass transition temperature and low hygroscopicity, so that the trehalose solution is less prone to form ice crystals; meanwhile, the number of unfrozen water molecules around the trehalose is the largest, so that a trehalose/water structure with higher rigidity can be formed, and the freeze dehydration resistance is higher. Although the glass transition temperature of trehalose is higher than that of sucrose, the glass transition temperature is relatively low, so that the glass transition temperature of the whole solution is very low, the trehalose is not easy to form during freeze-drying, and the protein activity loss can be caused although the trehalose is kept at a low temperature for a long time to avoid collapse.
Specifically, the hydroxyethyl starch is a macromolecular compound formed by hydroxyethylating glucose rings of amylopectin in corn or potato, is a macromolecular substance, can improve the glass transition temperature of a solution, and simultaneously can well protect the activity of protein and maintain the stability of the protein. In addition, the hydroxyethyl starch is a good freeze-drying excipient, and is beneficial to forming of products.
Hydroxyethyl starch has a higher glass transition temperature but does not retain the native configuration of the protein. By combining hydroxyethyl starch and trehalose, the hydroxyethyl starch can improve the glass transition temperature of the solution, thereby not only exerting the characteristic that trehalose protects protein, but also solving the problem that freeze-drying is not shaped due to low glass transition temperature, and further improving the freeze-drying activity and storage stability of the protein.
Specifically, the tween 20 is a nonionic surfactant, and is soluble in water, ethanol, methanol, and the like. In order to reduce protein freezing and dehydration denaturation caused by ice water interfacial tension in the freeze-drying process and protect protein as a lubricant in the redissolution process, Tween 20 is added into the freeze-drying protective agent at the same time. Tween 20 has relatively low critical micelle concentration, can satisfy protection effect at low concentration, and has solubilization effect to improve solubility of components.
The freeze-drying protective agent has various solvents, and in one embodiment, the freeze-drying protective agent also comprises sterile purified water, so that the influence of the solution on substances such as proteins and the like is reduced, bacteria or viruses are prevented from being brought in, the subsequent experimental result is prevented from being influenced, and interference factors are reduced.
The invention also provides a fluorescent PCR detection kit, which comprises the freeze-drying protective agent. Since the fluorescence PCR detection kit adopts all technical schemes of all the embodiments, at least all the beneficial effects brought by the technical schemes of the embodiments are achieved, and no details are repeated here. The fluorescent PCR detection kit can detect influenza A virus, influenza B virus, novel coronavirus and the like.
In one embodiment, the fluorescence PCR detection kit comprises a mixed solution, the mixed solution comprises a fluorescence PCR reagent and the lyoprotectant, and in the mixed solution, the final concentration of trehalose is 1-12% (W/V), the final concentration of hydroxyethyl starch is 1-10% (W/V), and the final concentration of tween 20 is 0.01-1.00%. The trehalose, the hydroxyethyl starch and the tween with specific contents enable the solution to have higher glass transition temperature, maintain the active conformation of the protein in the freeze-drying process, play a good role in protecting the protein in the fluorescent PCR reagent, and cannot inhibit the subsequent PCR reaction, thereby protecting the protein and not influencing the performance of the fluorescent PCR reagent. The mixture is a mixture of the lyoprotectant and the fluorescent PCR reagent before lyophilization.
Further, in one embodiment, the final concentration of trehalose in the lyoprotectant is 1.5-9.0% (W/V), the final concentration of hydroxyethyl starch is 1.2-9.0% (W/V), and the final concentration of tween 20 is 0.03-0.80%.
The invention also provides a freeze-drying process for freeze-drying the fluorescent PCR reagent by adopting the freeze-drying protective agent, which comprises the following steps:
s100, uniformly mixing a freeze-drying protective agent and a fluorescent PCR reagent to obtain a mixed solution;
s200, respectively loading the mixed solution into a bearing object, and carrying out freeze drying according to a preset freeze drying program to obtain a freeze-dried finished product.
The mixed solution is freeze-dried according to a preset freeze-drying program to obtain a freeze-dried finished product, so that freeze-drying of the fluorescent PCR reagent is realized, and finally, the freeze-dried fluorescent PCR detection kit is obtained, so that the freeze-dried fluorescent PCR detection kit can be sold and transported in the market.
In one embodiment, the preset lyophilization procedure is:
a pre-freezing stage: the temperature of the clapboard is-45 ℃ to-55 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, and the temperature is kept for 2.5 to 4 hours;
a sublimation drying stage: the temperature of the clapboard is-40 ℃ to-30 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, the vacuum is started, the vacuum degree is less than or equal to 10Pa, the temperature is started to rise to-18 ℃ to-12 ℃ after the vacuum is maintained for 6 to 8 hours, and the holding time is 1 to 2 hours;
a re-sublimation drying stage: continuously keeping the vacuum degree to be less than or equal to 10Pa, keeping the temperature of the cold trap at-65 ℃ to-75 ℃, increasing the temperature of the plate layer to 0-10 ℃, keeping the temperature for 1-2 hours, increasing the temperature of the plate layer to 15-25 ℃, and keeping the temperature for 3-6 hours.
In the step S100, the lyoprotectant and the fluorescent PCR reagent are prepared separately, and then mixed. Specifically, trehalose, hydroxyethyl starch and tween 20 are measured and dissolved in a solvent to obtain the freeze-drying protective agent. And uniformly mixing the buffer solution, the primer, the probe, the dNTPs, the enzyme and the like with the solvent to obtain the fluorescent PCR reagent. It is understood that the solvent of the lyoprotectant and the solvent of the fluorescent PCR reagent can be the same or different, and in one embodiment, the solvent of the lyoprotectant and the solvent of the fluorescent PCR reagent are sterile purified water.
In the step S200, the mixed solution may or may not be dispensed, so that the steps of dispensing and dispensing are not required when the user uses the mixed solution, thereby reducing the risk of reagent loss and reagent contamination. And putting the bearing substance and the mixed solution into a freeze dryer, and freeze-drying according to a preset freeze-drying program to obtain a freeze-dried finished product.
Specifically, the lyophilization process includes a prefreezing stage, a sublimation drying stage, and a re-sublimation drying stage, and a drying process in which primers, enzymes, and the like are frozen into a solid state at a low temperature and then water is directly sublimated into a gaseous state under vacuum to be dehydrated. The temperature rise in the re-sublimation drying stage enables the residual moisture in the solution to exist in a quantitative non-covalent bond form so as to maintain the stability of the activity of the protein and prolong the storage time, and is very important for maintaining the activity of the protein and the appearance form of the product.
There are various carriers for carrying the mixed solution, and in one embodiment, the carriers include a PCR tube, a vial, or a microfluidic chip. By adopting the freeze-drying protective agent and the freeze-drying process, freeze-drying in penicillin bottles, PCR tubes and microfluidic chips can be realized, the universality of the freeze-drying protective agent is further improved, the problem that the freeze-drying process of different carriers needs to be researched again is solved, the research and development period and workload of enterprises are reduced, and the research and development cost of products is reduced. Wherein, the PCR tube can be an eight-row PCR tube. By adopting the preset freeze-drying program and the carrier, the obtained freeze-dried finished product can be stored at 37 ℃ for at least 30 days, and at 4 ℃ for at least 8 months, and the performance of the product is kept unchanged.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
EXAMPLE 1 Freeze-drying protection of enzyme stocks from different manufacturers
(1) Preparation of 2 × lyoprotectant: weighing 8g of trehalose and 8g of hydroxyethyl starch, adding 100 mu L of tween 20, adding sterile purified water for dissolving, and fixing the volume to 100mL for later use.
(2) Preparing a fluorescent PCR reagent: three enzyme raw materials of freeze-drying Taq enzyme, reverse transcriptase and RNA enzyme inhibitor without glycerol or with low glycerol content (< 1%) of various reagent manufacturers are purchased to construct an RT-PCR system, and then primers, probes and the like for detecting influenza viruses are added.
Wherein, each reagent manufacturer is a commercially common enzyme raw material manufacturer of a fluorescent PCR reagent manufacturer, and comprises Roche diagnosis product (Shanghai) Co., Takara Bio Inc., Nanjing Nodezam Biotech Co., Ltd., Meridian Bioscience, Next Sheng Biotech (Shanghai) GmbH, Zhuhai Baoren Biotech Co., Ltd., and Wuhan Egyoka Biotech Co., Ltd.
(3) Preparing a test mixed solution: and mixing the 2X freeze-drying protective agent with a fluorescent PCR reagent to obtain a PCR reaction system. Wherein the final concentration of trehalose is 4% (W/V), the final concentration of hydroxyethyl starch is 4% (W/V), the final concentration of tween 20 is 0.05%, and the other components are shown in Table 1.
TABLE 1 concentration of each component in PCR reaction System
| Composition (I) | Concentration of |
| Taq enzyme (Freeze-drying) | 0.06U/μL |
| Reverse transcriptase (Freeze-drying) | 1.5U/μL |
| RNase inhibitor (Freeze-drying) | 0.5U/μL |
| MgCL2 | 2.5mM |
| Tris-Hcl(PH8.3) | 20mM |
| KCL | 80mM |
| dNTPs | 200nM |
| FluA-F (influenza A virus upstream primer) | 200nM |
| FluA-R (influenza A virus downstream primer) | 200nM |
| FluA-P (influenza A virus FAM fluorescent probe) | 200nM |
| FluB-F (upstream primer of influenza B virus) | 300nM |
| FluB-R (influenza B virus downstream primer) | 300nM |
| FluB-P (influenza B virus VIC fluorescent probe) | 300nM |
| RnaseP-F (human endogenous internal standard gene upstream primer) | 100nM |
| RnaseP-R (humanized internal standard gene downstream primer) | 100nM |
| RnaseP-P (human endogenous internal standard gene ROX fluorescent probe) | 100nM |
| 2 x freeze-drying protective agent | 1× |
| Sterilized purified water | Make up to 20 μ L per person |
Subpackaging the system prepared by the zymogen materials of each manufacturer into 20 mu L to the tube bottom of eight-row PCR tubes, transferring the system into an eight-row tube freeze-drying mould, putting the mould into a plate layer of a freeze-drying machine, and freeze-drying the system in the freeze-drying machine according to the following freeze-drying program:
table 2 lyophilization program parameters for each step
(4) Performance evaluation: after the freeze-drying is finished, the freeze-dried finished products of the enzyme freeze-dried of various manufacturers are evaluated for appearance, amplification performance and accelerated stability.
1) Appearance evaluation: referring to fig. 1, through appearance observation, freeze-dried finished products of enzyme freeze-dried products of various manufacturers are well formed, smooth and complete in appearance and free of cavity collapse.
2) Evaluation of amplification Performance: ct values were determined and the results are shown in Table 3.
TABLE 3 Ct values of PCR reaction systems for enzymes of different manufacturers
According to table 3, the performance of the reagent of the PCR reaction system after enzyme freeze-drying of each manufacturer is compared with the contrast amplification effect of the liquid PCR reagent, and the detection limit sample can be detected by 100%. Compared with the liquid control, the average Ct value of the amplified positive samples is within 1. The experimental results show that the amplification performance of the freeze-dried finished product can be maintained unchanged by using the freeze-drying protective agent and the freeze-drying process.
3) Evaluation of accelerated stability: the lyophilized reagents of the above groups were stored at 37 ℃ and the amplification performance was evaluated every 10 days, and the results are shown in Table 4.
TABLE 4 results of evaluation of amplification Performance of PCR reaction systems of enzymes of different manufacturers
As can be seen from Table 4, the Ct values of the lyophilized products of the enzymes of the manufacturers are within 1 after being placed at 37 ℃ for 40 days, and the target amplification fails after the liquid control is placed at 37 ℃ for 10 days. The result shows that the freeze-drying protective agent and the freeze-drying process can maintain that the amplification performance of the freeze-dried finished product is kept unchanged within 40 days at 37 ℃.
In conclusion, the freeze-drying protective agent can form freeze-dried finished products in eight-row consumable materials, the freeze-drying protective agent can well protect fluorescent PCR reagents consisting of raw materials of different manufacturers, the freeze-drying protective agent is well formed after freeze-drying, the amplification performance of the reagents is not damaged before and after freeze-drying, and the acceleration stability at 37 ℃ can reach 40 days.
(5) To verify the effect of the lyoprotectant of the invention, a comparative mixture was prepared: and preparing a contrast freeze-drying protective agent, and mixing the contrast freeze-drying protective agent with the fluorescent PCR reagent to obtain a PCR reaction system. The obtained PCR reaction system was dispensed 20. mu.L to eight rows of PCR tubes at the bottom and lyophilized in a lyophilizer according to the lyophilization procedure of Table 2.
Comparative example 1: trehalose was 4% (W/V) at the final concentration, hydroxyethyl starch was not contained, tween 20 was 0.05% at the final concentration, and the other components were the same as in table 1.
Comparative example 2: trehalose was 4% (W/V) at the final concentration, hydroxyethyl starch was 4% (W/V) at the final concentration, tween 20 was not contained, and the other components were the same as in table 1.
Comparative example 3: trehalose was not contained, the final concentration of hydroxyethyl starch was 4% (W/V), the final concentration of tween 20 was 0.05%, and the other components were the same as in table 1.
(6) Performance evaluation: after the freeze-drying is finished, the freeze-dried finished products of the enzyme freeze-dried of various manufacturers are evaluated for appearance, amplification performance and accelerated stability.
1) Appearance evaluation: referring to fig. 2 to 4, through appearance observation, most of the enzymes of the manufacturers in comparative example 1 cannot be molded after freeze-drying, and the cavities are obvious; the appearance of the enzyme of each manufacturer of the comparative example 2 is good after freeze-drying, and has no great difference with the enzyme of the example 1; the appearance of the freeze-dried enzyme of each manufacturer in the comparative example 3 is poor in forming, and obvious holes can be observed at the bottom of the freeze-dried powder.
2) Evaluation of amplification Performance: ct values were measured and the results are shown in tables 5 to 7.
TABLE 5 Ct values of PCR reaction systems for enzymes of different manufacturers in comparative example 1
TABLE 6 Ct values of PCR reaction systems for enzymes of different manufacturers in comparative example 2
TABLE 7 Ct values of PCR reaction systems for enzymes of different manufacturers in comparative example 3
According to tables 5 to 7, comparative examples 1 to 3 were poor in the protective effect on the enzyme lyophilization process of each manufacturer, and the samples with the detection limit concentration could not be stably detected. Therefore, the freeze-drying protective agent disclosed by the invention can play a good protection role on fluorescent PCR reagents consisting of enzyme materials of different manufacturers, the universality is strong, the reagents are not damaged before and after freeze-drying, and the stability of the product is good.
3) Evaluation of accelerated stability: as the amplification performance of the lyophilized reagents of comparative examples 1-3 does not reach the standard, the accelerated stability evaluation at 37 ℃ is not carried out subsequently.
Example 2 Freeze-drying experiment Using penicillin bottles as carriers
(1) Preparation of 5 × lyoprotectant: weighing 40g of trehalose and 10g of hydroxyethyl starch, adding 1.5mL of Tween 20, adding sterile purified water to dissolve, and diluting to 100mL for later use.
(2) Preparing a fluorescent PCR reagent: three enzyme raw materials of glycerol-free freeze-drying Taq enzyme, reverse transcriptase and RNA enzyme inhibitor of Nanjing Novozam Biotechnology limited are selected for construction of a PCR system, and then primers, probes and the like for detecting influenza viruses are added.
(3) Preparing a test mixed solution: and mixing the 5 Xlyophilized protectant with a fluorescent PCR reagent to obtain a PCR reaction system. The final concentrations of the components of the lyoprotectant are shown in Table 8, and the other components are shown in Table 9.
TABLE 8 Final concentrations of the components of the lyoprotectant
TABLE 9 concentration of each component in PCR reaction System
And subpackaging the prepared mixed solution into 500 mu L to 2mL penicillin bottles, putting the penicillin bottles into a plate layer of a freeze dryer, freeze-drying the penicillin bottles in the freeze dryer according to the freeze-drying program shown in the table 10, and plugging and capping the penicillin bottles after freeze-drying.
TABLE 10 Freeze drying procedure parameters for each step
(4) Performance evaluation: after the freeze-drying, evaluation of appearance, amplification performance and accelerated stability was performed.
1) Appearance evaluation: referring to fig. 5, by appearance observation, the freeze-dried powder of samples 1 to 3 is fluffy and smooth, and has good forming and no cavity collapse; the forming can not be carried out in comparison 1, the appearance is normal in comparison 2, and more cracks exist on the freeze-dried powder in comparison 3 and comparison 4.
2) Evaluation of amplification Performance: ct values were determined and the results are shown in Table 11.
TABLE 11 Ct values of PCR reaction systems for samples 1 to 3, comparative samples 1 to 4
According to Table 11, the detection rate of samples 1 to 3 with detection limits was 100%, whereas comparative sample 1 after lyophilization failed to amplify normally, and comparative samples 2 to 4 failed to stably detect the lowest detection limit although the medium positive sample was still detectable
3) Evaluation of accelerated stability: after the samples 1 to 3 and the comparisons 2 to 4 were stored at 37 ℃ for 30 days, the accelerated stability was examined, and the examination results are shown in Table 12.
TABLE 12 Ct values of PCR reaction systems for samples 1 to 3, and for comparisons 2 to 4
From table 12, it can be seen that after the freeze-dried finished products of samples 1 to 3 freeze-dried in the vial are stored at 37 ℃ for 30 days, the sample with the detection limit concentration can still be detected by 100%, and the Ct value of the amplification of the medium-positive sample is within 1 compared with the Ct value of the 0 th day, which indicates that the amplification performance is not changed. The components used in the comparison 2 to 4 are not in the protection range of the patent, the sample with the lowest detection limit concentration cannot be detected after being stored for 30 days at 37 ℃, and the difference value of the amplified Ct value of the medium-positive sample and the difference value of the amplified Ct value of the 0 th day are all more than 1, which shows that the formula of the freeze-drying protective agent is poor in stability and cannot be stored for a long time.
After the samples 1 to 3 were stored at 4 ℃ for 8 months, the Ct values thereof were measured, and the results are shown in Table 13.
TABLE 13 Ct values of PCR reaction systems for samples 1 to 3
According to table 13, after the samples 1 to 3 were stored at 4 ℃ for 8 months, the detection limit concentration sample could still be detected by 100%, the change of the amplification Ct value of the medium positive sample was within 1 compared to the day 0, and the performance remained unchanged, which indicates that the freeze-dried product lyophilized by penicillin bottle using the lyoprotectant of the present invention has good stability.
Example 3 Freeze-drying experiment Using microfluidic chip as support
(1) Preparation of 2.5 × lyoprotectant: weighing 5g of trehalose and 10g of hydroxyethyl starch, adding 125mL of Tween 20, adding sterile purified water, dissolving, and diluting to 100mL for later use.
(2) Preparing a fluorescent PCR reagent: three enzyme raw materials of glycerin-free freeze-drying Taq enzyme, reverse transcriptase and RNA enzyme inhibitor of Zhuhaibaorui Biotechnology Limited are selected for PCR system construction, and then primers, probes and the like for detecting influenza viruses are added. Preparing the following 8 systems, wherein the systems 1 to 6 are all fluorescence RT-PCR systems; systems 7 and 8 are DNA amplification systems without addition of reverse transcriptase and RNase inhibitors.
(3) Preparing a test mixed solution: and mixing the 2.5 Xfreeze-drying protective agent with a fluorescent PCR reagent to obtain a PCR reaction system. The final concentrations of the components of the lyoprotectant are shown in Table 14, and the other components are shown in Table 15.
TABLE 14 Final concentrations of the components of the lyoprotectant
| Components | Final concentration of trehalose | Final concentration of hydroxyethyl starch | Final concentration of Tween 20 |
| Sample No. 4 | 2％ | 4％ | 0.05％ |
| Comparative example 5 | 2％ | / | 0.05％ |
| Comparative example 6 | 2％ | 4％ | / |
| Comparative example 7 | / | 4％ | 0.05％ |
TABLE 15 concentrations of the components in the different PCR reaction systems
And (3) subpackaging 10 mu L of the prepared 8-individual mixed solution to each amplification hole position of the microfluidic chip, wherein the hole position 1 corresponds to the system 1, and the like. And (3) putting the micro-fluidic chip into a freeze dryer, freeze-drying according to the freeze-drying program in the table 16, and sealing the micro-fluidic chip with a film after freeze-drying, and vacuumizing, plastic-packaging and storing.
TABLE 16 lyophilization program parameters for each step
(4) Performance evaluation: evaluation of appearance, amplification performance, and accelerated stability was performed.
1) Appearance evaluation: referring to fig. 6, through appearance observation, the freeze-dried powder of sample 4 was well adhered to the microfluidic chip, had a smooth surface, and was free of cracks and slag. Referring to FIG. 7, the reagent in comparative example 5 failed to form, and the reagent in comparative example 6 formed more normally, similar to sample 4; compared with 7, the freeze-dried powder can be molded, but the freeze-dried powder has a loose structure and is easy to remove slag.
2) Evaluation of amplification Performance: the amplification performance of the lyophilized product (sample 4) lyophilized in the microfluidic chip was examined, and the results are shown in table 17.
TABLE 17 Ct values for PCR reaction systems for sample 4, comparative examples 5 to 7
According to Table 17, sample 4 was amplified for influenza A virus nucleic acid, influenza B virus nucleic acid, respiratory syncytial virus nucleic acid, human parainfluenza virus type 1 nucleic acid, human parainfluenza virus type 2 nucleic acid, human parainfluenza virus type 3 nucleic acid, adenovirus nucleic acid, and Mycoplasma pneumoniae nucleic acid.
Contrast 5, each reference sample cannot be amplified normally, and the reagent is invalid; although the reference products can still be amplified in comparison 6 and comparison 7, the Ct value of the amplified product is significantly greater than that of sample 4, and most of the differences are greater than 1, indicating that the protective effect of the cryoprotectant used in comparison 5 to comparison 7 on the fluorescent PCR reagent is not good.
3) Evaluation of accelerated stability: since the first performance test after lyophilization, comparison Nos. 5 to 7, did not achieve good amplification performance, the accelerated stability evaluation at 37 ℃ was not continued. Therefore, only sample 4 was evaluated for accelerated stability at 37 ℃. And (3) placing the freeze-dried chip at 37 ℃, carrying out performance detection every 15 days, and determining that the performance is unchanged if the difference value of the Ct value of the detection reference product and the Ct value of the detection reference product on the 0 th day is within 1. The reference substances for detection are inactivated cultures of influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus types 1, 2 and 3, adenovirus and mycoplasma pneumoniae to extract nucleic acids, and the detection results are shown in Table 18.
TABLE 18 Ct values of PCR reaction systems for sample 4
As can be seen from tables 17 and 18, the amplification performance of the lyophilized product (sample 4) lyophilized in the chip was good, and each reference product could be normally detected; the difference value between the Ct value of each reference product detected within 45 days after the freeze-drying protective agent is stored at 37 ℃ and the Ct value detected within 0 day is within 1, and the amplification performance is still not reduced at all, which indicates that the freeze-drying finished product freeze-dried by adopting the micro-fluidic chip freeze-drying protective agent has good stability.
In conclusion, it can be seen from example 1 that the cryoprotectant of the present invention can be used in a fluorescent PCR reagent composed of raw materials from different manufacturers, and has a good protection effect on proteins, strong versatility, no inhibition effect on the performance of the fluorescent PCR reagent, and good amplification performance and stability of the lyophilized product. In the embodiments 1 to 3, eight rows of PCR tubes, penicillin bottles and microfluidic chips are respectively adopted as carriers for freeze-drying, and the appearance, amplification performance and acceleration stability of freeze-dried finished products meet the requirements, so that the freeze-drying protective agent can be applied to different carriers, and the good universality of the freeze-drying protective agent is further demonstrated.
The above description is only an alternative embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. The fluorescent PCR detection kit is characterized by comprising a freeze-drying protective agent, wherein the freeze-drying protective agent comprises the following components: trehalose, hydroxyethyl starch and tween 20.
2. The fluorescence PCR assay kit of claim 1, wherein the fluorescence PCR assay kit comprises a mixed solution comprising a fluorescence PCR reagent and the lyoprotectant; in the mixed solution, the final concentration of the trehalose is 1-12% (W/V), the final concentration of the hydroxyethyl starch is 1-10% (W/V), and the final concentration of the tween 20 is 0.01-1.00%.
3. The fluorescence PCR detection kit according to claim 2, wherein the final concentration of trehalose in the mixture is 1.5-9.0% (W/V), the final concentration of hydroxyethyl starch is 1.2-9.0% (W/V), and the final concentration of Tween 20 is 0.03-0.80%.
4. The fluorescent PCR assay kit of claim 1, wherein the lyoprotectant further comprises sterile purified water.
5. A lyophilization process for lyophilizing fluorescent PCR reagents using the lyoprotectant of any one of claims 1-4, comprising the steps of:
uniformly mixing a freeze-drying protective agent and a fluorescent PCR reagent to obtain a mixed solution;
and (3) loading the mixed solution into a bearing object, and carrying out freeze drying according to a preset freeze drying program to obtain a freeze-dried finished product.
6. The lyophilization process according to claim 5, wherein the preset lyophilization program is:
a pre-freezing stage: the temperature of the clapboard is-45 ℃ to-55 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, and the temperature is kept for 2.5 to 4 hours;
a sublimation drying stage: the temperature of the clapboard is-40 ℃ to-30 ℃, the temperature of the cold trap is-65 ℃ to-75 ℃, the vacuum is started, the vacuum degree is less than or equal to 10Pa, the temperature is started to rise to-18 ℃ to-12 ℃ after the vacuum is maintained for 6 to 8 hours, and the holding time is 1 to 2 hours;
a re-sublimation drying stage: continuously keeping the vacuum degree to be less than or equal to 10Pa, keeping the temperature of the cold trap at-65 ℃ to-75 ℃, increasing the temperature of the plate layer to 0-10 ℃, keeping the temperature for 1-2 hours, increasing the temperature of the plate layer to 15-25 ℃, and keeping the temperature for 3-6 hours.
7. The lyophilization process of claim 5, wherein the support comprises a PCR tube, a vial, or a microfluidic chip.
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