PCR reagent kit and preparation method thereof and PCR reaction kit

Abstract

The present application relates to the technical field of molecular diagnosis, and particularly relates to a PCR primer probe reagent kit, a preparation method thereof and a PCR reaction kit, which comprises a wrapping layer, the wrapping layer is coated with primer probe reagents for detecting targets, and the wrapping layer can reduce the influence of light on the internal primer probe reagents. The present application introduces a wrapping layer with light-shielding properties, and separately configures the light-sensitive characteristic reagents in the PCR reagent kit to form special protection for the reagents, thereby improving the stability of the amplification reagent kit and the accuracy of subsequent detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnostics, in particular to a PCR reagent kit and a preparation method thereof and a PCR reagent kit. BACKGROUND

[0002] Nucleic acid quantification or qualitative analysis is widely used in biological research and clinical analysis, and is an important detection technology in the field of in vitro diagnostics. Some applications using this technology include measurement of gene expression, monitoring of biological responses to exogenous stimuli, quantification of genes at the genomic level, and pathogen detection. The most widely used is the polymerase chain reaction (PCR), which uses a fragment of the genetic material of an organism as a detection target. To detect a specific target analyte, real-time fluorescent PCR uses a signal generating device to generate a fluorescent signal that can be quantitatively displayed in proportion to the copy number of the target molecule to indicate the occurrence of target sequence amplification. By moving the fluorescence group and the quenching group away from each other during amplification, the fluorescence signal cannot be quenched and can be captured by the detector to be displayed. With the accumulation of amplification in each amplification cycle, a proportional fluorescence signal is generated in the detector to produce a progressively enhanced detection signal. By plotting the amplification cycles with the controller of the instrument, an amplification curve can be obtained.

[0003] To achieve amplification detection of target nucleic acid sequences, various types of amplification kits have been developed by different manufacturers. The amplification kit contains all the reagents needed to complete the PCR amplification reaction. To ensure the long-term effectiveness of the kit, multiple reagent bottles containing different reagents are usually configured in the amplification kit. After combining the multiple reagent bottles, the detection reagent kit for detecting target sequences is formed. In actual use, the various types of reagents in the different reagent bottles need to be transferred and mixed. After mixing, the purified nucleic acid extract obtained by extraction is added, and the amplification occurs in the amplification equipment under constant temperature or cyclic temperature rise and fall. This operation is relatively complex, and the total amount of the amplification reaction system is usually small, and the required reagent volume is also smaller. The use of existing kits requires the use of high-precision pipettors and experienced pipetting operators. Too much or too little reagent will affect the accuracy of the detection results. Too little reagent cannot obtain an accurate standard amplification curve, and too much reagent may cause insufficient sample injection or increase the reaction system, resulting in insufficient cyclic amplification heat.

[0004] In order to solve the defects of the multi-reagent bottle combination kit, many manufacturers have gradually explored a one-tube amplification kit that can be stored in the same amplification tube. In use, only the extraction and purification liquid needs to be added to complete the amplification. The invention patent CN 113373202 A discloses a reagent kit of constant temperature amplification type. Different types of reagents are configured in the reagent kit. In order to avoid the problem of invalidation caused by the pre-mixing of different reagents, a paraffin separation layer is arranged between the different reagents. During the amplification process, the paraffin melts due to the high temperature of constant temperature amplification or cyclic temperature amplification, and then all the reagents are mixed.

[0005] The invention patent CN 114703259 A optimizes the separation paraffin layer and designs a pre-packaged amplification reagent kit more suitable for a cyclic amplification system. By adding agarose in a specific proportion, the stability of the separation layer is higher, and it is suitable for longer-term reliable storage. Of course, in addition to the design idea of separating different types of reagents, the introduction of a new freeze-drying process also provides a new solution for the configuration in one tube.

[0006] The invention patent CN112760415A discloses a freeze-dried amplification reagent kit designed for influenza A and B viruses. In this scheme, all types of reagents required for amplification reaction are mixed in advance, and then the reagent kit is freeze-dried and packaged to obtain a freeze-dried pre-packaged reagent kit. Although this design can ensure that the stability of the mixed reagents is basically not affected, the subsequent operation needs a more complex mixing operation to ensure effective and pollution-free mixing operation.

[0007] From the current solutions, liquid reagents do not need efficient mixing to ensure the stability of the reaction system, but they do not distinguish between reagent types. Special reagents such as key components (such as fluorescent substances, enzymes, etc.) in PCR reagent kits are susceptible to light and have no special protection measures. In the freeze-drying scheme, mixing operation is required to ensure the uniformity of the reaction system, and the sealing state of the reagent kit directly affects the stability of the reagent kit. SUMMARY

[0008] The purpose of the present application is to provide a PCR primer probe reagent package, a preparation method thereof and a PCR reaction kit, which solves the technical problem of the prior art that specific reagent components need to be stored in a specific way, and multiple different reagents can be stored in the same reagent tube to simplify the complexity of amplification operation and reduce the dependence on professionals.

[0009] The present application discloses a PCR primer probe reagent package, which comprises a wrapping layer, the wrapping layer is coated with primer probe reagents for detecting target, and the wrapping layer can reduce the influence of light on the internal primer probe reagents.

[0010] Further, the wrapping layer comprises at least one barrier layer, and the barrier layer comprises hydroxypropyl methyl cellulose and polyvinyl alcohol.

[0011] Further, the barrier layer comprises 60-80 parts of hydroxypropyl methyl cellulose and 10-20 parts of polyvinyl alcohol by weight.

[0012] Further, the barrier layer further comprises 5-10 parts of a barrier agent by weight, and the barrier agent is at least one of montmorillonite, carbon nanotube, graphene oxide or nanosilica.

[0013] Further, the barrier agent is carbon nanotube, and the diameter of the carbon nanotube is 10-20 nm.

[0014] The use of the barrier agent can improve the oxygen barrier property of the barrier layer by 60%, and at the same time, the barrier layer has stronger antistatic property (surface resistance ≤ 109 Ω), avoiding reagent adsorption loss.

[0015] Further, the barrier layer further comprises 3-7 parts of triethyl citrate or glyceryl triacetate and 1-3 parts of an antibacterial agent by weight.

[0016] The addition of an appropriate amount of triethyl citrate or glyceryl triacetate (an environmentally friendly plasticizer) can improve the ductility of the film, and at the same time, the film strength is not affected due to the excessive amount of the plasticizer. Here, the elongation at break of the barrier layer is ≥ 300% by adding the plasticizer.

[0017] Further, the antibacterial agent is chitosan quaternary ammonium salt or lysozyme.

[0018] The addition of the antibacterial agent can inhibit microbial contamination, and is suitable for long-term storage scenarios. Even if the film is in contact with a liquid, contamination bacteria are not easy to breed. It is determined that the antibacterial agent is uniformly dispersed in the film according to the ratio of the present application, so that the colony inhibition rate of the film is ≥ 99% (GB / T20944.3-2008 standard).

[0019] Further, the wrapping layer has a three-layer structure, the barrier layer is located in the middle, the outer layer is a packaging layer, and the inner layer is a core layer, and the core layer is made of polypropylene or polycaprolactone.

[0020] Further, the wrapping layer has an oxygen transmission rate of ≤ 0.5 cc / (m 2 ·day), and the wrapping layer is in a stable state at 2-65°C.

[0021] In the second aspect of the present application, a preparation method of the PCR primer probe reagent package of the first aspect is provided. The barrier layer of the wrapping layer is prepared, and the thickness of the barrier layer is 30-80 μm. The primer probe reagent for detecting the target is wrapped, and the barrier layer is closed to form a closed internal space to accommodate the primer probe reagent for detecting the target.

[0022] Further, the barrier layer is closed by ultrasonic welding.

[0023] In the third aspect of the present application, a PCR reaction kit is provided, comprising a reaction tube, the reaction tube containing a PCR premix solution, and the PCR primer reagent package disclosed in the first aspect, the PCR primer reagent package being in contact with or separated from the PCR premix solution.

[0024] Further, the reaction tube is an octuple tube, and each octuple tube contains a PCR premix solution and a separated PCR primer reagent package in the reaction well unit.

[0025] Further, the reaction tube further comprises a reagent package storage joint shell matched with the octuple tube, 8 PCR primer reagent packages are arranged in the reagent package storage joint shell, and the reaction tube can be arranged in a centrifugal device to make the PCR primer reagent packages in the reagent package storage joint shell separate by centrifugal action and contact with the PCR premix solution.

[0026] Compared with the prior art, the present application has the beneficial effects that:

[0027] 1. The present application separately arranges the light-sensitive reagent in the PCR reagent kit by introducing a wrapping layer with light-shielding properties to form special protection for the reagent, thereby improving the stability of the amplification reagent kit and the accuracy of subsequent detection. The wrapping layer comprises a barrier layer containing 60%-80% of hydroxypropyl methylcellulose and 10%-20% of polyvinyl alcohol, so that the barrier layer meets the light-shielding requirement and also has sufficient strength and toughness. The barrier layer further contains 5%-10% of a barrier agent, 3%-7% of triethyl citrate or glyceryl triacetate, and 1%-3% of an antibacterial agent, so that the barrier layer has higher reliable water and gas sealing performance, and the addition of the antibacterial agent enables the barrier layer to be stably stored for a long time.

[0028] 2. The wrapping layer of the present application has an oxygen permeability of ≤0.5 cc / (m 2 ·day), and the wrapping layer is in a stable state at 2℃-65℃, so that the reagent in the wrapping layer does not have the problem of contamination and failure even if it is stored in a liquid state;

[0029] 3. This invention also discloses a PCR reaction kit. The reaction tubes of the kit contain PCR premix and the aforementioned PCR probe kit. The PCR probe kit is either in contact with or separated from the PCR premix. This allows for the independent preparation of various types of reagents in different reaction tubes, while providing special protection for photosensitive reagents. The reagents are prepared in a single tube without the need for additional operations such as pipetting. The kit is designed as an 8-tube kit, enabling rapid detection of more identical or different targets. The PCR probe kit is more effectively housed within the reagent pack storage shell, creating a more reliable physical isolation. It detaches from the reagent pack storage shell and comes into contact with the PCR premix under centrifugation. The operation is simple and essentially identical to current mature PCR amplification reactions. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the cross-sectional structure of the PCR probe kit of the present invention.

[0032] Figure 2 This is a schematic diagram of various single-tube reaction tubes containing the PCR probe kit of the present invention, wherein (a) is a 0.2ml PCR single tube and (b) is a 2ml cryopreservation tube.

[0033] Figure 3 This is a structural diagram of an eight-tube kit containing a PCR probe kit provided by the present invention, wherein (a) is an eight-tube kit with a cap and (b) is an eight-tube kit with a single cap.

[0034] Figure 4 This is a schematic diagram of the contact configuration of the PCR probe kit and PCR premix in the eight-tube kit provided by the present invention, wherein (a) is stored in the capped eight-tube kit and (b) is stored in the single-capped eight-tube kit.

[0035] Figure 5 This is a schematic diagram of the reagent kit structure including a reagent pack storage shell provided by the present invention.

[0036] Figure 6 This is a verification diagram of HBV target amplification after the kit of the present invention has been stored and transported.

[0037] Figure 7 This is a verification diagram of HCV target amplification using the kit of the present invention after storage and transportation.

[0038] Figure 8 This is a graph showing the test results of a commercially available test kit.

[0039] Figure 9 This is a graph showing the test results of a paraffin-based reagent kit.

[0040] Figure 10 The diagram shows the control test results for a barrier layer prepared according to a formulation not conforming to the present invention.

[0041] Figure 11 The diagram shows the control test results for a barrier layer prepared according to a different formulation than that of the present invention.

[0042] Figure 12 This is a graph showing the test results of the reagent kit within the component ratio range of this invention.

[0043] Figure 13 In order to be in Figure 12 The results of boundary value testing were obtained by changing the content of the barrier layer components based on the reagent kit.

[0044] Figure 14 In order to be in Figure 12 The test results are shown in the figure after the content of the barrier layer component was changed to another boundary value based on the reagent kit.

[0045] Figure 15 This is a schematic diagram of the three-layer structure of the coating layer of the present invention.

[0046] In the above figures, the meanings of each mark are as follows: 10-coating layer, R1-primer probe reagent, 11-reagent cap, 12-storage casing, 13-tube body. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0048] This invention discloses a PCR probe kit, its preparation method, and a PCR reaction kit, such as... Figure 1 As shown, the internal space is formed by the encapsulation layer 10 and the encapsulation layer 10. The internal space contains primer and probe reagent R1 for detecting the target. The encapsulation layer 10 can reduce the influence of light on the internal primer and probe reagent. In other embodiments, the encapsulation layer 10 of the reagent package can be shaped into various shapes, such as spheres, droplets, capsules, and biomimetic small fish, etc.

[0049] The wrapping layer 10 is configured in a specific ratio to make the whole brown or other types of colored types, so as to absorb light, achieve similar light shielding effect similar to storing primer probe reagents in a brown or other colored bottle, the PCR primer probe reagent package of the application can be produced by using more precise pipetting equipment, accurately quantifying the appropriate amount of primer probe in each reaction tube, and packaging as a whole. After that, only the reagent package as a whole is configured in each reaction tube of the reagent box, and no additional pipetting and mixing operations are required in user use, and the overall operation efficiency is higher. In order to effectively protect other active ingredients (such as polymerase or other types of enzymes) sensitive to light, the mixture of primer probe reagents is configured in the internal space of the wrapping layer 10. By configuring primer probe reagents for different targets in the internal space, PCR primer probe reagent packages for detecting different targets can be formed. The wrapping layer 10 is a multilayer structure, and the wrapping layer 10 comprises a barrier layer comprising 60-80 parts by weight of hydroxypropyl methylcellulose and 10-20 parts by weight of polyvinyl alcohol. The hydroxypropyl methylcellulose (HPMC) has excellent biocompatibility, is resistant to water and oil, and can form a dense barrier. The polyvinyl alcohol (PVA) can enhance the flexibility and heat sealing performance of the formed film material.

[0050] In the experiment, the inventors found that adjusting the ratio of HPMC to PVA can produce a composite film with light shielding properties. The closed cavity formed by the composite film with light shielding properties can specifically protect light-sensitive reagents in fluorescence amplification, and through the exploration of suitable proportions, it can effectively isolate water and other influencing factors. By placing such a reagent package in the reaction tube of the reagent box, the light-sensitive reagent can be more effectively preserved. Through electron microscopy scanning, the light shielding component particle size of the application is ≤50 nm, that is, the effective molecular particle size of the HPMC and PVA composite film formed according to the ratio of the application. A larger particle size will actually affect the light shielding properties, mainly due to the larger gap between large particles. The barrier layer mainly composed of HPMC and PVA has a soil degradation rate of 75% in 6 months (ASTM D5338 standard), which is suitable for the EU green policy and meets the requirements of relevant standards in China.

[0051] In order to ensure that the barrier layer has sufficient water and oxygen isolation performance, the barrier layer further comprises 5%-10% by weight of a barrier agent, and the barrier agent is selected from at least one of montmorillonite, carbon nanotubes, graphene oxide or nanosilica. In one case, the barrier agent is mainly selected from nanosilica with a particle size range of 20-50 nm. This design can effectively fill the pores with the barrier agent, so that the oxygen transmission rate of the obtained barrier layer is ≤0.5 cc / (m 2 ·day), and the water blocking rate of the barrier layer is improved to ≤0.3 g / (m 2·day), mechanical strength +40% or so, to ensure that the barrier layer excellent water blocking gas characteristics, otherwise, in the case of carbon nanotubes as a barrier agent, the optimal selection of its diameter is 10-20nm, using it to make the barrier layer oxygen barrier properties to improve 60%, while giving the barrier layer stronger antistatic properties (surface resistance ≤ 109Ω), to avoid reagent adsorption loss.

[0052] The barrier layer also contains 3%-7% of triethyl citrate or glyceryl triacetate, and 1%-3% of an antibacterial agent. Adding an appropriate amount of triethyl citrate or glyceryl triacetate, an environmentally friendly plasticizer, can improve the ductility of the film, without affecting the film strength due to excessive amount. Here, the elongation at break of the barrier layer is ≥300% through the addition of triethyl citrate or glyceryl triacetate. The antibacterial agent can be selected from chitosan quaternary ammonium salt or lysozyme, which can inhibit microbial contamination and is suitable for long-term storage scenarios. Even if the film is in contact with a liquid, it is not easy to breed contaminating bacteria. It is determined that the antibacterial agent of the present application is uniformly dispersed in the film, so that the final detection of the film has a bacterial inhibition rate of ≥99% (GB / T20944.3-2008 standard). The composite film obtained by using the above-mentioned materials has high density, heat sealing strength ≥2.0N / 15mm, can withstand a temperature difference of-20℃ to 80℃, and can make the barrier layer made of it stable at 2℃-65℃, still maintaining the physical properties such as strength and ductility.

[0053] The preparation process of the composite film can use a flow casting film scheme, and the implementation process is as follows: 1) solution preparation: add HPMC with a proportion of 60%-80% and PVA with a proportion of 10%-20% to deionized water, stir at 60℃ until completely dissolved, form a 10% solid content base film solution, then add nano-silicon dioxide with a proportion of 5%-10% and triethyl citrate or glyceryl triacetate with a proportion of 3%-7% to the base film solution, ultrasonic dispersion for 30 minutes, filtration and impurity removal, and of course, chitosan quaternary ammonium salt or lysozyme with a proportion of 1%-3% can also be added before filtration; 2) flow casting film: uniformly coat the filtered solution on a PET carrier film, control the thickness to be 30-100μm, dry in a 45℃ oven for 12 hours, and then peel off to obtain a composite film; 3) functional treatment (optional): if a quick dissolving type is needed, immerse the film in step 2 in a 5% Tween-80 solution for 10 minutes, and then dry to obtain, and of course, other similar functional treatment steps can be used to obtain a composite film with changed functional properties in order to improve its acid-base sensitivity or salt ion sensitivity, etc., which will not be described here;

[0054] Another extrusion film forming process is as follows: 1) component preparation HPMC specific gravity 60%-80%, PVA specific gravity 10%-20%, nano-montmorillonite (also can choose ordinary type montmorillonite) specific gravity 5%, triethyl citrate specific gravity 3%-7%; 2) extrusion molding montmorillonite is modified by silane coupling agent, and is extruded into a film with HPMC / PVA at 170°C, and the thickness is controlled at 30-100 μm, of course, triethyl citrate is also mixed with it after extrusion, and other components falling within the range disclosed before can also be extruded into a film, which will not be repeated here.

[0055] The wrapping layer 10 can be configured as a more reliable multi-layer structure. In an optimized case, the wrapping layer 10 is a three-layer structure, including an inner core layer of polypropylene or polycaprolactone material, a middle barrier layer, and an outermost packaging layer. The inner core layer is used to form a package for the probe reagent, and the internal space of the package has a volume of not more than 15 μL, so as to be able to accommodate various viral detection reagents of the same program (such as multi-union detection primers, probe buffer), and the inner core layer can achieve an enzyme activity retention rate of ≥98% at -20°C (experimental data: 4°C storage for 12 months, Taq enzyme activity loss ≤1.5%), thereby achieving effective protection of the reagent; the barrier layer is more preferably formed into 30-80 μm, and an independent chamber is formed by ultrasonic welding, and the puncture strength is ≥1.2 N (simulated pipette tip impact test), so as to ensure that the strength of the wrapping layer 10 is sufficient and also does not make the wrapping layer difficult to melt, thereby causing the problem of inaccurate amplification detection results; the packaging layer is configured as a thin film layer doped with europium (Eu 3+ ) rare earth complex, and the whole can be configured as an ellipsoidal contour (length 5-10 mm, short diameter 3-6 mm), drop impact absorption energy ≥0.5 J (1 m drop to cement floor without damage), and the probe reagent configured in the internal space enclosed by the wrapping layer is optimally configured in a liquid form, so that no additional mixing operation is required, and the liquid probe reagent can be released by melting the wrapping layer during use. Of course, under general requirements, it can also be configured in a freeze-dried form, and such a form of reagent does not affect the core protection point of the present application, that is, the effect of light on the characteristics of the photosensitive reagent in the amplification reaction is reduced or eliminated by the setting of the wrapping layer.

[0056] Example 1

[0057] A PCR probe reagent package is disclosed in this embodiment, which includes a wrapping layer 10 and a packaging layer 20. Figure 1As shown, the package layer 10 and the internal space enclosed by the package layer 10 contain the primer probe reagent R1 for detecting the target, the package layer 10 can reduce the influence of light on the primer probe reagent in the internal space, the package layer 10 contains a barrier layer, the barrier layer contains 65% of hydroxypropyl methyl cellulose and 15% of polyvinyl alcohol, the barrier layer further contains 10% of a barrier agent, the barrier agent is mainly selected from nano-silicon dioxide with a particle size range of 20-50 nm, the barrier layer further contains 7% of triethyl citrate and 3% of chitosan quaternary ammonium salt, the oxygen permeability of the package layer is ≤0.5 cc / (m 2 ·day), the thickness of the barrier layer is 60 μm, the package contains a specific amount of primer probe reagent for detecting the target, and the barrier layer is closed by ultrasonic welding.

[0058] Embodiment 2

[0059] This embodiment describes a PCR reaction kit containing the PCR primer probe reagent package disclosed in Embodiment 1 in detail, please refer to Figure 2 , the kit is configured as a single reaction well unit, the PCR primer probe reagent package of Embodiment 1 is arranged in the well units of two different types of kits, and other reagents required for PCR reaction, such as PCR premix, can be filled in the well units in liquid form (here, the PCR premix can contain dNTPs, K + / Na + balance ion solution, Mg 2+ ion solution, pH balance solution, etc.), the package layer of the PCR primer probe reagent package can effectively block the primer probe reagent and the PCR premix, and due to its unique light shielding property, the light-sensitive reagents such as the primer probe reagent can be optimally protected during storage, the package layer 10 can be ablated at a high temperature, for example, it can be dissolved at a temperature above 80℃, and the high temperature in amplification can meet the dissolution conditions of the package layer 10, so that the kit can be directly used in amplification.

[0060] Embodiment 3

[0061] As shown in Figure 3 and Figure 4 , this embodiment provides a PCR reaction kit of eight-pipe type, the kit contains eight connected reaction well units, the PCR primer probe reagent package of Embodiment 1 is arranged in each well unit, and the reaction premix needs to be added when used, and then the sample is filled to perform the amplification reaction, of course, in a special case, a tank part for packaging the reaction premix can be arranged outside the reagent package to form a sandwich type structure to centrally arrange all reagents required for the amplification reaction, Figure 4The implementation scheme includes a PCR probe kit for each reaction well unit, and a premixed solution is prepared within the reaction well unit. Here, the PCR probe kit can directly contact the premixed solution to form a ready-to-use amplification kit.

[0062] Example 4

[0063] like Figure 5 As shown, this embodiment provides a kit containing PCR premix and a PCR probe kit with separate configuration. The reaction tube is an eight-tube bundle. The kit also includes a reagent kit storage shell 12 that works with the eight-tube bundle. The kit includes a tube body 13 connected to eight reaction well units. Premix is ​​placed in each well unit of the tube body. The opening of the reaction well unit can be connected to the reagent storage chamber of the reagent kit storage shell 12. The PCR probe kit from Embodiment 1 is placed in the reagent storage chamber. Finally, the top is sealed with the kit cap 11. In this way, the PCR probe kit and the premix in the eight-tube reaction well units are physically separated, which can ensure reliable protection of the probe reagent inside by the encapsulation layer 10. Before use, the reaction tube can be placed in a centrifuge device. Centrifugation will cause the PCR probe kit in the reagent kit storage shell to detach and come into contact with the PCR premix. The figure shows the state of the PCR probe kit detached from the reaction well unit under centrifugal force. After centrifugation, the reaction tube can perform the amplification reaction. During the amplification, the encapsulation layer 10 of the reagent kit is dissolved to release the probe reagent and complete the automated amplification reaction.

[0064] Example 5

[0065] A method for preparing the PCR primer and probe reagent package in Example 1 is provided. A barrier layer with a thickness of 60 μm is prepared, encapsulating a specific amount of primer and probe reagents for the detection target. The barrier layer is sealed using ultrasonic welding to form a closed internal space to accommodate the primer and probe reagents for the detection target. The barrier layer can be prepared using either hot extrusion or casting. The barrier layer film itself has high density and a heat-sealing strength ≥2.0 N / 15 mm. The double encapsulation of "film wrapping + ultrasonic welding" ensures absolute isolation of the reagents from the outside environment. The film has an elastic modulus of 100-300 MPa, can withstand a 1-meter drop impact, and is adaptable to transportation bumps, allowing the internal reagents to be more reliably preserved.

[0066] like Figure 15 As shown, in some embodiments where the encapsulation layer is a multilayer composite structure, a core layer is formed by encapsulating a probe reagent with a volume not exceeding 15 μL using polypropylene (PP) or polycaprolactone (PCL). A barrier layer with a thickness of 30-80 μm is disposed outside the core layer, and an intermediate barrier layer is formed by ultrasonic welding to encapsulate the core layer. Europium doping (Eu) is then coated outside the barrier layer. 3+The thin film forming encapsulation layer of the rare earth complex is shaped as an ellipsoid. The physical isolation of the multilayer design can significantly improve the stability of the reagents, especially the activity of the enzyme and the integrity of the probe, and can also avoid the mutual reaction in the premix solution.

[0067] Example 6

[0068] In order to verify whether the packaging layer 10 of the present application can protect different types of fluorescent probes, the same batch of kits produced in this example and the commercially available separate storage kits (Hepatitis B Virus Nucleic Acid Detection Kit (fluorescent PCR method) (ultra-sensitive) and Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (fluorescent PCR method) (ultra-sensitive) produced by Suzhou Tianlong Biotechnology Co., Ltd. (P101 / P174 and P100 / P175) were used to detect two different types of targets, HBV (Hepatitis B) and HCV (Hepatitis C). The comparison chart of the detection results is shown in FIG. 6. The Ct values of the amplification results corresponding to two different targets and four groups of standard samples were counted, wherein the control group was the commercially available kit, and the side view group was the reagent kit in the form of the reagent package of the present application. The comparison results of different types of targets are shown in Tables 2 and 3.

[0069] Table 1. Comparison of detection results of the kit of this example and the commercially available kit

[0070]

[0071] In the table, PC is the positive control. From the comparison table of the above detection results, it can be seen that there is no significant difference between the test group and the control group in amplifying the blood nucleic acid extraction product and the positive control. The detection results of different fluorescent labels are very small, which also shows that the packaging layer 10 of the present application can protect various types of fluorescently labeled probe reagents. The kit of the present application can adapt to storage and transportation verification test, and the kit has high reliability.

[0072] Figure 6 and Figure 7 In order to verify whether the packaging layer 10 of the present application can protect different types of fluorescent probes, the same batch of kits produced in this example and the commercially available separate storage kits (Hepatitis B Virus Nucleic Acid Detection Kit (fluorescent PCR method) (ultra-sensitive) and Hepatitis C Virus (HCV) Nucleic Acid Detection Kit (fluorescent PCR method) (ultra-sensitive) produced by Suzhou Tianlong Biotechnology Co., Ltd. (P101 / P174 and P100 / P175) were used to detect two different types of targets, HBV (Hepatitis B) and HCV (Hepatitis C). The comparison chart of the detection results is shown in FIG. 6. The Ct values of the amplification results corresponding to two different targets and four groups of standard samples were counted, wherein the control group was the commercially available kit, and the side view group was the reagent kit in the form of the reagent package of the present application. The comparison results of different types of targets are shown in Tables 2 and 3.

[0073] Table 2. Statistics of detection results for HBV targets

[0074]

[0075] Table 3. Statistics of detection results for HCV targets

[0076]

[0077] Figure 6 and Figure 7 It can be seen from the amplification curves that the kit of the present embodiment and the commercially available kit can achieve good amplification for all samples and internal standards and four standard samples, and the amplification curves can present a shape close to an S-shaped curve. Tables 2 and 3 also show that for DNA virus detection for HBV targets and RNA virus detection for HCV, the two kits can obtain very high similarity of Ct value results, which also indicates that the kit of the present embodiment can be suitable for efficient and accurate detection of different types of viruses of RNA and DNA.

[0078] Example 7

[0079] In order to further verify the optimal composition ratio of the wrapping layer 10 of the present application, especially the barrier layer contained therein, and the protection effect of the optimal ratio on the internal probe and other photosensitive reagents, the inventors further designed a detection comparison experiment under the same storage conditions of different samples positive for HBV for different storage times. The comparison of the statistical detection of Ct values of samples for different times is shown in Figures 8-12 .

[0080] Figure 8 It is a commercially available scheme for storing premixed liquid and probe reagents in multiple reagent bottles respectively, and mixing and configuring immediately before testing on site, and then detecting. Here, the illumination conditions (daylight lamp / ultraviolet lamp irradiation) of different experimental conditions are the same, and the kit is removed from the outer packaging for condition verification under different storage times (3d / 7d / 15d / 30d). It can be seen that the commercially available kit is stored separately, and the probe reagent with photosensitivity is in a brown reagent bottle, so that after detection after different time intervals, relatively similar accurate detection results can be obtained.

[0081] Figure 9 It is a scheme for separating and configuring the probe reagent and the premixed reagent in the same reagent tube well unit by using paraffin as a separation layer. In this scheme, since paraffin only plays a separation role and does not effectively attenuate light, the activity of the probe reagent with photosensitivity or certain photosensitive enzymes is affected after long-term storage. Therefore, as time goes on, the difference between the detection results becomes larger and larger, and the detection results become less and less accurate. The overall detection results present an error type of clustering phenomenon.

[0082] Figure 10 The wrapping layer 10 similar to the present application is adopted, but the material ratio of the wrapping layer 10, especially the barrier layer, is not the same as the present application. The PCR probe reagent kit is changed only that the proportion of hydroxypropyl methyl cellulose is 55%, the proportion of polyvinyl alcohol is 30%, the proportion of barrier agent is 8%, the proportion of triacetin is 5%, and the proportion of antibacterial agent is 2% based on the embodiment 1. It can be known from the detection result that the detection result accuracy of the kit is also worse with the increase of time, and the detection reliability is low. Compared with the ratio of the present application, the proportion of the core materials hydroxypropyl methyl cellulose and polyvinyl alcohol is greatly different. The core material does not adopt the best proportion range of the present application. On the one hand, the stability of the film may be insufficient to cause the problem of insufficient isolation. On the other hand, the proportion of the core material is also greatly different for the attenuation of light, so that the probe reagent in the wrapping layer 10 is not effectively protected. These factors alone or in combination cause the unreliability of the detection kit.

[0083] Figure 11 The wrapping layer 10 similar to the present application is adopted, but the material ratio of the wrapping layer 10, especially the barrier layer, is not the same as the present application. The PCR probe reagent kit is changed only that the proportion of hydroxypropyl methyl cellulose is 55%, the proportion of polyvinyl alcohol is 30%, the proportion of barrier agent is 8%, the proportion of triacetin is 5%, and the proportion of antibacterial agent is 2% based on the embodiment 1. It can be known from the detection result that the detection result accuracy of the kit is also worse with the increase of time, and the detection reliability is low. Compared with the ratio of the present application, the proportion of the core materials hydroxypropyl methyl cellulose and polyvinyl alcohol is greatly different. The core material does not adopt the best proportion range of the present application. On the one hand, the stability of the film may be insufficient to cause the problem of insufficient isolation. On the other hand, the proportion of the core material is also greatly different for the attenuation of light, so that the probe reagent in the wrapping layer 10 is not effectively protected. These factors alone or in combination cause the unreliability of the detection kit. Figure 10

[0084] Figure 12 ​The figure shows the test results of the kit within the component ratio range of the present invention. In this embodiment, the barrier layer of the present invention was manufactured using the component ratios in Example 1. The proportions of hydroxypropyl methylcellulose and polyvinyl alcohol were 65% and 15% respectively within the protection range. The test results show that the results are highly consistent with the test results of commercially available separation and preservation kits, indicating that the ratio of the present invention can form a reliable and stable barrier layer that meets the requirements for light attenuation. The operation of the kit is also simpler. Users only need to add elution buffer to perform conventional PCR amplification reaction, without the need for pipetting and other complicated operations. During the PCR amplification process, the coating layer 10 can automatically dissolve under the action of temperature, so that the amplification system solution can be automatically mixed.

[0085] Figure 13 The kit used in Figure 12 The only change is that the barrier layer contains 60% hydroxypropyl methylcellulose and 20% polyvinyl alcohol by weight. The content of both important components is verified using the boundary content method. The test results show that the results are highly consistent with those of commercially available separation and preservation kits, indicating that the formulation of the present invention can form a reliable and stable barrier layer that meets the requirements for light attenuation. The operation of the kit is also simpler. The low content of hydroxypropyl methylcellulose in the 30-day verification results showed certain aggregation characteristics in the 25-30 Ct value range, indicating that the boundary content, especially the low content of hydroxypropyl methylcellulose, causes a certain degree of attenuation in the water-proof properties of the barrier layer after light exposure and long-term storage. However, the overall results are still within the acceptable range.

[0086] Figure 14 The kit used was validated using a barrier layer with different component boundary contents. This barrier layer contained 80% hydroxypropyl methylcellulose and 10% polyvinyl alcohol. Further, the barrier layer contained 5% barrier agent, 3% triacetyl ester, and 2% antibacterial agent. The test results showed high consistency with those of commercially available separation and preservation kits, demonstrating that the formulation of this invention can form a reliable and stable barrier layer that meets the requirements for light attenuation. The kit is also simpler to operate. The higher content of hydroxypropyl methylcellulose in the barrier layer results in a more robust barrier layer compared to... Figure 13 Despite its better water-blocking properties, the product still exhibited some agglomeration after long-term storage, but the reliability of the test results remained acceptable.

[0087] Figures 10-14The detection result verifies the influence of the content of the two most important components in the barrier layer on the detection result, and the detection result shows that the barrier layer containing 60-80 parts of hydroxypropyl methyl cellulose and 10-20 parts of polyvinyl alcohol by weight can make the barrier layer play the best light-shielding, water-blocking and antioxidant properties, and the values of the remaining components in each example and the comparative example are verified within the component content range defined in the application. Of course, the optimal values of other components can also be obtained by using a similar verification method, and the process will not be described here.

[0088] In addition, other performance verifications related to the reliability of the kit are also carried out. Under the condition of 4℃, the reagent activity retention rate of the reagent bag of the kit is ≥98% (12 months), and under room temperature (25℃), it is ≥95% (6 months), which is better than the traditional packaging (only 3 months at room temperature). The experimental preparation time is shortened by more than 50%, avoiding the cross contamination risk and inaccurate pipetting problem caused by multi-tube dispensing. The reagent kit can resist extreme temperature: -20℃ freezing or 60℃ constant temperature, the wrapping layer 10 is not brittle and does not deform, the reagent bag in the kit is strong against chemical corrosion: the permeability of organic solvents such as ethanol and chloroform is ≤0.01 mg / (m 2 ·24h), and the wrapping layer 10 of the reagent bag is environmentally friendly and biodegradable: HPMC / PVA are biodegradable materials, which can be decomposed by soil microorganisms within 6 months after being discarded. The wrapping layer 10 of the reagent bag is detected: phthalate ≤0.05 ppm (GC-MS method, detection limit 0.01 ppm), which meets the requirements of green laboratory.

[0089] The above is the embodiment of the embodiment, but the embodiment is not limited to the above optional embodiment, and those skilled in the art can obtain other various embodiments by arbitrarily combining the above methods. Any person can obtain other various forms of embodiments under the inspiration of the embodiment. The above specific embodiments should not be understood as limiting the protection scope of the embodiment, and the protection scope of the embodiment should be defined by the claims, and the specification can be used to explain the claims.

Claims

1. A PCR probe kit, characterized in that: It includes a coating layer, which encapsulates primer and probe reagents for detecting targets. The coating layer can reduce the impact of light on the internal primer and probe reagents. The encapsulation layer has a three-layer structure, including a core layer made of polypropylene or polycaprolactone, a barrier layer with a thickness of 30-80μm disposed outside the core layer, an intermediate barrier layer encapsulating the core layer by ultrasonic welding, and a film doped with europium rare earth complexes wrapped around the barrier layer to form an encapsulation layer. The barrier layer contains 60-80 parts by weight of hydroxypropyl methylcellulose; Polyvinyl alcohol with a specific gravity of 10-20 parts; A barrier agent with a specific gravity of 5%-10%, wherein the barrier agent is selected from at least one of montmorillonite, carbon nanotubes, graphene oxide or nano silica. Triethyl citrate or glyceryl triacetate with a specific gravity of 3%-7%; And an antibacterial agent with a specific gravity of 1%-3%, which can be chitosan quaternary ammonium salt or lysozyme.

2. The PCR probe kit according to claim 1, characterized in that: The oxygen permeability of the coating layer is ≤0.5cc / (m 2 ·day), and the wrapping layer is stable at 2℃-65℃.

3. A method for preparing a PCR probe kit according to any one of claims 1-2, characterized in that: A barrier layer with a thickness of 30-80 μm is prepared to encapsulate the primer and probe reagents of the detection target. The barrier layer is then sealed to form a closed internal space to accommodate the primer and probe reagents of the detection target.

4. A PCR reaction kit, characterized in that: The PCR probe kit is prepared by the method described in any one of claims 1-2, or by the method described in claim 3.

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