CN111826427B

CN111826427B - Freeze-drying protective agent and product of PCR amplification reagent, and preparation method and application thereof

Info

Publication number: CN111826427B
Application number: CN202010728670.0A
Authority: CN
Inventors: 欧格; 邓京; 何绮婷; 勾宏娜
Original assignee: Zhuhai Livzon Diagnostics Inc
Current assignee: Zhuhai Livzon Diagnostics Inc
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2023-09-26
Anticipated expiration: 2040-07-24
Also published as: CN111826427A

Abstract

The invention relates to the technical field of biology, and in particular provides a freeze-drying protective agent of a reagent for PCR amplification, a product, a preparation method and application thereof. The freeze-drying protective agent of the reagent for PCR amplification provided by the invention comprises trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan. The freeze-drying protective agent can obviously prolong the effective period of the reagent for PCR amplification, simultaneously can effectively reduce the possibility of aerosol formation of a PCR amplification reaction system during use, and improves the sensitivity and accuracy of PCR.

Description

Freeze-drying protective agent and product of PCR amplification reagent, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a freeze-drying protective agent of a reagent for PCR amplification, a product, a preparation method and application thereof.

Background

The polymerase chain reaction (Polymerase Chain Reaction, PCR) is an in vitro controlled nucleic acid technique that provides a large number of specific gene fragments in a short period of time in a suitable buffer system, through repeated cycles of denaturation-renaturation-extension in the presence of specific primers, deoxyribonucleic acid, a suitable polymerase and a cation.

Because many components in the PCR system, especially enzymes, primer probes, dNTPs and the like are sensitive to temperature, the fluorescent PCR reagent has very strict requirements on use, storage and transportation conditions. At present, more reagents for PCR amplification are applied in a liquid state, the stability is poor, the reagent is easy to lose efficacy, and the effective period of the reagent stored at the temperature of 2-8 ℃ is only about 3 months. In recent years, there have been many researchers who have performed freeze-drying treatment on PCR systems, but the results are rarely effective due to the influence (e.g., stability) of the freeze-drying technique and the freeze-drying process on enzymes. In addition, many formulations are difficult to re-dissolve after freeze-drying, and great inconvenience is brought to users.

In the existing drug freeze-drying technology, common auxiliary materials used as supporting function in the freeze-drying protective agent are sugar, alcohols, polymers, amino acids and the like, such as mannitol, polyvinylpyrrolidone, trehalose, sucrose, gelatin, alginate, cyclodextrin and the like. Although the fillers are widely applied to drug lyophilization, the requirements of PCR reagents on the fillers are obviously different from those of drugs: for example, drugs are required to be harmless to the body (e.g., non-lethal, teratogenic, mutagenic, etc.), while PCR reagents are more important to the requirements of the packing materials and have no effect on the detection reaction (sensitivity, specificity, duration), e.g., no substances inhibiting the reaction such as cations, reaction competitors or inhibitors, etc. can be introduced. Therefore, the application scenarios of fillers in medicine and PCR systems are very different.

For lyophilization of PCR systems, it is desirable to first maintain the activity of the enzyme, dNTP and primer probes during lyophilization and secondly to take into account the limitations of the glass transition temperature (Tg') of the filler on the lyophilization temperature conditions and the shortened shelf life due to the hygroscopicity of the protectant.

The data speculate that the hydroxyl groups of disaccharides can be connected with hydrogen bonds of the disaccharides in time to form hydration films in the protein dehydration process, thereby protecting the advanced structure and the functional integrity of biological macromolecules. Therefore, sugar, especially trehalose and sucrose, are common in the lyophilization of PCR systems, but sucrose is liable to cause mella reaction during the high temperature denaturation of PCR reaction, which affects the detection result, and generally results in deterioration of the sensitivity and uniformity of reagents. The trehalose is combined with water, moisture absorption of the trehalose is easy to occur, the validity period of the freeze-dried reagent can be directly influenced by moisture absorption of the freeze-dried powder in the storage process, the stability of the freeze-dried reagent is closely related to the residual water of the freeze-dried powder, the more the water is, the worse the stability is, therefore, the stability of the reagent cannot be optimal by simply using the trehalose.

In view of this, the present invention has been made.

Disclosure of Invention

The first object of the present invention is to provide a lyoprotectant for a reagent for PCR amplification.

The second object of the present invention is to provide a method for preparing a reagent for freeze-dried PCR amplification.

The third object of the present invention is to provide a lyophilized PCR amplification reagent prepared by the above preparation method.

A fourth object of the present invention is to provide the use of a reagent for freeze-dried PCR amplification.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

a freeze-drying protective agent for PCR amplification reagent comprises trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan.

Further, the composition comprises 2-6w/v% of trehalose, 0.4-2w/v% of hydroxypropyl-beta-cyclodextrin, 0.05-0.20w/v% of inulin and 0.005-0.020w/v% of carrageenan.

Further, it comprises 4w/v% trehalose, 1w/v% hydroxypropyl-beta-cyclodextrin, 0.1w/v% inulin and 0.010w/v% carrageenan.

Further, the PCR amplification reagent comprises at least one of an enzyme, an RNase inhibitor, dNTPS, a primer, a probe and a buffer.

A preparation method of a freeze-dried PCR amplification reagent comprises the step of freeze-drying a mixed solution of the PCR amplification reagent and the freeze-drying protective agent to obtain the freeze-dried PCR amplification reagent.

Further, the reagent for PCR amplification includes an enzyme;

preferably, the enzyme comprises at least one of a reverse transcriptase, a DNA polymerase and a UNG enzyme.

Further, the PCR amplification reagent further comprises at least one of an RNase inhibitor, dNTPS, a primer, a probe and a buffer solution;

preferably, the buffer comprises at least one of Tris-HCl, potassium ions, magnesium ions and ammonium sulfate.

Further, the process sequence of freeze drying is as follows: -50 to-45 ℃ for 180-220min; -35 to-30 ℃,15-20Pa,550-650min; 3-7deg.C, 1-5Pa,50-70min;20-30 ℃,1-5Pa,180-220min.

The freeze-dried PCR amplification reagent prepared by the preparation method.

The freeze-dried PCR amplification reagent is applied to nucleic acid amplification reaction or preparation of nucleic acid amplification reaction products.

Compared with the prior art, the invention has the beneficial effects that:

the freeze-drying protective agent of the reagent for PCR amplification provided by the invention comprises trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan. The freeze-drying protective agent has the advantages that on one hand, through mutual coordination and action among the components, the damage to active ingredients of the freeze-drying PCR amplification reagent in the processes of preparation, split charging and the like is reduced to the greatest extent, the activity of the freeze-drying PCR amplification reagent is kept not to be reduced, moisture absorption is difficult to occur, good re-solubility is achieved, the sensitivity, the precision and the like of PCR amplification reaction are not interfered, meanwhile, the validity period of the PCR amplification reagent can be remarkably prolonged, and the prepared freeze-drying PCR amplification reagent can be placed for at least 2 years at the temperature of 2-8 ℃ without freezing preservation and dry ice transportation. On the other hand, the freeze-drying protective agent can also enable the reagent for PCR amplification to form colloid at normal temperature, can effectively reduce the possibility of aerosol formation of a PCR amplification reaction system during use, and improves the sensitivity and accuracy of PCR. In addition, the lyoprotectant has wide application scenes, has good protection effect on reagents for PCR amplification (such as various enzymes, dNTPS, primers or probes, and the like, and a mixture of various combinations of the reagents), and particularly can realize one-tube lyoprotection when all active components in a PCR reaction system and the lyoprotectant are used for preparing a lyoprotectant, so that a user does not need to temporarily prepare the reaction system, the operation is simple and convenient, and errors caused by manual operation are eliminated.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.

The invention provides a freeze-drying protective agent of a reagent for PCR amplification, which comprises trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan.

In the present invention, trehalose protects proteins from denaturation and inactivation during lyophilization; inulin has the ability to bind free water and thereby reduce water activity; hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is an ideal excipient, has high water solubility, and can enhance the stability and long-acting property of the active ingredient; the carrageenan is formed by alternately connecting sulfuric acid-based or non-sulfuric acid-based galactose and 3, 6-dehydrated galactose through alpha-1, 3 glycosidic bonds and beta-1, 4 bonds, and the carrageenan is a flowing solution in hot water without influencing PCR reaction and detection, and the carrageenan is a colloid after cooling, so that the carrageenan has good supporting effect, provides loose intermolecular pore diameters, accelerates the sublimation of water in the freeze-drying process, and reduces water residues.

The freeze-drying protective agent has the advantages that on one hand, through the mutual coordination and the interaction among the components, the damage to active ingredients of the freeze-drying PCR amplification reagent in the processes of preparation, split charging and the like is reduced to the greatest extent, the activity of the freeze-drying PCR amplification reagent is kept not to be reduced, moisture absorption is not easy to occur, good re-solubility is achieved, the PCR amplification reaction is not interfered, meanwhile, the validity period of the PCR amplification reagent can be remarkably prolonged, and the prepared freeze-drying PCR amplification reagent can be placed for at least 2 years at the temperature of 2-8 ℃ without freezing preservation and dry ice transportation. On the other hand, the freeze-drying protective agent can also enable the reagent for PCR amplification to form colloid at normal temperature, can effectively reduce the possibility of aerosol formation of a PCR amplification reaction system during use, and improves the sensitivity and accuracy of PCR. In addition, the lyoprotectant has wide application scenes, has good protection effect on reagents for PCR amplification (such as various enzymes, dNTPS, primers or probes, and the like, and a mixture of various combinations of the reagents), and particularly can realize one-tube lyoprotection when all active components in a PCR reaction system and the lyoprotectant are used for preparing a lyoprotectant, so that a user does not need to temporarily prepare the reaction system, the operation is simple and convenient, and errors caused by manual operation are eliminated.

In a preferred embodiment, the lyoprotectant comprises 2-6w/v% trehalose, 0.4-2w/v% hydroxypropyl-beta-cyclodextrin, 0.05-0.20w/v% inulin, and 0.005-0.020w/v% carrageenan. Further preferred are: 4w/v% trehalose, 1w/v% hydroxypropyl-beta-cyclodextrin, 0.1w/v% inulin and 0.010w/v% carrageenan.

The contents of trehalose, hydroxypropyl-beta-cyclodextrin, inulin and carrageenan provided by the invention are all the use contents when preparing a pre-freeze-dried solution for the lyophilized PCR amplification reagent, for example, the content of trehalose in the pre-freeze-dried solution is 2w/v%, and the pre-freeze-dried solution is subjected to freeze drying to obtain the lyophilized PCR amplification reagent. Wherein, "w/v%" refers to the grams of material contained in each 100ml of solution. The trehalose content may be, but is not limited to, 2w/v%, 3w/v%, 4w/v%, 5w/v% or 6w/v%; the content of hydroxypropyl-beta-cyclodextrin may be, but is not limited to, 0.4w/v%, 0.6w/v%, 0.8w/v%, 1w/v%, 1.2w/v%, 1.4w/v%, 1.6w/v%, 1.8w/v% or 2w/v%; the inulin content may be, but is not limited to, 0.05w/v%, 0.07w/v%, 0.1w/v%, 0.13w/v%, 0.15w/v%, 0.18w/v% or 0.2w/v%; the carrageenan content may be, but is not limited to, 0.005w/v%, 0.007w/v%, 0.01w/v%, 0.013w/v%, 0.015w/v%, 0.017w/v% or 0.02w/v%.

In some embodiments, the reagents for PCR amplification include at least one of an enzyme, an rnase inhibitor, dNTPS, a primer, a probe, and a buffer. It will be understood that reagents for PCR amplification refer to the various active ingredients of the PCR amplification reaction system, such as buffers, various enzymes, dNTPS, primers, etc., as well as probes, RNase inhibitors, etc., used in some particular reaction systems, and that the lyoprotectant may be used for the lyoprotection of one or more of the active ingredients, preferably the reaction system other than the amplification template.

The invention also provides a preparation method of the freeze-dried PCR amplification reagent, which comprises the step of freeze-drying a mixed solution of the PCR amplification reagent and the freeze-dried protective agent to obtain the freeze-dried PCR amplification reagent. The reagent for freeze-dried PCR amplification is stable and has long effective period, and aerosol pollution is basically not existed in the prepared reaction system.

In some embodiments, the reagent for PCR amplification includes an enzyme, which may be at least one of a reverse transcriptase, a DNA polymerase, and a UNG enzyme. RNase inhibitors, dNTSs, primers, probes, or buffers, and combinations thereof, may also be included, wherein the buffers include at least one of Tris-HCl, potassium ions, magnesium ions, and ammonium sulfate. It will be appreciated that the person skilled in the art can select the PCR amplification reagents of different active components to prepare the lyophilized PCR amplification reagents according to the specific PCR type (reverse transcription PCR, real-time fluorescent quantitative PCR, loop-mediated isothermal amplification reaction, etc.), and then use the reagents together with other components in PCR amplification reaction.

In the preparation method, the freeze drying process is preferably the following process, and the operations are sequentially as follows: -50 to-45 ℃ for 180-220min; -35 to-30 ℃,15-20Pa,550-650min; 3-7deg.C, 1-5Pa,50-70min;20-30 ℃,1-5Pa,180-220min. Further preferred is the following operation:

the invention also protects the freeze-dried PCR amplification reagent prepared by the preparation method, and the reagent is applicable to various nucleic acid amplification reactions or is used for preparing nucleic acid amplification reaction products.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

The research idea of the invention is to add primer probes and dNTPs protective agents on the basis of trehalose serving as a protein freeze-drying protective agent, and optimize the formula of the freeze-drying protective agent according to conditions such as the effective period of an accelerated environment, the re-solubility and the like under the condition that the sensitivity, the precision and the like of a PCR reaction system are not affected.

Taking a Hepatitis B Virus (HBV) nucleic acid measuring system as an example, the effects of four fillers and protective agents of trehalose, hydroxypropyl-beta-cyclodextrin (HP-beta-CD), inulin and carrageenan are tested at a time, and the test stages are evaluated by checking three aspects of appearance, resolubility and accelerated stability at-45 ℃ for 3 days, 6 days, 9 days, 12 days, 15 days, 18 days and 21 days (if necessary for a prolonged period of time) respectively, and the optimal concentration ranges are selected respectively. Then, the high concentration reference (reference H), the medium concentration reference (reference M) and the low concentration reference (reference L) were detected at 6, 12, 15, 18, 21, 24, 27 months (as needed, or longer), respectively, and the real-time stability effect by the check filler was observed for the Ct value change thereof to check the real-time stability effect by the filler.

Acceptable standards:

1. appearance: complete white loose blocky freeze-dried powder without collapse and atrophy;

2. and (3) re-dissolving: the freeze-dried powder is completely clarified by adding water until the water is dissolved, and the time is less than or equal to 60 sec;

3. acceleration stability assessment criteria:

3.1 reference L: detecting that 4 positive plasmids should all be detected as positive (4/4);

3.2 reference M: the Ct value is less than or equal to 1.66 after the comparison (the concentration is detected in 0 day) (the Ct value is calculated according to the requirement that the accuracy deviation of a quantitative reagent of ' kit for detecting nucleic acid amplification ' YY/T1182-2010 ' is not more than 0.5 order of magnitude);

4. real-time stability (2-8 ℃) assessment criteria:

4.1 reference L: detecting that 4 positive plasmids should all be detected as positive (4/4);

4.2 reference M and reference H: the Ct value is less than or equal to 1.66 (according to the above) after the Ct value of the reagent control group (the concentration is detected in 0 days).

5. Optimum condition selection criteria: on the basis of meeting the appearance, the resolubility, the acceleration stability and the real-time stability, the relationship between the theoretical conditions and the results shows normal distribution, so that the numerical value of the intermediate embodiment meeting the range of the standard is selected as the optimal condition.

Reagent:

1. trehalose: purchasing from Shanghai workers;

2. inulin (inulin): purchasing from Shanghai workers;

3. hydroxypropyl beta-cyclodextrin: derived from Zibo Qian Hui organism;

4. carrageenan: purchasing from aledine;

5. 10 XHBV primer probe mix (10 XHBVbmix): purchasing Yingweiji;

6. 10 XPCR Buffer (containing 10mM Tris-HCl, 10mM ammonium sulfate, 50mM potassium chloride, 25mM magnesium chloride, bai. Mu.M dNTPs, etc.): self-matching;

7. enzyme (containing Taq DNA polymerase and UNG enzyme): purchasing a Sharp organism from Zhuhuabao;

8. HBV plasmid: constructed on Yingweiji.

The device comprises:

1. vacuum freeze dryer: LYO-0.5 part of Dongfulong;

2. real-time fluorescent quantitative PCR instrument: shanghai macrostone;

3. water jacket type thermostat: shanghai extract macro;

4. refrigerator with temperature of 2-8℃: sea medicine preservation cabinet.

Preparing reference materials for examination:

1. reference L preparation: HBV plasmid was diluted to 1 copy/. Mu.L with nucleic acid dilution, and the DNA and RNase free screw tube aliquots were kept at-20℃as low value reference. When in use, 50 mu L of sample is directly added for machine detection.

2. Reference M preparation: HBV plasmid was diluted to 1E+03 copies/. Mu.L with nucleic acid dilution, and the DNA and RNA enzyme free screw tube aliquots were kept at-20℃as high value reference. When in use, 50 mu L of sample is directly added for machine detection.

3. Reference H preparation: the HBV plasmid was diluted to 1E+06 copies/. Mu.L with nucleic acid dilution, and the DNA and RNA enzyme free screw tube branches were aliquoted as high value reference and stored at-20 ℃. When in use, 50 mu L of sample is directly added for machine detection.

EXAMPLE 1 trehalose concentration selection

1. Preparing 40% trehalose mother solution: weighing 40g of trehalose, dissolving to 100mL with ultrapure water, fully dissolving and uniformly mixing, sub-packaging, and freezing for preservation.

2. PCR reaction solutions with different trehalose contents (1%, 2%, 4%, 6% and 8%) were prepared as in Table 1-1, and 50. Mu.L of each well was dispensed into PCR eight-tube tubes and placed into a freeze dryer.

Table 1-1: trehalose system formulation table units at different concentrations: mu L (mu L)

3. Freeze-drying was performed. The freeze-drying conditions were as follows:

table 1-2: freeze drying conditions

4. After lyophilization was completed, the reagents were removed and capped. Appearance results of all groups were recorded, bagged, tested for 0 days and redissolved results were recorded.

Tables 1-3: appearance and redissolution results of trehalose PCR systems with different concentrations

5. The reagents were placed in 45℃incubators and the high and low concentration references were removed at 3 days, 6 days, 9 days and 12 days, respectively (as required for the results). The results were as follows:

tables 1 to 4: accelerated stability results of different concentration trehalose PCR systems

Tables 1 to 5: summarizing results of PCR (polymerase chain reaction) systems with trehalose at different concentrations

Group of	1% trehalose	2% trehalose	4% trehalose	6% trehalose	8% trehalose
						Appearance of	Loose cake shape	Loose cake shape	Loose cake shape	Loose cake shape	Bottom collapse
Time of reconstitution	For 10 seconds	15 seconds	15 seconds	For 20 seconds	25 seconds
						Conclusion of apparent resolubility	Qualified product	Qualified product	Qualified product	Qualified product	Failure to pass
Stability at 45 DEG C	Day 0	9 days	9 days	9 days	Day 0

6. Conclusion:

the HBV systems containing trehalose with different concentrations are examined from three aspects of appearance, resolubility and acceleration stability, and the results show that 2% -6% of trehalose has a qualified loose cake shape, the resolubilization time is less than 60sec, and the acceleration stability Ct value is minimum in the post-processing and meets the requirement that the post-processing is less than or equal to 1.66. Therefore, it is considered that the concentration of trehalose in the PCR system is preferably 2% -6%.

In the next step, when the concentration of hydroxypropyl-beta-cyclodextrin is selected, 4% is preferred as the concentration of trehalose in the PCR system.

Example 2 concentration selection of hydroxypropyl-cyclodextrin (hereinafter referred to as HP-beta-CD)

1. Preparing 20% HP-beta-CD mother solution: weighing 20g of hydroxypropyl-beta-cyclodextrin, dissolving to 100mL by using ultrapure water, fully dissolving and uniformly mixing, sub-packaging, and freezing for preservation:

2. preparing PCR reaction solutions with 4% trehalose and different HP-beta-CD contents according to the table 2-1, subpackaging 50 mu L of each group of each hole into PCR eight-connecting tubes, and putting into a freeze dryer;

table 2-1: different concentrations of HP-beta-CD system recipe units: mu L (mu L)

3. Lyophilization was performed under the conditions of tables 1-2.

Table 2-2: appearance and reconstitution results of different concentrations of HP-beta-CD PCR System

Group of

0.2％

0.4％

0.8％

1.6％

2.0％

2.4％

Appearance of

Loose cake shape

Bottom collapse

Time of reconstitution

25 seconds

40 seconds

Conclusion(s)

Qualified product

Failure to pass

5. The reagents were placed in 45℃incubators and high and low concentration references were removed at 6 days, 12 days and 15 days, respectively (as required for the results). The results were as follows:

table 2-3: accelerated stability results of different concentrations of HP-beta-CD PCR System

Tables 2 to 4: summarizing PCR system results of HP-beta-CD with different concentrations

6. Analysis and conclusion:

from the two aspects of appearance and redissolution, 0.2% -2% of hydroxypropyl-beta-cyclodextrin has a qualified loose cake shape, and the redissolution time is less than 60 sec; when the concentration of hydroxypropyl- β -cyclodextrin was further increased, the appearance was not satisfactory, and it was presumed that too high a concentration affected the effect of freeze-drying.

As shown by the results of trehalose concentration screening, the 45 ℃ stability of HBV PCR system containing 2% -6% of trehalose is 9 days, the data of the group is increased by 0.4% -2.0% of hydroxypropyl-beta-cyclodextrin, the 45 ℃ accelerated stability is improved to 12 days, and the stability of HBV PCR system without hydroxypropyl-beta-cyclodextrin is longer. Therefore, the PCR system contains 0.4% -2.0% of hydroxypropyl-beta-cyclodextrin, which is considered to have an effect on the stability of HBV PCR system. When 0.2% and 2.4% of hydroxypropyl-beta-cyclodextrin with concentration of 0.4% -2.0% are added, the PCR acceleration stability is 6 days, and it is presumed that the freeze-drying effect of the system is affected or the concentration is insufficient due to the increase of the concentration of the filler, and the water residue is excessive.

And in the next step, the HBV PCR system preferentially uses 4% of trehalose and 1% of hydroxypropyl-beta-cyclodextrin as basic freeze-drying protective agents, different concentrations of inulin are added, and the effect of the HBV PCR system on the system stability is examined.

Example 3 selection of inulin concentration

1. Preparing 5% inulin mother liquor: weighing 5g of inulin, adding ultrapure water to 100mL, fully dissolving and uniformly mixing, subpackaging the small branches, and freezing for preservation:

2. preparing PCR reaction solutions of 4% trehalose, 1% hydroxypropyl-beta-cyclodextrin and different contents (0.02%, 0.05%, 0.10%, 0.20% and 0.50%) of inulin according to Table 3-1, packaging 50 μl of each group of each hole into PCR eight-connecting tubes, and placing into a freeze dryer;

table 3-1: formulation table of different concentration inulin system

3. Lyophilization was performed under the conditions of tables 1-2.

Table 3-2: appearance and redissolution results of different-concentration inulin PCR system

Group of	0.02% inulin	0.05% inulin	0.10% inulin	0.20% inulin	0.40% inulin
						Appearance of	Loose cake shape	Loose cake shape	Loose cake shape	Loose cake shape	Collapse of
Time of reconstitution	30 seconds	30 seconds	30 seconds	30 seconds	2 minutes
						Conclusion(s)	Qualified product	Qualified product	Qualified product	Qualified product	Failure to pass

5. The reagents were placed in 45℃incubators and the high and low concentration references were removed at 12 days, 15 days, 18 days and 21 days, respectively (as the results require). The results were as follows:

table 3-3: accelerated stability results of different concentration inulin PCR systems

Tables 3 to 4: summarizing results of PCR systems of different concentrations of inulin

Group of	0.02% inulin	0.05% inulin	0.10% inulin	0.20% inulin	0.40% inulin
						Appearance of	Loose cake shape	Loose cake shape	Loose cake shape	Loose cake shape	Collapse of
Time of reconstitution	30 seconds	30 seconds	30 seconds	30 seconds	2 minutes
						Conclusion of apparent resolubility	Qualified product	Qualified product	Qualified product	Qualified product	Failure to pass
Stability at 45 DEG C	For 12 days	For 15 days	For 15 days	For 15 days	Day 0

6. Conclusion:

from the three aspects of appearance, resolubility and acceleration stability, the HBV system containing 4% of trehalose, 1% of hydroxypropyl-beta-cyclodextrin and inulin with different concentrations is examined, and the result shows that 0.05% -0.20% of inulin has a qualified loose cake shape, the resolubilization time is less than 60sec, and the acceleration stability Ct value is minimum after the 15 days which are consistent with less than or equal to 1.66 after the delay, and the stability is longer than that when the system does not contain inulin. Therefore, the concentration of inulin in the PCR system is 0.05% -0.20%, which is considered to have an effect of promoting the stability of HBV PCR system.

And in the next step, the HBV PCR system preferentially uses 4% of trehalose, 1% of hydroxypropyl-beta-cyclodextrin and 0.1% of inulin as basic freeze-drying protective agents, carrageenan with different concentrations is added, and the effect of the HBV PCR system on the stability of the HBV system is examined.

Example 4 selection of carrageenan concentration

1. 0.5% carrageenan: weighing 0.50g carrageenan, adding ultrapure water to 100mL, carrying out water bath at 80 ℃ for 20min, reversing and mixing for 50 times every 5min, fully dissolving and mixing, subpackaging small branches, and preserving at 2-8 ℃;

2. preparing a system: heating carrageenan to 37deg.C for dissolving, and preparing PCR reaction solution of 4% trehalose, 1% hydroxypropyl-beta-cyclodextrin, 0.1% inulin and carrageenan with different contents according to table 4-1, wherein after carrageenan is added into the system, mixing thoroughly. 50 mu L of each group of each hole is split into eight PCR connecting tubes and put into a freeze dryer;

table 4-1: formulation table of carrageenan systems with different concentrations

3. Lyophilization was performed under the conditions of tables 1-2.

Table 4-2: appearance and redissolution results of carrageenan PCR systems with different concentrations

Group of	0.002％	0.005％	0.010％	0.020％	0.040％
						Appearance of	Loose cake shape	Loose cake shape	Loose cake shape	Loose cake shape	Relatively knot(s)Cake shape
Time of reconstitution	30 seconds	For 35 seconds	45 seconds	54 seconds	5 minutes 30 seconds
						Conclusion(s)	Qualified product	Qualified product	Qualified product	Qualified product	Failure to pass

5. The reagents were placed in a 45℃incubator and the high and low concentration references were removed at 15 days, 18 days and 21 days, respectively. The results were as follows:

table 4-3: accelerated stability results of carrageenan PCR systems with different concentrations

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Tables 4-4: summarizing PCR system results of carrageenans with different concentrations

6. Conclusion:

the HBV system containing 4% of trehalose, 1% of hydroxypropyl-beta-cyclodextrin, 0.1% of inulin and carrageenan with different concentrations is examined from the aspects of appearance and redissolution, when the carrageenan concentration is 0.002% -0.020%, the appearance is qualified loose cake shape, and the redissolution time is less than 60 seconds; when the carrageenan concentration of the system reaches 0.040%, the freeze-dried powder has a firm appearance, and the redissolution time is as long as five minutes and half minutes, which is not satisfactory in daily experiments and application of products in clients. Compared with the 15-day result of the previous screening of the concentration of the inulin, the method has the advantages that the stability of the system with the concentration of 0.002 percent of carrageenan is kept unchanged for 15 days, and the stability of the system with the concentration of 0.002 to 0.040 percent of carrageenan is greatly improved. The standard stability of less than or equal to 1.66 is 21 days after the Ct value is pushed, but the result shows that the longer the storage time is, the more obvious the Ct value is pushed. And Ct values of the 0.040% carrageenan concentration group reference M are obviously pushed compared with other groups, and the system is supposed to be inhibited by too high carrageenan concentration.

In consideration of the comprehensive appearance, the redissolution time and the 45 ℃ accelerated stability result, the carrageenan has a positive effect on the stability of the HBV PCR system, and the optimal concentration is 0.005% -0.020%.

Example 5 real-time stability verification

According to the initially optimized PCR freeze-drying protective agent formula (2-6% (w/v) trehalose, 0.4-2% (w/v) hydroxypropyl beta-cyclodextrin, 0.05-0.20% (w/v) inulin and 0.005-0.020% (w/v) carrageenan), the test comprises the protective agents with the four components. The method is applied to HBV PCR system, and the real-time stability of the HBV PCR system is checked at the temperature of 2-8 ℃. To verify the effect of the protectant of the invention and the method of lyophilization of the reagents on the system, a set of liquid systems was specially designed as a control.

1. Preparing a system: heating carrageenan to 37 ℃ in advance for dissolution, and preparing PCR reaction liquid according to the table 5-1, wherein after the carrageenan is added into the system, the mixture is fully and uniformly mixed immediately.

Table 5-1: formulation table of filler systems with different concentrations

Units: mu L (mu L)

2. Control group (15 μl/human): 145 μL (9 parts) are packed into centrifuge tubes, and are placed at 2-8deg.C for 3 days, 6 days and 9 days for examination;

3. subpackaging 50 mu L of each other group of holes into PCR eight-connecting tubes, and putting into a freeze dryer; lyophilization was performed under the conditions of tables 1-2.

4. After lyophilization was completed, the reagents were removed and capped. To avoid that the subsequent tests involved stability of unsealing, it was decided to seal the package to a specification of 8 persons/bag per test check.

5. Appearance results of all groups were recorded, bagged, tested for 0 days and redissolved results were recorded.

Table 5-2: appearance and redissolution results of different-concentration filler PCR system

Group of	①	②	③	④	⑤
						Appearance of	Loose cake shape	Loose cake shape	Loose cake shape	Loose cake shape	Stronger cake shape
Time of reconstitution	30 seconds	30 seconds	40 seconds	55 seconds	9 minutes
						Conclusion(s)	Qualified product	Qualified product	Qualified product	Qualified product	Failure to pass

6. The reagents in the sealed package were placed in a 2-8deg.C refrigerator, and high concentration (reference H), medium concentration (reference M) and low concentration (reference L) references were detected at 6, 12, 15, 18, 21, 24, 27 months (as needed, or longer), respectively, to examine the effect of the filler on real-time stability. The results were as follows:

table 5-3: real-time stability results of HBV PCR systems with different concentrations of protective agents at 2-8 DEG C

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7. Summarizing the results:

tables 5 to 4: summarizing results of PCR (polymerase chain reaction) system with fillers of different concentrations

In summary, compared with the stability of the liquid system at 2-8 ℃ less than 3 days, the optimized freeze-drying protective agent has obvious protective effect on the PCR freeze-drying system, the stability at 2-8 ℃ is as long as 27 months, and the effect is very obvious.

Example 6PCR System Adaptation verification

According to the optimized PCR freeze-drying protective agent formula (4% w/v trehalose, 1% w/v hydroxypropyl-beta-cyclodextrin, 0.1% w/v inulin and 0.01% w/v carrageenan), the PCR freeze-drying protective agent is applied to different DNA amplification systems (a Tubercule Bacillus (TB) detection system, a hepatitis C virus (HBV) liver detection system) and RNA amplification systems (influenza virus (Flu), human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) liver detection systems) and the real-time stability of the PCR freeze-drying protective agent is checked at 2-8 ℃. To verify the effect of the protective agent containing the invention and the agent lyophilization process on the system.

1. Preparing a system: heating carrageenan to 37deg.C for dissolving, and preparing PCR reaction solution according to table 5-1(3), wherein after carrageenan is added into the system, mixing thoroughly.

6. The sealed and packed reagent was placed in a refrigerator at 2-8 c, and the real-time stability effect of the filler was examined at 27 months for high concentration reference (reference H), medium concentration reference (reference M) and low concentration reference (reference L). Summarizing the results:

table 6-1: summarizing results of different PCR systems

In summary, compared with the stability of a liquid system at 2-8 ℃ less than 3 days, the freeze-drying protective agent provided by the invention has obvious protective effect on different PCR freeze-drying systems (mainly divided into a DNA system for direct amplification and an RNA system containing reverse transcription), has the stability of 2-8 ℃ as long as 27 months, and has very obvious effect.

While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. The application of the freeze-drying protective agent of the reagent for PCR amplification in nucleic acid amplification reaction or preparation of nucleic acid amplification reaction products; the freeze-drying protective agent consists of 2-6w/v% of trehalose, 0.4-2w/v% of hydroxypropyl-beta-cyclodextrin, 0.05-0.20w/v% of inulin and 0.005-0.020w/v% of carrageenan;

the PCR amplification reagent comprises enzyme, dNTPS, a primer, a probe and a buffer solution.

2. The use according to claim 1, wherein the lyoprotectant consists of 4w/v% trehalose, 1w/v% hydroxypropyl- β -cyclodextrin, 0.1w/v% inulin and 0.010w/v% carrageenan.

3. The use according to claim 1, wherein the reagents for PCR amplification comprise enzymes, rnase inhibitors, dNTPS, primers, probes and buffers.

4. The use according to claim 3, wherein the enzyme comprises at least one of reverse transcriptase, DNA polymerase and UNG enzyme.

5. The use according to claim 3, wherein the buffer comprises at least one of Tris-HCl, potassium ions, magnesium ions and ammonium sulphate.

6. The use according to any one of claims 1 to 5, wherein the lyophilized PCR amplification reagent is obtained by freeze-drying a mixture of the PCR amplification reagent and the lyoprotectant.

7. The use according to claim 6, wherein the process sequence of freeze-drying is in turn: -50 to-45 ℃ for 180-220min; -35 to-30 ℃,15-20Pa,550-650min; 3-7deg.C, 1-5Pa,50-70min;20-30 ℃,1-5Pa,180-220min.