Freeze-dried microspheres of STR multiplex fluorescent composite amplification detection reagent and preparation method thereof

Abstract

The application provides a freeze-dried microsphere of a STR multiplex fluorescence composite amplification detection reagent and a preparation method thereof. The freeze-dried microsphere comprises a STR multiplex fluorescence composite amplification PCR reaction system and a freeze-drying protective agent. Raw material components of the freeze-drying protective agent include dextran, trehalose, bovine serum albumin, gelatin, glycerol, dimethyl sulfoxide, a surfactant, an antifoaming agent and a preservative. The application solves the problem that the PCR reaction reagent cannot be stored and transported at room temperature in the prior art, and avoids repeated freeze-thaw of the PCR reagent, so that the reagent has higher stability. The freeze-dried product comprises main reagents of the PCR reaction, is easy to use, improves the detection rate of trace samples by full-volume sample adding, effectively avoids pollution introduced in the experimental operation process and reduces the error of adding reaction components.

Description

Technical Field

[0001] This invention relates to lyophilized microspheres of a STR multiplex fluorescent multiplex amplification detection reagent and its preparation method, belonging to the field of molecular biology technology. Background Technology

[0002] Short tandem repeats (STRs), also known as microsatellites or simple sequence repeats (SSRs), are a class of DNA tandem repeat sequences widely found in eukaryotic genomes, with a core sequence consisting of 2-6 base repeat units. STR loci are numerous, widely distributed, accounting for approximately 3% of the entire genome, and exhibit high polymorphism. This polymorphism primarily stems from individual differences in the number of core sequence repeats, and this variation follows Mendelian inheritance laws during the genetic process.

[0003] STR multiplex fluorescent multiplex amplification refers to the simultaneous PCR detection of two or more STR gene loci by adding two or more pairs of fluorescent primers to the same PCR reaction system. Subsequent analysis of the PCR products at each STR locus yields the required data for individual identification. Therefore, STR multiplex fluorescent multiplex amplification technology is widely used for individual identification, kinship testing, and population genetics research.

[0004] However, since biological reagents are active substances, and the key reagent Taq enzyme is a protein product, it is particularly sensitive to temperature. Enzyme inactivation leads to the inability to amplify. At the same time, the fluorescent primers in the mixture are very sensitive to temperature and light intensity. High temperature and high pressure can easily cause primer fluorescence shedding and degradation, resulting in reduced amplification efficiency. Due to these characteristics, the product needs to be stored at low temperature and transported in a cold chain throughout the transportation process. Poor transportation and storage conditions can easily lead to product failure, so the transportation cost is very high and the risks cannot be controlled.

[0005] Currently, the main forms of lyophilization include vials, eight-tube strips, irregularly shaped tubes, and lyophilized microspheres. Each lyophilization technology has its own advantages and disadvantages. Compared with other lyophilization methods, lyophilized microspheres have significant advantages: precise quantification, single-use per person, and convenient product use; they can undergo special processing to prevent moisture absorption, have no requirements for packaging materials, and can be packaged into eight-tube strips, microfluidic chips, various biochips, cards, flow cytometry tubes, 96-well plates, etc., and can be stored at room temperature; because there are no packaging material limitations, its production capacity can be maximized to the limit of the lyophilizer.

[0006] Lyophilized microspheres can transform unstable chemical reagents at room temperature into high-quality, stable lyophilized microspheres that can be stored for extended periods and transported at room temperature. However, different substances require different lyophilization protectants. Inappropriate selection of the protectant can lead to lyophilized microsphere preparation failure, or even if lyophilized microspheres are obtained, it may adversely affect enzymes and fluorescent primers, thus impacting test performance and accuracy. This invention uses lyophilization technology to freeze-dry the primer-enzyme mixture, requiring only room temperature transport during transit. This avoids the repeated freeze-thaw cycles that affect reagent performance and shelf life. For immediate use, only dilution with water is needed. This not only simplifies user operation but also reduces the demands on operators. For sales to remote areas, conventional transportation is possible, saving significant transportation costs and aiding local DNA database construction and case investigation. Summary of the Invention

[0007] This invention provides a lyophilized microsphere for STR multiplex fluorescence amplification detection reagent and its preparation method. The lyophilized microsphere has strong stability and good protection effect. After being lyophilized into microspheres from PCR amplification mixture, there is no difference in amplification efficiency, amplification uniformity, and amplification sensitivity. Moreover, it can be stored at room temperature for a longer time.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The raw material components of the lyophilized microspheres of a STR multiplex fluorescence multiplex amplification detection reagent include: STR multiplex fluorescence multiplex amplification PCR reaction system and lyophilization protectant; the raw material components of the lyophilization protectant include: dextran, trehalose, bovine serum albumin, gelatin, glycerol, dimethyl sulfoxide, surfactant, defoamer and preservative.

[0010] Inappropriate selection of lyophilization protectants can lead to failure in the preparation of lyophilized microspheres, or even if lyophilized microspheres are obtained, they may adversely affect enzymes and fluorescent primers, thus impacting test performance and accuracy. Through the specific selection of lyophilization protectants, the inventors not only prepared the "STR multiplex fluorescent amplification PCR reaction system" into lyophilized microspheres, improving their storage stability at room temperature, but also maintained the enzyme protein activity essentially unchanged during pre-freezing and lyophilization, and prevented fluorescence shedding from the dual-fluorescent labeled primers. The lyophilized microspheres exhibit good hygroscopic resistance and high rigidity, and are not easily broken or shed powder during the post-lyophilization packaging process.

[0011] The aforementioned lyophilization protectant, while being able to be formed into lyophilized microspheres, does not affect the amplification efficiency of enzymes and fluorescent primers.

[0012] To better minimize the impact of the cryoprotectant on amplification detection, the cryoprotectant composition is as follows: dextran 0.5-6%, trehalose 2-20%, bovine serum albumin 0.5-3.0 mg / ml, gelatin 0.1-0.5%, glycerol 0.5-2.0%, dimethyl sulfoxide 0.06-1.5%, surfactant 0.2-3.5%, defoamer 0.04-0.4%, and preservative 0.02-0.15%. All percentages are by mass. The content of each ingredient refers to the content of each ingredient after the cryoprotectant is added to the PCR amplification reaction reagent in step ① during the preparation process described below.

[0013] To improve material homogeneity without affecting test performance, the surfactants include at least one of: Tween 20, Tween 80, polyethylene glycol octylphenyl ether (Triton X-100), or ethyl phenyl polyethylene glycol (NP-40). The defoamers include one or more combinations of isopropanol, ethanol, and Foamban.

[0014] To ensure the storage performance of the material without affecting the testing performance, the preservatives include one or more combinations of potassium sorbate, sodium azide, and Proclin-300.

[0015] The above-mentioned STR multiplex fluorescent complex amplification PCR reaction system includes: PCR buffer, STR multiplex fluorescent complex primers, and Taq polymerase.

[0016] The PCR buffer mentioned above includes: Tris-HCl, KCl, DMSO, betaine, (NH4)2SO4, dNTPs, and Mg. 2+ Preferably, the PCR buffer contains 85mM Tris-HCl (pH 8.5), 95mM KCl, 5mM DMSO, 1.5M betaine, 10mM (NH4)2SO4, 6.5mM dNTPs, and 8mM MgCl2.

[0017] A method for preparing lyophilized microspheres of an STR multiplex fluorescent multiplex amplification detection reagent includes the following steps:

[0018] ① After preparing the STR multiplex fluorescent complex amplification PCR amplification reaction reagent, add a certain amount of lyophilization protectant, dissolve it completely, and store it at 2-8℃ for later use;

[0019] ② Pour liquid nitrogen into a sterile medicine cup, and use a pipette to drop the mixed solution into the liquid nitrogen in 20 μL volumes, which will condense into small round balls;

[0020] ③ Seal the medicine cup with a sterile film and punch holes in the sterile film;

[0021] ④ Place the medicine cup into a pre-cooled freeze dryer and prepare STR multiplex fluorescent composite amplification freeze-dried microspheres according to the set freeze-drying program.

[0022] The freeze-drying procedure is as follows: ① Pre-freezing: Set the partition temperature to -55℃ and maintain at 1 atmosphere for 1 hour; ② Sublimation drying: Set the partition temperature to -45℃ and the vacuum degree to 2 Pa for 10 hours, then adjust the partition temperature to -25℃ and the vacuum degree to 12 Pa for 4 hours; ③ Desorption drying: Set the partition temperature to 25℃ and the vacuum degree to 0 Pa for 7 hours.

[0023] The aforementioned lyophilized microsphere technology maximizes enzyme activity, and the loose network structure of the lyophilized microspheres allows for rapid reconstitution. The lyophilized microspheres of the STR multiplex fluorescent multiplex amplification assay reagent offer advantages such as high speed, high specificity, high sensitivity, strong resistance to impurities, and good stability, significantly improving the reagent's stability, ease of use, and transport convenience.

[0024] Any techniques not mentioned in this invention are directly referenced from existing technologies.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. The lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent of the present invention showed no difference in amplification efficiency, amplification uniformity, and amplification sensitivity compared to the unlyophilized microspheres.

[0027] 2. The storage requirements for the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent of this invention are significantly reduced. Not only is room temperature transportation achieved, but the shelf life is also extended from 2 years of low-temperature storage to 3 years of room-temperature storage. Even if the surface of the lyophilized microspheres is slightly damaged due to excessive jolting during transportation, it will not affect the amplification effect.

[0028] 3. The lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent of this invention correspond to one reaction per microsphere. Each microsphere is individually packaged to avoid repeated freeze-thaw cycles, making it more convenient to use and ensuring higher reagent stability.

[0029] 4. The lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent of this invention improve the detection rate of extremely small sample volumes by adding the whole volume of sample.

[0030] 5. The lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent of the present invention contain a reaction buffer for isothermal amplification, Taq polymerase, dNTPs, and Mg. 2+ With primer mixes, PCR amplification can be performed simply by adding water and template, reducing the preparation steps of the amplification system, lowering the risk of human error and contamination, and greatly improving work efficiency. Attached Figure Description

[0031] Figure 1 This is a photograph of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent of this invention.

[0032] Figure 2 This is a flowchart illustrating the preparation method of the lyophilized microspheres for the STR multiplex fluorescence multiplex amplification detection reagent of the present invention.

[0033] Figure 3 This is a genotyping pattern of STR multiplex fluorescence multiplex amplification detection reagent before lyophilization and after lyophilization of lyophilized microspheres amplifying 1 ng / μL of positive 9948.

[0034] Figure 4 This is a genotyping chart of the STR multiplex fluorescence multiplex amplification detection reagent before lyophilization and after lyophilization of the lyophilized microspheres amplifying a trace sample of 30 pg / μL positive 9948.

[0035] Figure 5 The image shows the appearance of the lyophilized microspheres of the STR multiplex fluorescence amplification detection reagent after simulated transport testing in this embodiment of the invention.

[0036] Figure 6 This is the genotyping pattern of 1 ng / μL positive 9948 amplified by lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent in this embodiment of the invention after simulated transport testing.

[0037] Figure 7 This is a genotyping spectrum of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent in an embodiment of the present invention, obtained after being dissolved and subjected to accelerated stability testing.

[0038] Figure 8 This is the genotyping spectrum of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent in this embodiment of the invention, accelerated at 45°C.

[0039] Figure 9 This is the genotyping pattern of 1 ng / μL positive 9948 amplified by lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent in the comparative example of this invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in complete and detailed manner below with reference to specific embodiments and corresponding drawings. The described embodiments are only some embodiments of this invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The technical solutions and practical applications provided by the various embodiments of this invention will be described below with reference to the accompanying drawings.

[0041] On one hand, this invention provides lyophilized microspheres of a STR multiplex fluorescent multiplex amplification detection reagent, comprising: an STR multiplex fluorescent multiplex amplification PCR reaction system and a lyophilization protectant. The STR multiplex fluorescent multiplex amplification PCR reaction system includes: PCR buffer, STR multiplex fluorescent multiplex primers, and Taq polymerase; the PCR buffer includes: Tris-HCl, KCl, DMSO, betaine, (NH4)2SO4, dNTPs, and Mg2+. The lyophilization protectant includes: dextran, trehalose, bovine serum albumin, gelatin, glycerol, dimethyl sulfoxide, surfactant, defoamer, and preservative. The surfactant includes at least one of: Tween 20, Tween 80, polyethylene glycol octylphenyl ether (Triton X-100), or ethylphenyl polyethylene glycol (NP-40). The defoamer includes one or more combinations of isopropanol, ethanol, and Foamban. The preservative includes one or more combinations of potassium sorbate, sodium azide, and Proclin-300.

[0042] During the research and development process, this invention conducted tests and combinations of different concentrations of various components of the freeze-drying protectant, including dextran, trehalose, defoamer, and preservatives. This not only improved the storage stability of the freeze-dried microspheres at room temperature but also maintained the enzyme protein activity essentially unchanged during pre-freezing and freeze-drying, and prevented fluorescence shedding from the dual-fluorescent labeled primers. Simultaneously, these improvements enhanced the hygroscopic resistance and rigidity of the freeze-dried microspheres, making them less prone to breakage and powder loss during the post-freeze packaging process. The freeze-drying protectant composition is as follows: glucose 0.5-6%, trehalose 2-20%, bovine serum albumin 0.5-3.0 mg / ml, gelatin 0.1-0.5%, glycerol 0.5-2.0%, dimethyl sulfoxide 0.06-1.5%, surfactant 0.2-3.5%, defoamer 0.04-0.4%, and preservative 0.02-0.15%.

[0043] On the other hand, the present invention provides a method for preparing lyophilized microspheres of STR multiplex fluorescence multiplex amplification detection reagent, comprising the following steps: ① After preparing the STR multiplex fluorescence multiplex amplification PCR amplification reaction reagent, a certain amount of lyophilization protectant is added, fully dissolved, and placed at 2-8℃ for later use; ② Liquid nitrogen is poured into a sterile medicine cup, and the mixed solution is dropped into the liquid nitrogen in 20 μL volumes using a pipette, which solidifies into spherical microspheres; ③ The medicine cup is sealed with a sterile membrane, and several holes are punched in the sterile membrane; ④ The medicine cup is placed in a pre-cooled freeze dryer, and the lyophilized microspheres of STR multiplex fluorescence multiplex amplification are prepared according to the set freeze-drying program. The freeze-drying procedure is as follows: ① Pre-freezing: Set the partition temperature to -55℃ and maintain at 1 atmosphere for 1 hour; ② Sublimation drying: Set the partition temperature to -45℃ and the vacuum degree to 2 Pa for 10 hours, then adjust the partition temperature to -25℃ and the vacuum degree to 12 Pa for 4 hours; ③ Desorption drying: Set the partition temperature to 25℃ and the vacuum degree to 0 Pa for 7 hours.

[0044] The lyophilization method of this invention significantly shortens the lyophilization time and achieves energy conservation and emission reduction. It also facilitates the subsequent expansion and improvement of lyophilized reagent production capacity. Furthermore, the formulation of the lyophilization protectant in this invention further enhances the sensitivity and stability of the lyophilized detection reagent. The flowchart of the preparation method of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent described in this invention is as follows: Figure 2 As shown.

[0045] The present invention will be described below through specific embodiments.

[0046] Example 1:

[0047] A method for preparing lyophilized microspheres of an STR multiplex fluorescent multiplex amplification detection reagent includes the following steps:

[0048] Measure the raw materials as shown in Table 2, mix them thoroughly to obtain a mixture, and store at 2-8℃ for later use. The primers for A33 STR multiplex fluorescent amplification are those from the Jiangsu Subo A33 Plex fluorescent detection kit, and the Taq DNA polymerase was purchased from Takara. The manufacturers and models of the reagents used are shown in Table 1.

[0049] Table 1. Reagent information used in Example 1

[0050]

[0051] Table 2. Components and content of lyophilized microspheres in the STR multiplex fluorescence amplification detection reagent in Example 1.

[0052]

[0053]

[0054] Liquid nitrogen was poured to two-thirds full into a sterile medicine cup. A pipette was used to add 20 μL of the mixed solution (a mixture of the STR multiplex fluorescence multiplex amplification PCR reaction system and the lyophilization protectant, composition shown in Table 2 above) to the liquid nitrogen, causing it to solidify into small spherical spheres. The medicine cup was sealed with a sterile membrane, and holes were punched in the membrane. The medicine cup was then placed in a pre-cooled freeze dryer, and the freeze-drying program was set as follows: Pre-freezing: separator temperature set to -55℃, 1 atmosphere, for 1 h; Sublimation drying: separator temperature set to -45℃, vacuum degree 2 Pa, for 10 h, then the separator temperature was adjusted to -25℃, vacuum degree 12 Pa, for 4 h; Desorption drying: separator temperature set to 25℃, vacuum degree 0 Pa, for 7 h. STR multiplex fluorescence multiplex amplification lyophilized microspheres were thus prepared.

[0055] The obtained STR multiplex fluorescence multiplex amplification lyophilized microspheres were spherical, with smooth surfaces and good integrity. The lyophilized microspheres were aliquoted into eight-tube bundles (one microsphere per tube). The aliquoted microspheres showed no surface damage and good integrity. The lyophilized microspheres aliquoted into the eight-tube bundles were as follows: Figure 1 As shown.

[0056] Example 2: Performance Testing

[0057] The performance of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent obtained in Example 1 was tested.

[0058] The lyophilized microspheres, aliquoted into 8-tube PCR units, were centrifuged to the bottom of the tube (ensuring each microsphere was at the bottom, one microsphere per tube). Each microsphere was dissolved in 9 μL of water, and 1 μL of Positive 9948 was added as the amplification template. The undried liquid reagent from the amplification kit served as a control. The amplification templates were 1 ng / μL Positive 9948 and 30 pg / μL Positive 9948. The amplification program was as follows: 95℃ for 3 min; 94℃ for 10 s, 60℃ for 90 s, 28 cycles; 60℃ for 12 min; and stored at 4–16℃. The amplification products were analyzed for fluorescence signal detection using a genetic analyzer. Fluorescence signal data were collected, and the DNA genotyping results from the A33 fluorescence detection kit were analyzed using gene analysis software. In the example, the genotyping patterns of the STR multiplex fluorescent multiplex amplification detection reagent before lyophilization (liquid reagent) and after lyophilization (lyophilized microspheres) are shown below. Figure 3 , Figure 4 As shown.

[0059] Depend on Figure 3 , Figure 4It can be seen that after preparing lyophilized microspheres from the mixed mix system of the A33 fluorescence detection kit using the lyophilization protectant ratio and lyophilization scheme of this invention, the amplification efficiency, amplification uniformity and sensitivity of the 1ng / μL positive 9948 sample and the 30pg / μL positive 9948 sample were repeatedly detected three times, and there was no difference between them and the undried liquid reagent.

[0060] Example 3: Simulated Transportation Test

[0061] The lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent obtained in Example 1 were subjected to simulated transport tests.

[0062] The lyophilized microspheres, aliquoted into 8-tube strips, were fixed in a shaker with parameters set to 25°C, 600 RPM, and 96 hours to simulate transport conditions. Amplification was performed using 1 ng / μL of positive 9948 as a template, compared to normally stored lyophilized microsphere detection reagents. The amplification program was as follows: 95°C for 3 min; 94°C for 10 s, 60°C for 90 s, 28 cycles; 60°C for 12 min; and storage at 4–16°C. The amplified products were analyzed for fluorescence signal detection using a genetic analyzer; the fluorescence signal data were collected, and the DNA genotyping results of the A33 fluorescence detection kit were analyzed using gene analysis software. The appearance of the lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent before and after simulated transport testing, along with their amplified genotyping patterns, are shown in the figures below. Figure 5 , Figure 6 As shown.

[0063] Depend on Figure 5 , Figure 6 It can be seen that the lyophilized STR multiplex fluorescent multiplex amplification detection reagent has good integrity and is basically undamaged after simulated transportation. Even if the surface of some lyophilized microspheres is slightly damaged due to excessive bumps during transportation, the amplification efficiency and amplification uniformity of the 1ng / μL positive 9948 sample are not significantly different from those before transportation when the sample is tested three times.

[0064] Example 4: Stability test after dissolution

[0065] The stability of the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent obtained in Example 1 was tested after being dissolved into a liquid reagent.

[0066] After dissolving the lyophilized microspheres in 9 μL of water and centrifuging with shaking, the mixture was placed at 4°C and amplified for 1, 3, 5, and 7 days. Amplification was then performed simultaneously with the immediately dissolved lyophilized microsphere detection reagent using 1 ng / μL of positive 9948 as a template for comparison. The amplification program was as follows: 95°C for 3 min; 94°C for 10 s, 60°C for 90 s, 28 cycles; 60°C for 12 min; and stored at 4–16°C. The amplification products were analyzed for fluorescence signal detection using a genetic analyzer. The fluorescence signal data were collected, and the DNA genotyping results of the A33 fluorescence detection kit were analyzed using gene analysis software. The stability test and genotyping pattern of the lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent after dissolving into liquid reagent in the example are shown below. Figure 7 As shown.

[0067] Depend on Figure 7 It can be seen that after the lyophilized STR multiplex fluorescent multiplex amplification detection reagent was dissolved and accelerated stability test was performed, the amplification efficiency and amplification uniformity of the 1ng / μL positive 9948 sample were not significantly different from those before the test when the sample was tested three times.

[0068] Example 5: Accelerated Stability Test

[0069] Accelerated stability tests were performed on the lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent obtained in Example 1.

[0070] The lyophilized microspheres, aliquoted into 8-tube packs, were placed in a 45°C oven and accelerated for 15, 30, 45, and 60 days, respectively. Amplification was then performed simultaneously with lyophilized microsphere detection reagents stored at room temperature, using 1 ng / μL of positive 9948 as a template, for comparison testing. The amplification program was as follows: 95°C for 3 min; 94°C for 10 s, 60°C for 90 s, 28 cycles; 60°C for 12 min; and stored at 4–16°C. The amplification products were analyzed for fluorescence signal detection using a genetic analyzer. Fluorescence signal data were collected, and the DNA genotyping results of the A33 fluorescence detection kit were analyzed using gene analysis software. The accelerated stability test genotyping pattern of the lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent in this example is shown below. Figure 8 As shown.

[0071] Depend on Figure 8 It can be seen that after the lyophilized STR multiplex fluorescent multiplex amplification detection reagent was accelerated at 45℃ for 60 days, three repeated tests were performed on a 1ng / μL positive 9948 sample. The amplification efficiency and amplification uniformity were not significantly different from those before the test. This indicates that the lyophilized microsphere preparation method of the STR multiplex fluorescent multiplex amplification detection reagent of the present invention can improve the stability of the detection reagent and can be used for room temperature transportation and storage.

[0072] In summary, lyophilization of primer-enzyme mixtures provides extremely high stability. During transportation, only room temperature transport and storage are required, avoiding the impact of repeated freeze-thaw cycles on reagent performance and shelf life. The shelf life can be extended to 3 years at room temperature. For immediate use, only dilution with water is needed, which is convenient for users and reduces the skill requirements for operators.

[0073] Comparative Example 1

[0074] Unlike Example 1, dextran was omitted; all other steps were the same as in Example 1. The resulting lyophilized microspheres were used to perform three replicate tests on a 1 ng / μL positive 9948 sample. The amplification patterns of the lyophilized microspheres showed that, without dextran as a protective agent, the amplification of large fragment sites was inhibited, affecting overall amplification efficiency and amplification uniformity. The amplification patterns are shown below. Figure 9 As shown.

[0075] Comparative Example 2

[0076] Unlike Example 1, trehalose was omitted; all other steps were the same as in Example 1. The resulting lyophilized microspheres were used to perform three replicate tests on a 1 ng / μL positive 9948 sample. The amplification patterns of the lyophilized microspheres showed that, without trehalose as a protective agent, the amplification of large fragment sites was inhibited, affecting overall amplification efficiency and amplification uniformity. The amplification patterns are shown below. Figure 9 As shown.

[0077] Comparative Example 3

[0078] Unlike Example 1, bovine serum albumin was omitted; all other procedures were followed as in Example 1. The resulting lyophilized microspheres were used to perform three replicate tests on a 1 ng / μL positive 9948 sample. The amplification patterns of the lyophilized microspheres showed that, without bovine serum albumin as a protective agent, the amplification of large fragment sites was inhibited, affecting overall amplification efficiency and amplification uniformity. The amplification patterns are shown below. Figure 9 As shown.

[0079] Comparative Example 4

[0080] Unlike Example 1, the surfactant was omitted, but all other aspects were the same as in Example 1. The results showed that the freeze-dried microspheres exhibited an overall collapsed state with a rough surface.

[0081] Comparative Example 5

[0082] The difference from Example 1 is that Tween 80 was replaced with polyethylene glycol octylphenyl ether (Triton X-100), Tween 20, and ethyl phenyl polyethylene glycol (NP-40), respectively, while the rest were the same as in Example 1. The results showed that the freeze-dried microspheres all exhibited non-roundness and wrinkled surfaces.

[0083] Comparative Example 6

[0084] Unlike Example 1, the freeze-drying process was adjusted and tested. Pre-freezing: The separator temperature was set to four test gradients: -65℃, -60℃, -50℃, and -45℃, under 1 atmosphere, for 1 hour. Sublimation drying: The separator temperature was set to three gradients: -40℃, -50℃, and -55℃, with a vacuum of 2 Pa, for 10 hours. Then, the separator temperature was adjusted to three gradients: -20℃, -30℃, and -35℃, with a vacuum of 12 Pa, for 4 hours. Desorption drying: The separator temperature was set to 25℃, with a vacuum of 0 Pa, for 7 hours. The rest of the process followed the same procedure as in Example 1. The results showed that changing the temperature at different stages of the process led to the failure of freeze-dried microsphere preparation, resulting in microspheres that were not formed or were not round.

[0085] The embodiments of the present invention described above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A lyophilized microsphere for a STR multiplex fluorescent multiplex amplification detection reagent, characterized in that, Its raw material components include the STR multiplex fluorescent multiplex amplification PCR reaction system and lyophilization protectant; The raw material components of the freeze-drying protectant include: 0.5-6% dextran, 2-20% trehalose, 0.5-3.0 mg / ml bovine serum albumin, 0.1-0.5% gelatin, 0.5-2.0% glycerol, 0.06-1.5% dimethyl sulfoxide, 0.2-3.5% surfactant, 0.04-0.4% defoamer, and 0.02-0.15% preservative. All percentages mentioned above are by mass. The surfactant is Tween 80, the defoamer is isopropanol, and the preservative is sodium azide. The STR multiplex fluorescent complex amplification PCR reaction system includes PCR buffer, STR multiplex fluorescent complex primers, and Taq polymerase.

2. The lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent according to claim 1, characterized in that, PCR buffer includes Tris-HCl, KCl, DMSO, betaine, (NH4)2SO4, dNTPs, and Mg. 2+ .

3. The lyophilized microspheres of the STR multiplex fluorescent multiplex amplification detection reagent according to claim 2, characterized in that, The PCR buffer consists of 85 mM Tris-HCl (pH 8.5), 95 mM KCl, 5 mM DMSO, 1.5 M betaine, 10 mM (NH4)2SO4, 6.5 mM dNTPs, and 8 mM MgCl2.

4. A method for preparing lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent according to any one of claims 1-3, characterized in that, Includes the following steps: ① After preparing the STR multiplex fluorescent multiplex amplification PCR reaction system, add the lyophilization protectant, dissolve it completely, and store it at 2-8℃ until use; ② Pour liquid nitrogen into a sterile medicine cup, and use a pipette to drop 20 μL of the mixture of STR multiplex fluorescence amplification PCR reaction system and lyophilization protectant into the liquid nitrogen, which will solidify into small round balls; ③ Seal the medicine cup with a sterile film and punch holes in the sterile film; ④ Place the medicine cup into a pre-cooled freeze dryer and freeze-dry to prepare PCR reaction reagent lyophilized microspheres.

5. The method for preparing lyophilized microspheres of the STR multiplex fluorescence multiplex amplification detection reagent according to claim 4, characterized in that, The freeze-drying process is as follows: Pre-freezing: Set the partition temperature to -50~-55℃, maintain at 1 atmosphere for 0.8~1h; Sublimation drying: Set the partition temperature to -40~-45℃ and the vacuum degree to 2Pa, maintain for 8~10h, then adjust the partition temperature to -20~-25℃ and the vacuum degree to 12Pa, maintain for 3~4h; Drying: Set the partition temperature to 20~25℃, the vacuum degree to 0Pa, and maintain for 6~7h.

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