Multi-volume and multi-form STR (short tandem repeat) multiplex fluorescence multiplex amplification detection freeze-drying reagent as well as preparation method and application thereof

Abstract

The invention discloses a multi-volume and multi-form STR (short tandem repeat) multiplex fluorescence multiplex amplification detection freeze-drying reagent as well as a preparation method and application thereof. The freeze-drying reagent consists of an STR multiplex fluorescence multiplex amplification system and a freeze-drying protective agent system, the STR multiplex fluorescent multiplex amplification system is composed of a 5X amplification buffer, a 5X primer mixture and Taq DNA polymerase; the 5X amplification buffer is prepared from Tris-HCl, ammonium sulfate, 20% Tween, magnesium chloride, bovine serum albumin, potassium chloride, ethylenediamine tetraacetic acid and dithiothreitol; the 5X primer mixture is a mixture of primers in a DNATperTM 30 kit of the Ministry of Public Security Identification Center; the freeze-drying protective agent system is composed of cane sugar, trehalose, polyethylene glycol 8000 and D-mannitol. The raw materials are freeze-dried in an in-situ freeze-drying or microsphere freeze-drying mode. According to the invention, the technical problems that the stability of the STR multiple fluorescent multiplex amplification detection freeze-drying reagent is poor and the detection accuracy is influenced by a freeze-drying protective agent due to the incoordination of an STR multiple fluorescent multiplex amplification system and a freeze-drying protection system can be solved.

Description

Technical Field

[0001] This invention relates to the field of STR multiplex fluorescence multiplex amplification detection technology. Specifically, it relates to a multi-volume, multi-form lyophilized reagent for STR multiplex fluorescence multiplex amplification detection, its preparation method, and its application. Background Technology

[0002] STRs (short tandem repeats) are highly polymorphic genetic markers in the human genome, possessing significant individual identification characteristics. DNA from different tissues within the same individual shows complete consistency in STR typing, while the core repeat count at the same STR locus varies significantly between individuals, with a uniqueness probability on the order of one in a trillion. Based on this characteristic, forensic medicine can accurately identify individuals and determine kinship by examining the STR patterns of biological samples left at crime scenes.

[0003] STR multiplex fluorescent amplification refers to the addition of two or more pairs of fluorescent primers to the same PCR amplification system to simultaneously amplify two or more STR loci. After amplification, the samples are subjected to capillary electrophoresis to obtain STR patterns, achieving the purpose of individual identification.

[0004] Freeze-drying technology uses the freezing effect at low temperatures to freeze the water in a sample into ice crystals. Then, by applying appropriate heat under vacuum conditions, the ice crystals are directly transformed from a solid state to a gaseous state, bypassing the liquid state, thereby achieving the removal of water.

[0005] The combination of lyophilization technology and STR multiplex fluorescent multiplex amplification assay reagents can remove moisture, maintain the stability of biological samples, and achieve stable preservation at room temperature. Although lyophilization technology removes moisture, the low temperature can affect the enzyme activity in the STR multiplex fluorescent multiplex amplification assay reagents. Therefore, a lyophilization protection system is needed to maintain enzyme activity and ensure that it retains good activity after lyophilization.

[0006] Therefore, an ideal lyophilized reagent for STR multiplex fluorescence multiplex amplification (MLFA) detection requires harmony between the MLFA system and the lyophilization protectant system, as their components can influence each other. If the type or ratio of protectant in the lyophilization system is inappropriate, it will not only affect the MLFA system, leading to problems with genotyping, but also result in incomplete lyophilization and insufficient stability.

[0007] Patent document CN 117286228 A discloses a lyophilized microsphere for STR multiplex fluorescence amplification detection reagent and its preparation method. The lyophilized microspheres prepared by this patent exhibit no difference in amplification efficiency, amplification uniformity, or amplification sensitivity compared to the un-lyophilized microspheres, enabling room temperature transport and improving the detection rate of extremely small samples with full-volume loading. However, this lyophilized microsphere has the following drawbacks: the preparation process is complex, the freezing conditions are more stringent, and the lyophilized microspheres often escape from the octet due to electrostatic adsorption during dispensing into the octet array.

[0008] Patent document CN 120210379 A discloses an anti-contamination lyophilized eight-color Mini-STR multiplex amplification kit, which involves in-situ lyophilization using trehalose, mannitol, glycine, sucrose, and / or polyethylene glycol. This lyophilized kit significantly improves the detection rate of degraded sample DNA and exhibits higher stability, making it suitable for a wide range of applications. However, the glycine used in the lyophilization protectant system of this patent inhibits the STR multiplex fluorescence multiplex amplification system, leading to a decrease in amplification efficiency. Summary of the Invention

[0009] Therefore, the technical problem to be solved by the present invention is to provide a multi-volume, multi-form lyophilized STR multiplex fluorescence multiplex amplification detection reagent, its preparation method and application, so as to solve the technical problems such as poor stability of STR multiplex fluorescence multiplex amplification detection lyophilized reagent and the influence of lyophilization protectant on detection accuracy caused by the incompatibility between the STR multiplex fluorescence multiplex amplification system and the lyophilization protection system.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0011] A multi-volume, multi-form STR multiplex fluorescent multiplex amplification detection lyophilized reagent consists of an STR multiplex fluorescent multiplex amplification system and a lyophilization protectant system. The STR multiplex fluorescent multiplex amplification system comprises a 5X amplification buffer, a 5X primer mixture, and Taq DNA polymerase. The 5X amplification buffer consists of Tris-HCl, ammonium sulfate, Tween-20, dNTPs (a mixture of nitrogenous bases), magnesium chloride, bovine serum albumin, potassium chloride, ethylenediaminetetraacetic acid, and dithiothreitol. The 5X primer mixture is a mixture of primers from the DNATyper™ 30 kit from the Ministry of Public Security Identification Center. The lyophilization protectant system consists of sucrose, trehalose, polyethylene glycol 8000 (polyethylene glycol with a relative molecular mass of 8000), and D-mannitol. Sucrose serves two purposes: firstly, as a component of the lyophilization protectant to reduce damage to polymerase activity during the lyophilization process; and secondly, as a component of the amplification buffer.

[0012] In the 5X amplification buffer, Tris-HCl and ammonium sulfate are mainly used to adjust the pH of the 5X amplification buffer; Tween-20, bovine serum albumin, and dithiothreitol synergistically protect Taq DNA polymerase; dNTPs provide nitrogenous bases for the PCR reaction; magnesium chloride activates Taq DNA polymerase during amplification; ethylenediaminetetraacetic acid inhibits nucleases and prevents DNA template degradation; by selecting appropriate components and controlling the ratio of each component, the amplification efficiency of the STR multiplex fluorescent complex amplification system can be significantly improved.

[0013] In the freeze-drying protectant system, sucrose and trehalose act as excipients, which can protect the stability of Taq DNA polymerase components and facilitate the formation of a loose and porous structure during freeze-drying; polyethylene glycol 8000 and D-mannitol act as fillers, ensuring a stable physical structure after freeze-drying; during the freeze-drying process, the excipients and fillers work together to maintain the activity of Taq DNA polymerase, and ensure a stable and good freeze-dried form while avoiding a decrease in enzyme activity.

[0014] The STR multiplex fluorescence multiplex amplification detection system of the present invention can be freeze-dried in situ or by microsphere freeze-drying, neither of which will affect the amplification efficiency of the amplification system.

[0015] The above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent has a mass ratio of 1:(4-8) between the STR multiplex fluorescence multiplex amplification system and the lyophilization protectant system. Excessive use of the lyophilization protectant system will inhibit the amplification efficiency of the STR multiplex fluorescence multiplex amplification system; insufficient use will hinder lyophilization and fail to achieve the desired enzyme activity protection. Controlling the amounts of both within the above-mentioned range achieves ideal enzyme activity protection without affecting the amplification efficiency of the amplification system, while also facilitating lyophilization. In the STR multiplex fluorescence multiplex amplification system: 5X amplification buffer, 5X primer mixture, and Taq... The mass ratio of DNA polymerases is (8000-10000):(0.5-2):(5-18). Within this range, it is beneficial to improve amplification efficiency and reduce non-specific amplification. In the 5X amplification buffer, the molar ratio of Tris-HCl, ammonium sulfate, dNTPs, magnesium chloride, potassium chloride, ethylenediaminetetraacetic acid, and dithiothreitol is (300-400):(80-120):(3-6):(7-13):(10-20):(0.03- 0.08):(0.8-1.3), the amount of Tris-HCl is calculated based on the amount of tris(hydroxymethyl)aminomethane, and the mass ratio of ammonium sulfate, Tween-20, and bovine serum albumin is (1.0-1.5):(0.3-0.6):(0.03-0.06); in the lyophilization protectant system: the mass ratio of sucrose, trehalose, polyethylene glycol 8000, and D-mannitol is (25-35):(20-30):(3-8):(5-10).

[0016] A method for preparing a multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent includes the following steps:

[0017] Step A: Prepare the STR multiplex fluorescence multiplex amplification mixture and the lyophilization protectant mixture separately;

[0018] Step B: Shake and mix the STR multiplex fluorescence multiplex amplification mixture and the lyophilization protectant mixture until homogeneous to obtain the STR multiplex fluorescence multiplex amplification detection reagent solution;

[0019] Step C: According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, the STR multiplex fluorescence multiplex amplification detection lyophilized reagent solution is freeze-dried under the set freeze-drying program using in-situ freeze-drying or microsphere freeze-drying methods; after freeze-drying, the above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent is obtained.

[0020] The preparation method of the above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, in step A, the STR multiplex fluorescence multiplex amplification mixture consists of 5X amplification buffer, 5X primer mixture, and Taq DNA polymerase dispersion; the 5X amplification buffer consists of Tris-HCl, ammonium sulfate, Tween-20, dNTPs, magnesium chloride, bovine serum albumin, potassium chloride, ethylenediaminetetraacetic acid, dithiothreitol, and water; the 5X primer mixture is the primer mixture from the DNATyper™ 30 kit of the Ministry of Public Security Identification Center; the volume ratio of 5X amplification buffer, 5X primer mixture, and Taq DNA polymerase dispersion is (1.4-2.6):(1.5-2.5):(0.05-0.15); the lyophilization protectant mixture consists of sucrose, trehalose, polyethylene glycol 8000, D-mannitol, and water; in step B, the volume ratio of the STR multiplex fluorescence multiplex amplification system to the lyophilization protectant system is 1:(1-2).

[0021] In the above-mentioned method for preparing lyophilized reagents for multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection, in step A, the volume ratio of 5X amplification buffer, 5X primer mixture and Taq DNA polymerase dispersion is 1.9:2.0:0.1; in step B, the volume ratio of STR multiplex fluorescence multiplex amplification system and lyophilization protectant system is 1:1.5.

[0022] The above-mentioned method for preparing multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, in step B, the STR multiplex fluorescence multiplex amplification detection reagent solution contains: Tris-HCl with a molar concentration of 71.25 mmol / L (the molar concentration of Tris-HCl is calculated based on the molar concentration of tris(hydroxymethyl)aminomethane); ammonium sulfate with a molar concentration of 19 mmol / L; Tween-20 with a mass fraction of 0.095 wt%; dNTPs with a molar concentration of 0.95 mmol / L; and magnesium chloride with a molar concentration of... The concentrations of the following components were as follows: 1.9 mmol / L for potassium chloride, 0.095 mg / mL for bovine serum albumin, 2.85 mmol / L for potassium chloride, 0.0095 mmol / L for ethylenediaminetetraacetic acid, and 0.19 mmol / L for dithiothreitol; 0.18 g / mL for sucrose, 0.15 g / mL for trehalose, 0.03 g / mL for polyethylene glycol 8000, 0.048 g / mL for D-mannitol, and 0.1 U / μL for Taq DNA polymerase; the total concentration of the 5X primer mixture ranged from 0.046 to 0.908 μmol / L.

[0023] The preparation method of the above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, the lyophilization program set in step C is as follows:

[0024] (1) Pre-freezing: Set the shelf temperature of the freeze dryer to -45℃ and maintain it for 6 hours under one standard atmosphere;

[0025] (2) First drying: The temperature is increased from -45℃ to -28℃ at a heating rate of 0.02℃ / min and kept under vacuum for 8 hours;

[0026] (3) Secondary drying: Under vacuum conditions, the temperature is raised from -28℃ to 35℃ at a heating rate of 0.16℃ / min and held for 4 hours. In the primary and secondary drying processes, if the heating rate is too low, it will affect enzyme activity. If the heating rate is too fast, it will affect freeze-drying properties. By controlling the heating rate during the drying process, the influence of the drying process on enzyme activity can be effectively reduced and good freeze-drying properties can be ensured.

[0027] The preparation method of the above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, specifically the in-situ lyophilization method, includes the following steps:

[0028] Step (1-1): After thoroughly shaking and mixing the STR multiplex fluorescence amplification system and the lyophilization protectant system, centrifuge to obtain the mixture;

[0029] Steps (1-2): According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, drop the mixture into each of the eight-tube PCR tubes and centrifuge.

[0030] Steps (1-3): Place the centrifuged eight-unit stack in a metal cryogenic rack that has been pre-frozen at -45°C; then transfer it to a freeze dryer and freeze dry it according to the set freeze-drying program.

[0031] Steps (1-4): After freeze-drying, remove the eight-pack and cover it with the lid under an environment with an air humidity of less than or equal to 20%. After vacuum packaging, store it in an aluminum foil bag.

[0032] The preparation method of the above-mentioned multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, specifically the microsphere lyophilization method, includes the following steps:

[0033] Step (2-1): After thoroughly shaking and mixing the STR multiplex fluorescence amplification system and the lyophilization protectant system, centrifuge to obtain the mixture;

[0034] Step (2-2): According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, drop the mixture into a liquid nitrogen pot containing liquid nitrogen;

[0035] Steps (2-3): After the mixture is frozen into microspheres, pour it into a tray that has been pre-frozen at -45°C; then transfer it to a freeze dryer and freeze dry it according to the set freeze-drying program.

[0036] Steps (2-4): After freeze-drying, the obtained freeze-dried microspheres are aliquoted into eight-tube PCR tubes, and the tubes are capped with the eight-tube caps under an environment with an air humidity of less than or equal to 20%, vacuum-packed, and then stored in aluminum foil bags.

[0037] This invention discloses the application of a multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent. The multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent prepared using the aforementioned method is used for the detection of trace samples with full-volume loading. In this invention, "trace sample" refers to a low-copy DNA (LCN) sample, i.e., a sample with a genomic content of less than 100 pg (low-copy DNA).

[0038] The technical solution of the present invention achieves the following beneficial technical effects:

[0039] 1. This invention discloses a multi-volume, multi-form lyophilized STR multiplex fluorescence multiplex amplification detection reagent, comprising two parts: an STR multiplex fluorescence multiplex amplification system and a lyophilization protectant system. By selecting a suitable lyophilization protectant system, a high-performance lyophilized STR multiplex fluorescence multiplex amplification system is formed. This invention utilizes a multi-target multiplex amplification design to improve detection efficiency while retaining the advantages of lyophilized reagents. Through innovative lyophilization processes, it achieves room-temperature storage and transportation, avoiding repeated freeze-thaw cycles. Use only requires reconstitution with water, shortening operation time and reducing contamination risks and labor costs. Furthermore, the lyophilized reagent allows for template expansion, making it suitable for trace or degraded samples, thus improving detection rates. In emergency and disaster response, its portability makes it suitable for use in extreme environments such as disaster areas and battlefields.

[0040] 2. Compared with existing lyophilized reagent products (such as SureID® S6 Human DNA Identification Kit, NHID® 25A Case Kit (lyophilized version), Microreader™ 36A-S ID System, etc.), the lyophilized STR multiplex fluorescent multiplex amplification detection reagent of the present invention can achieve in-situ lyophilization of the STR multiplex fluorescent multiplex amplification system, and can also achieve lyophilization of microspheres that are not easily broken or stuck to containers during dispensing. This greatly reduces the escape phenomenon of lyophilized microspheres detaching from the octet due to electrostatic adsorption during dispensing, which is convenient for production.

[0041] 3. The lyophilization protectant system developed in this invention is suitable for multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilization reagents. It is morphologically stable after lyophilization and can be stored at room temperature. This invention allows for full-volume sample loading, offering advantages in detecting trace samples. When researchers do not wish to perform purification steps on trace samples, they can directly add the extracted trace DNA template in its entirety to the lyophilization reagent for amplification. Attached Figure Description

[0042] Figure 1 The physical image of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent prepared in the embodiments of the present invention (the two octets on the left are in situ lyophilized, and the two octets on the right are microsphere lyophilized).

[0043] Figure 2 The genotyping patterns of blood cards amplified by the STR multiplex fluorescence multiplex amplification detection lyophilized reagent prepared in this embodiment of the invention (the first image is of the lyophilized reagent, and the second image is of the liquid reagent).

[0044] Figure 3 In this embodiment of the invention, the liquid reagent amplification and... Figure 2 The typing pattern of the same blood type;

[0045] Figure 4 The genotyping pattern of a 10 pg / μL 9947A positive sample amplified by the STR multiplex fluorescence multiplex amplification lyophilized reagent prepared in this embodiment of the invention (the first image is of the lyophilized reagent, and the second image is of the liquid reagent).

[0046] Figure 5 In this embodiment of the invention, the genotyping spectrum of a 10 pg / μL 9947A positive sample amplified with the liquid reagent before lyophilization of the STR multiplex fluorescence multiplex amplification detection reagent is shown. Detailed Implementation

[0047] 1. Composition of raw materials for lyophilized reagents for STR multiplex fluorescence multiplex amplification detection

[0048] This embodiment of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent consists of an STR multiplex fluorescence multiplex amplification system and a lyophilization protectant system. The STR multiplex fluorescence multiplex amplification system consists of 5X amplification buffer, 5X primer mixture, and Taq DNA polymerase dispersion. The 5X amplification buffer consists of Tris-HCl solution, ammonium sulfate (NH4)2SO4, Tween-20, a mixture of nitrogenous bases (dNTPs), magnesium chloride (MgCl2), bovine serum albumin (BSA), potassium chloride (KCl), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and double-distilled water. The Taq DNA polymerase was purchased from Suzhou Xinhai Biotechnology Co., Ltd. The lyophilization protectant system consists of trehalose, sucrose, polyethylene glycol 8000 (PEG8000), D-mannitol, and double-distilled water.

[0049] The raw material information used in this embodiment is shown in Table 1.

[0050]

[0051] The 5X amplification buffer contained: Tris-HCl at a concentration of 375 mmol / L (the concentration of Tris-HCl is calculated based on the concentration of tris(hydroxymethyl)aminomethane), ammonium sulfate at a concentration of 100 mmol / L, Tween-20 at a mass fraction of 0.5 wt%, dNTPs at a concentration of 5 mmol / L, magnesium chloride at a concentration of 10 mmol / L, bovine serum albumin at a mass concentration of 0.5 mg / mL, potassium chloride at a concentration of 15 mmol / L, ethylenediaminetetraacetic acid at a concentration of 0.05 mmol / L, and dithiothreitol at a concentration of 1 mmol / L; the 5X primer mixture was prepared by the DNATyper of the Ministry of Public Security Identification Center. TM The kit contains a mixture of primers for 30 gene loci; the concentration of the 5X primer mixture is 0.23-4.54 μM; the concentration of Taq DNA polymerase in the Taq DNA polymerase dispersion is 10 U / μL.

[0052] In the freeze-drying protectant system: sucrose has a mass fraction of 30 wt%, trehalose has a mass fraction of 25 wt%, polyethylene glycol 8000 has a mass fraction of 5 wt%, D-mannitol has a mass fraction of 8 wt%, and the balance is water.

[0053] In the composition of the lyophilized reagent, the ratio of the STR multiplex fluorescence multiplex amplification system to the lyophilization protectant system is 1:1.5. The amounts of the STR multiplex fluorescence multiplex amplification system and the lyophilization protectant system can be adjusted according to different systems. For example, in a 10 μL lyophilized reagent, 4 μL of the STR multiplex fluorescence multiplex amplification system (1.9 μL of 5X amplification buffer, 2 μL of 5X primer mixture, and 0.1 μL of Taq DNA polymerase dispersion) and 6 μL of the lyophilization protectant system are used to prepare the STR multiplex fluorescence multiplex amplification detection reagent solution (the final concentrations of each raw material are shown in Table 2), and then lyophilized. Testing showed that the freezing point of this STR multiplex fluorescence multiplex amplification detection reagent solution is approximately -25℃, and lyophilization ensures that the Tris-HCl content in the system remains unchanged, avoiding HCl loss due to poor compatibility of system components during lyophilization. This ensures that the pH of the multiplex fluorescence multiplex amplification detection reagent after reconstitution is within the predetermined range, thereby guaranteeing its amplification efficiency.

[0054] Table 2 Final concentrations of each component in the STR multiplex fluorescence multiplex amplification detection reagent solution

[0055]

[0056] 2. Preparation of lyophilized reagents for STR multiplex fluorescence multiplex amplification detection

[0057] Take the component raw materials as shown in Table 2, dissolve them thoroughly, and then shake to mix them well to obtain the STR multiplex fluorescence multiplex amplification mixture and the lyophilization protectant mixture.

[0058] A. The procedure for in-situ lyophilization of a 10 μL volume of an octet is as follows: Take 32 μL of the STR multiplex fluorescence amplification system and 48 μL of the lyophilization protectant system into an EP tube, shake and centrifuge thoroughly. Then, use a pipette to take 10 μL of the mixture dropwise into each tube of the octet. After centrifugation, place the octet in a metal cryogenic rack pre-frozen at -45°C and put it into a freeze dryer for lyophilization. After lyophilization, remove the octet, cap it, vacuum pack it, and store it in an aluminum foil bag under conditions where the air humidity is below 20% (see [link to documentation]). Figure 1 ).

[0059] B. The procedure for lyophilizing a 10 μL volume of an eight-pack of microspheres is as follows: Take 32 μL of the STR multiplex fluorescence amplification system and 48 μL of the lyophilization protectant system into an EP tube, shake and centrifuge thoroughly. Then, use a pipette to take 10 μL of the mixture dropwise into a liquid nitrogen bath containing liquid nitrogen. After stable microspheres have formed, pour the liquid nitrogen into a tray pre-frozen at -45°C and place it in a freeze dryer for lyophilization. After lyophilization, use tweezers to remove the microspheres one by one, place them into an eight-pack, and cover the eight-pack with the cap under conditions of air humidity below 20%. Vacuum pack and store in an aluminum foil bag (see [link to documentation]). Figure 1 ).

[0060] The freeze-drying process described above is as follows: ① Pre-freezing: The shelf temperature is set to -45℃ and maintained at one standard atmosphere for 6 hours; ② First drying: The temperature is slowly raised from -45℃ to -28℃ and maintained under vacuum for 8 hours; ③ Second drying: Under vacuum, the temperature is raised from -28℃ to 35℃ and maintained for 4 hours.

[0061] Instructions for using the lyophilized reagent: Remove the lyophilized reagent from the aluminum foil bag. Determine the number of eight-tube strips to use based on the number of samples. Redissolve the required number of tubes in water, then add the DNA template or directly redissolve the extracted DNA solution. After vortexing and centrifuging, place the tubes in an amplification instrument for amplification.

[0062] 3. Performance Testing

[0063] (1) Compare the amplification effect of STR multiplex fluorescent multiplex amplification detection reagent before and after lyophilization (taking 10μL in situ lyophilization system as an example).

[0064] Tear open the aluminum foil bag, remove the eight-pack, open the cap, add 9 μL of nucleus-free water to reconstitute, then add 1 μL of 1 ng / μL 9947A positive standard; use the same composition of undried liquid reagent as the control group, also adding 1 μL of 1 ng / μL 9947A positive standard. The amplification program is as follows: 95℃ for 8 min; 95℃ for 10 s, 60℃ for 2 min, 28 cycles; 60℃ for 30 min; store at 4℃. Fluorescence signal detection was performed using a genetic analyzer, data were collected, and analyzed using gene analysis software to obtain the STR genotype map (…). Figure 2 ).

[0065] Depend on Figure 2 It can be seen that, compared with liquid reagents, the amplification efficiency and amplification uniformity of the lyophilized STR multiplex fluorescent composite amplification detection reagent using the present invention are reduced, but the overall amplification efficiency is still acceptable.

[0066] (2) The test of the lyophilized STR multiplex fluorescent multiplex amplification detection reagent has the advantage of full-volume sample addition (taking 10μL in situ lyophilized system as an example).

[0067] Tear open the aluminum foil bag, remove the eight-pack, open the cap, and add 10 μL of 10 pg / μL 9947A positive standard for reconstitution. Use the unprotected liquid reagent as the control group. The maximum volume of the liquid reagent is 6 μL, so add 6 μL of 10 pg / μL 9947A positive standard to the liquid reagent. The amplification program is as follows: 95℃ for 8 min; 95℃ for 10 s, 60℃ for 2 min, 28 cycles; 60℃ for 30 min; store at 4℃. Detect fluorescence signals using a genetic analyzer, collect data, and analyze with gene analysis software to obtain the STR genotype map. Figure 3 ).

[0068] Depend on Figure 3 It can be seen that the lyophilized STR multiplex fluorescent multiplex amplification detection reagent has a better detection efficiency for trace samples under full-volume sample addition conditions.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A multi-volume, multi-form STR multiplex fluorescent multiplex amplification detection lyophilized reagent, characterized in that, The system consists of an STR multiplex fluorescent multiplex amplification system and a lyophilization protectant system. The STR multiplex fluorescent multiplex amplification system comprises a 5X amplification buffer, a 5X primer mixture, and Taq DNA polymerase. The 5X amplification buffer consists of Tris-HCl, ammonium sulfate, Tween-20, dNTPs, magnesium chloride, bovine serum albumin, potassium chloride, ethylenediaminetetraacetic acid, and dithiothreitol. The 5X primer mixture is from the DNATyper of the Ministry of Public Security Identification Center. TM 30. The primer mixture in the kit; the lyophilization protectant system consists of sucrose, trehalose, polyethylene glycol 8000 and D-mannitol.

2. The multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 1, characterized in that, The mass ratio of the STR multiplex fluorescent multiplex amplification system to the lyophilization protectant system was 1:(4-8); in the STR multiplex fluorescent multiplex amplification system: 5X amplification buffer, 5X primer mixture, and Taq... The mass ratio of DNA polymerase is (8000-10000):(0.5-2):(5-18); in the 5X amplification buffer: the molar ratio of Tris-HCl, ammonium sulfate, dNTPs, magnesium chloride, potassium chloride, ethylenediaminetetraacetic acid and dithiothreitol is (300-400):(80-120):(3-6):(7-13):(10-20):(0.03-0.08):(0.8-1.3), and the mass ratio of ammonium sulfate, Tween-20 and bovine serum albumin is (1.0-1.5):(0.3-0.6):(0.03-0.06); in the lyophilization protectant system: the mass ratio of sucrose, trehalose, polyethylene glycol 8000 and D-mannitol is (25-35):(20-30):(3-8):(5-10).

3. A method for preparing a multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, characterized in that, Includes the following steps: Step A: Prepare the STR multiplex fluorescence multiplex amplification mixture and the lyophilization protectant mixture separately; Step B: Shake and mix the STR multiplex fluorescence multiplex amplification mixture and the lyophilization protectant mixture until homogeneous to obtain the STR multiplex fluorescence multiplex amplification detection reagent solution; Step C: According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, the STR multiplex fluorescence multiplex amplification detection lyophilized reagent solution is freeze-dried under the set freeze-drying program by in-situ freeze-drying or microsphere freeze-drying; after freeze-drying, the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent as described in claim 1 or 2 is obtained.

4. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 3, characterized in that, In step A, the STR multiplex fluorescent complexation mixture consists of 5X amplification buffer, 5X primer mixture, and Taq DNA polymerase dispersion; the 5X amplification buffer consists of Tris-HCl, ammonium sulfate, Tween-20, dNTPs, magnesium chloride, bovine serum albumin, potassium chloride, ethylenediaminetetraacetic acid, dithiothreitol, and water; the 5X primer mixture is from the DNATyper of the Ministry of Public Security Identification Center. TM The volume ratio of the primer mixture in kit 30; the volume ratio of 5X amplification buffer, 5X primer mixture and Taq DNA polymerase dispersion is (1.4-2.6):(1.5-2.5):(0.05-0.15); the lyophilization protectant mixture consists of sucrose, trehalose, polyethylene glycol 8000, D-mannitol and water; in step B, the volume ratio of the STR multiplex fluorescent complex amplification system and the lyophilization protectant system is 1:(1-2).

5. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 4, characterized in that, In step A, the volume ratio of 5X amplification buffer, 5X primer mixture, and Taq DNA polymerase dispersion is 1.9:2.0:0.1; in step B, the volume ratio of STR multiplex fluorescent complex amplification system and lyophilization protectant system is 1:1.

5.

6. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 5, characterized in that, The STR multiplex fluorescent multiplex amplification detection reagent solution prepared in step B contains: Tris-HCl at a concentration of 71.25 mmol / L; ammonium sulfate at a concentration of 19 mmol / L; Tween-20 at a mass fraction of 0.095 wt%; dNTPs at a concentration of 0.95 mmol / L; magnesium chloride at a concentration of 1.9 mmol / L; bovine serum albumin at a mass concentration of 0.095 mg / mL; potassium chloride at a concentration of 2.85 mmol / L; ethylenediaminetetraacetic acid at a concentration of 0.0095 mmol / L; dithiothreitol at a concentration of 0.19 mmol / L; sucrose at a mass concentration of 0.18 g / mL; trehalose at a mass concentration of 0.15 g / mL; polyethylene glycol 8000 at a mass concentration of 0.03 g / mL; D-mannitol at a mass concentration of 0.048 g / mL; and Taq... The DNA polymerase activity concentration was 0.1 U / μL; the total molar concentration of the 5X primer mixture was 0.046-0.908 μmol / L.

7. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 3, characterized in that, The freeze-drying program set in step C is as follows: (1) Pre-freezing: Set the shelf temperature of the freeze dryer to -45℃ and maintain it for 6 hours under one standard atmosphere; (2) First drying: The temperature is raised from -45℃ to -28℃ at a heating rate of 0.02℃ / min and kept under vacuum for 8 hours; (3) Secondary drying: Under vacuum conditions, the temperature is raised from -28℃ to 35℃ at a heating rate of 0.16℃ / min and then held for 4 hours.

8. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 7, characterized in that, The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent as described in claim 1 or 2 by in-situ lyophilization includes the following steps: Step (1-1): After thoroughly shaking and mixing the STR multiplex fluorescence amplification system and the lyophilization protectant system, centrifuge to obtain the mixture; Steps (1-2): According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, drop the mixture into each of the eight-tube PCR tubes and centrifuge. Steps (1-3): Place the centrifuged eight-unit stack in a metal cryogenic rack that has been pre-frozen at -45°C; then transfer it to a freeze dryer and freeze dry it according to the set freeze-drying program. Steps (1-4): After freeze-drying, remove the eight-pack and cover it with the lid under an environment with an air humidity of less than or equal to 20%. After vacuum packaging, store it in an aluminum foil bag.

9. The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent according to claim 7, characterized in that, The method for preparing the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent as described in claim 1 or 2 by microsphere lyophilization includes the following steps: Step (2-1): After thoroughly shaking and mixing the STR multiplex fluorescence amplification system and the lyophilization protectant system, centrifuge to obtain the mixture; Step (2-2): According to the specifications of the STR multiplex fluorescence multiplex amplification detection lyophilized reagent, drop the mixture into a liquid nitrogen pot containing liquid nitrogen; Steps (2-3): After the mixture is frozen into microspheres, pour it into a tray that has been pre-frozen at -45°C; then transfer it to a freeze dryer and freeze dry it according to the set freeze-drying program. Steps (2-4): After freeze-drying, the obtained freeze-dried microspheres are aliquoted into eight-tube PCR tubes, and the tubes are capped with the eight-tube caps under an environment with an air humidity of less than or equal to 20%, vacuum-packed, and then stored in aluminum foil bags.

10. An application of a multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent, characterized in that, The multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent prepared by the preparation method of the multi-volume, multi-form STR multiplex fluorescence multiplex amplification detection lyophilized reagent as described in claim 3 will be used for the detection of trace samples with full-volume loading.

Images