QPCR (quantitative polymerase chain reaction) amplification reaction composition and application thereof

Abstract

The invention provides a qPCR (quantitative polymerase chain reaction) amplification reaction composition and application thereof. Specifically, the invention relates to a qPCR (quantitative polymerase chain reaction) amplification reaction composition which comprises Tris-HCl, ammonium sulfate, KCl, dNTPs (deoxyribonucleoside triphosphates), BSA (bovine serum albumin), MgCl2, DNA (deoxyribonucleic acid) polymerase, polyacrylic acid and a freeze-drying protective agent. The invention also relates to a kit comprising a reagent for preparing the composition and a nucleic acid releasing agent matched with the composition for use. The invention also relates to a method for quantitative or qualitative analysis of nucleic acids in a biological sample using the composition, kit or release agent. The nucleic acid releasing agent can rapidly and effectively release DNA under the room temperature condition, the composition can directly apply a nucleic acid crude sample to qPCR amplification in a freeze-drying form or a liquid form, nucleic acid detection is achieved under the condition that a nucleic acid extraction step is not needed, the detection efficiency is remarkably improved, and the composition is suitable for molecular POCT application scenes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a freeze-dried, directly amplifiable nucleic acid amplification reaction composition and an application thereof. Background Art

[0002] Detection of nucleic acids in biological samples usually requires extraction and purification of the nucleic acids from the sample before PCR is performed. This is because the components of the biological sample (e.g., hemoglobin in a blood sample) and preservatives (e.g., anticoagulants) tend to interfere with PCR amplification. However, extracting nucleic acids from biological samples is time-consuming and presents a high risk of contamination. Given the high complexity involved in separating and detecting nucleic acid molecules in biological samples, it is increasingly desirable to directly detect nucleic acids in biological samples without any upstream nucleic acid extraction or a large number of pretreatment steps.

[0003] Direct molecular amplification, also known as Direct PCR technology or extraction-free amplification technology, is a type of PCR technology. Direct molecular amplification can directly add the original sample without DNA extraction and purification into the PCR reaction system and obtain the amplified product, reducing the inspection steps, reducing the risk of contamination and inspection costs. Before the emergence of this technology, sample pretreatment was an unavoidable and headache task for scientific researchers. The grinding of animal and plant tissue samples, the extraction and purification of nucleic acids involved many steps and took a long time, which not only easily caused cross-contamination and degradation of the samples, but also increased the reagents, instruments and time costs of the experiment.

[0004] With the advancement of technology, molecular direct amplification and POCT (point-of-care testing) have made great progress in recent years. The molecular POCT technology of direct amplification can perform rapid nucleic acid amplification on site. However, the traditional PCR reaction-related reagents have extremely high requirements for storage temperature. Once the temperature is too high during storage or transportation, the related reagents, especially protein raw materials such as enzymes, are easily inactivated, resulting in loss of reagent function. Therefore, the conventional method requires transportation through cold chain. However, cold chain transportation does not meet the application scenarios of molecular POCT. The PCR reaction reagents must be freeze-dried through vacuum freeze-drying technology to achieve the purpose of room temperature transportation and storage to meet the use scenarios of molecular POCT.

[0005] Chinese patent CN117004694A discloses a system and method for preventing aerosol contamination caused by nucleic acid amplification products. The system combines a nucleic acid releaser with a freeze-dryable isothermal amplification reagent, which can be well applied to molecular POCT. However, the article mainly describes the role of UDG (Uracil-DNA Glycosylase) in the system, and does not systematically study freeze-drying reactions and direct amplification reactions. In addition, the sensitivity of isothermal amplification is still far behind that of fluorescent quantitative PCR, and the false positive and false negative rates are high.

[0006] Chinese patent CN116590475A discloses a direct amplification freeze-dried detection kit based on loop-mediated isothermal amplification technology and its application, and develops a LAMP system suitable for freeze-drying, and finally achieves freeze-drying of all components to meet the needs of room temperature transportation, instant testing and home self-testing. However, the LAMP technology itself has the problems of low specificity and low sensitivity.

[0007] In summary, the prior art fails to solve the technical problem of combining direct molecular amplification with fluorescent quantitative PCR freeze-drying. Summary of the invention

[0008] In order to overcome the above-mentioned technical problems existing in the prior art, the present invention provides a qPCR amplification reaction composition in the first aspect, wherein the composition comprises: Tris-HCl, ammonium sulfate, KCl, dNTPs, BSA, MgCl2, hot-start DNA polymerase, polyacrylic acid and a lyophilization protective agent, wherein the lyophilization protective agent comprises cyclodextrin, mannitol, sucrose and polyvinyl alcohol.

[0009] In a second aspect, the present invention provides a nucleic acid releaser for use with the composition of the first aspect of the present invention, wherein the nucleic acid releaser comprises potassium hydroxide, dioxane, sodium dodecyl sulfate, Brij 58, lithium chloride and water.

[0010] The present invention provides a nucleic acid detection kit in a third aspect, the kit comprising a first reagent and / or a second reagent, the first reagent being a reagent for preparing the composition described in the first aspect of the present invention; the second reagent being a reagent for preparing the nucleic acid releasing agent described in the second aspect of the present invention.

[0011] In a fourth aspect, the present invention provides a method for quantitative or qualitative analysis of DNA in a biological sample, the method being performed using the composition described in the first aspect of the present invention, the nucleic acid releasing agent described in the second aspect of the present invention, and / or the kit described in the third aspect of the present invention.

[0012] Compared with the prior art, the present invention has the following technical effects:

[0013] The nucleic acid releaser and qPCR amplification reaction composition of the present invention are used to treat body fluid samples such as whole blood, serum, plasma, and swabs, which can quickly and effectively release DNA under room temperature conditions. The crude sample after lysis can be directly used for qPCR amplification (lyophilized or liquid form) without the need for nucleic acid extraction steps, greatly improving the detection efficiency and being suitable for molecular POCT application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is a photo of the freeze-dried product obtained after the qPCR amplification reaction composition provided in Preparation Example 3 was freeze-dried in Example 1.

[0015] Figure 2 This is a photograph of the freeze-dried product obtained after freeze-drying the qPCR amplification reaction composition provided in Preparation Example 4 in Example 1.

[0016] Figure 3 This is a photograph of the freeze-dried product obtained after the qPCR amplification reaction composition provided in Preparation Example 5 was freeze-dried in Example 1.

[0017] Figure 4 The amplification curves of different amounts of crude extract products were shown in Example 3 using sample S1 to test the qPCR lyophilized powders of Preparation Examples 3 and 5. DETAILED DESCRIPTION

[0018] In a first aspect, the present invention provides a qPCR amplification reaction composition. The qPCR amplification reaction composition can be prepared, sold or used in a lyophilized form or a liquid form. For example, when used, whether in a liquid form or a lyophilized form, direct amplification of nucleic acids can be performed.

[0019] In some embodiments, the qPCR amplification reaction composition comprises Tris-HCl, ammonium sulfate, KCl, dNTPs, BSA, MgCl2, hot-start DNA polymerase, polyacrylic acid, and a lyophilization protectant.

[0020] Preferably, the lyoprotectant comprises cyclodextrin, mannitol, sucrose, and polyvinyl alcohol. More preferably, the lyoprotectant consists of cyclodextrin, mannitol, sucrose, and polyvinyl alcohol.

[0021] In some preferred embodiments, the composition comprises 20-100 mM (e.g., 30, 40, 50, 60, 70, 80, or 90 mM) of Tris-HCl, 10-50 mM (e.g., 20, 30, or 40 mM) of ammonium sulfate, 20-100 mM (e.g., 30, 40, 50, 60, 70, 80, or 90 mM) of KCl, 100-500 μM (e.g., 200, 300, or 40 mM) of 400μM) of dNTPs, 100-500μg / mL (e.g., 200, 300 or 400μg / mL) of BSA, 1-5mM (e.g., 2, 3 or 4mM) of MgCl2, 0.1-0.2U / μL (e.g., 0.15U / μL) of hot start DNA polymerase, 0.1-10mM (e.g., 2, 3, 4, 5, 6, 7, 8 or 9mM) of polyacrylic acid and a lyophilization protectant. Preferably, the lyophilization protectant is as described above.

[0022] In some more preferred embodiments, the composition further comprises 0.25-0.5% (W / v) (e.g., 0.30, 0.35, 0.40 or 0.45% (W / v)) cyclodextrin, 6-10% (W / v) (e.g., 7, 8 or 9% (w / v)) mannitol, 6-12% (W / v) (e.g., 7, 8, 9, 10 or 11% (W / V)) sucrose and 0.025-0.4% (W / v) (e.g., 0.05, 0.075, 0.1, 0.2 or 0.3% (w / V)) polyvinyl alcohol. For example, the composition comprises 20-100 mM Tris-HCl, 10-50 mM ammonium sulfate, 20-100 mM KCl, 100-500 μM dNTPs, 100-500 μg / mL BSA, 1-5 mM MgCl2, 0.1-0.2 U / μL hot start DNA polymerase, 0.1-10 mM polyacrylic acid, 0.25-0.5% (w / v) cyclodextrin, 6-10% (w / v) mannitol, 6-12% (w / v) sucrose and 0.025-0.4% (W / v) polyvinyl alcohol.

[0023] In the present invention, the hot start DNA polymerase can be a mutant of Taq DNA polymerase, and can achieve hot start. Therefore, in some preferred embodiments, the hot start DNA polymerase is hot start Taq DNA polymerase.

[0024] In some more preferred embodiments, the hot-start DNA polymerase is an antibody-modified, ligand-modified or chemically-modified hot-start Taq DNA polymerase. Further preferably, the hot-start DNA polymerase is an antibody-modified hot-start Taq DNA polymerase.

[0025] In the present invention, BSA can neutralize impurities such as hemoglobin in the crude sample, and polyacrylic acid can bind to denatured hemoglobin, albumin, bile acid, antibodies, and other PCR inhibitors, so that they no longer inhibit the activity of DNA polymerase. The inventors have found that the composition system of the present invention contains both BSA and polyacrylic acid, and qPCR detection can be performed when a large sample amount is used and without the need for a nucleic acid extraction step.

[0026] In some preferred embodiments, the molecular weight of the polyacrylic acid is 2000-500000 (for example, 5000, 8000, 10000, 12000, 20000, 50000, 80000, 100000, 200000, 300000 or 400000). Preferably, the molecular weight of the polyacrylic acid is 50000-500000.

[0027] In some preferred embodiments, the cyclodextrin is γ-cyclodextrin.

[0028] In some preferred embodiments, the molecular weight of the polyvinyl alcohol is 5000-20000 (eg, 10000 or 15000).

[0029] In some embodiments, the qPCR amplification reaction composition can be stored at room temperature for at least 3 months (e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 months) after being lyophilized, and more preferably at least 12 months. The room temperature condition is 20-25° C. (e.g., 21, 22, 23, or 24° C.), preferably 25° C.

[0030] The present invention has no particular limitation on the freeze-drying procedure of the qPCR amplification reaction composition. However, in some preferred embodiments, the freeze-drying procedure may adopt the following rapid vacuum freeze-drying procedure, which includes one or more of a pre-freezing procedure, a primary drying procedure, a secondary drying procedure, and an analytical drying procedure.

[0031] The present invention provides a rapid vacuum freeze-drying process for the qPCR amplification reaction composition. The vacuum freeze-drying technology includes one or more of a pre-freezing process, a primary drying process, a secondary drying process, and an analytical drying process.

[0032] In a second aspect, the present invention provides a nucleic acid release agent for use with the composition of the first aspect of the present invention, the nucleic acid release agent comprising: potassium hydroxide, dioxane, sodium dodecyl sulfate, Brij 58, lithium chloride and water. The qPCR amplification reaction composition can be in liquid form or in freeze-dried powder form.

[0033] The nucleic acid releaser of the present invention can quickly release DNA in various biological samples and remove most PCR inhibitors, thereby achieving direct amplification without complicated nucleic acid extraction steps.

[0034] In some preferred embodiments, the nucleic acid releasing agent comprises 5-50 mM (e.g., 10, 20, 30 or 40 mM) potassium hydroxide, 5-20% (w / v) (e.g., 10 or 15% (w / v)) dioxane, 0.01-0.5% (w / v) (e.g., 0.05, 0.1, 0.2, 0.3 or 0.4% (w / v)) sodium dodecyl sulfate, 0.05-1% (w / v) (0.06, 0.07, 0.08 or 0.09, 0.1, 0.5 or 0.8% (w / v)) Brij 58, 5-100 mM (e.g., 10, 20, 30, 40, 50, 60, 70, 80 or 90 mM) lithium chloride and the remainder water.

[0035] In some more preferred embodiments, the nucleic acid releasing agent comprises 10-25 mM potassium hydroxide, 5-10% (w / v) dioxane, 0.01-0.25% (w / v) sodium dodecyl sulfate, 0.1-0.5% (w / v) Brij 58, 10-25 mM lithium chloride and the remainder water.

[0036] In some further preferred embodiments, the nucleic acid releasing agent comprises 10 mM potassium hydroxide, 5% (w / v) dioxane, 0.05% (w / v) sodium dodecyl sulfate, 0.2% (w / v) Brij 58, 25 mM lithium chloride and the balance water.

[0037] In the nucleic acid releaser, the potassium hydroxide, sodium dodecyl sulfate and Brij 58 can quickly destroy the cell membrane and release nucleic acid. The dioxane can dissolve the cell membrane and allow the releaser to penetrate into the cell membrane faster. The sodium dodecyl sulfate is a high-performance anionic surfactant that exhibits very good stability in a strong alkaline solution, can lyse cells at room temperature, and denature proteins. Unexpectedly, the inventors found that under weak alkaline conditions, sodium dodecyl sulfate interacts with Brij 580 to quickly lyse cells and release DNA into the solution, wherein the Brij 58 is a nonionic surfactant that can quickly lyse cells and release nucleic acids with sodium dodecyl sulfate under weak alkalinity, and Brij 58, as a nonionic surfactant, has little effect on PCR and will not affect downstream amplification reactions.

[0038] The present invention provides a nucleic acid detection kit in a third aspect, the kit comprising a first reagent and / or a second reagent, the first reagent being a reagent for preparing the composition described in the first aspect of the present invention; the second reagent being a reagent for preparing the nucleic acid releasing agent described in the second aspect of the present invention.

[0039] In a fourth aspect, the present invention provides a method for quantitative or qualitative analysis of DNA in a biological sample, the method being performed using the composition described in the first aspect of the present invention, the nucleic acid releasing agent described in the second aspect of the present invention, and / or the kit described in the third aspect of the present invention.

[0040] In some preferred embodiments, the method comprises the following steps:

[0041] (1) adding a nucleic acid releasing agent to a biological sample to release nucleic acid from the biological sample, thereby preparing a crude sample;

[0042] (2) adding a qPCR amplification reaction composition to the crude sample, and then adding primers and probes of corresponding detection targets to obtain a reaction working solution;

[0043] (3) Using the reaction working solution to perform quantitative or qualitative analysis on the DNA in the biological sample through qPCR.

[0044] In some preferred embodiments, in step (2), if the qPCR amplification reaction composition is in liquid form, the volume of the crude sample after release accounts for 10-50% (for example, 20, 30 or 40%) of the volume of the reaction working solution (i.e., the entire amplification reaction solution). If the qPCR amplification reaction composition is in a lyophilized form, the volume of the crude sample after release accounts for 20-100% (for example, 30, 40, 50, 60, 70, 80 or 90%) of the volume of the reaction working solution (i.e., the entire amplification reaction solution). If the proportion of the qPCR amplification reaction composition to the reaction working solution is too low, it means that the sample amount is small and the sensitivity is low. Therefore, if a high proportion of the qPCR amplification reaction composition can be used, the detection sensitivity can be improved. The present invention can adopt a ratio of even up to 100%, which can significantly improve the detection sensitivity and is particularly suitable for application scenarios such as POCT.

[0045] In some preferred embodiments, the biological sample can be a liquid sample. In some more preferred embodiments, the liquid sample is selected from one of whole blood, saliva, plasma, serum and swab eluate.

[0046] In other preferred embodiments, when the biological sample is a liquid sample, a crude sample can be obtained by taking 50-200 μL (e.g., 100 or 150 μL) of the liquid sample, then adding an equal volume of a nucleic acid release agent, mixing evenly at room temperature (e.g., by vortexing), and letting stand for 5 minutes.

[0047] Example

[0048] In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. Different embodiments can be replaced or combined, and for those of ordinary skill in the art, other implementation methods can also be obtained based on these embodiments without paying creative work.

[0049] The experimental methods described in the following examples are conventional methods unless otherwise specified; the materials described are commercially available unless otherwise specified. The solvent of the qPCR amplification reaction composition prepared in the preparation example is water.

[0050] Preparation Example 1

[0051] This preparation example provides a nucleic acid releaser, which is composed of: 10 mM potassium hydroxide, 5% (w / v) dioxane, 0.05% (w / v) sodium dodecyl sulfate, 0.2% (w / v) Brij58, 25 mM lithium chloride and the balance water.

[0052] Preparation Example 2

[0053] This preparation example provides a nucleic acid releaser, which is composed of: 15mM potassium hydroxide, 10% (w / v) dioxane, 0.02% (w / v) sodium dodecyl sulfate, 0.5% (w / v) Brij 58, 10mM lithium chloride and the balance water.

[0054] Preparation Example 3

[0055] This preparation example provides a qPCR amplification reaction composition, which is provided in a 2X form and is also called 2XqPCR Mix, and is composed of: 100mM Tris-HCl, 50mM ammonium sulfate, 40mM KCl, 100-500μM dNTPs, 100μg / mL BSA, 2mM MgCl2, 0.2U / μL hot start DNA polymerase and 2.5mM polyacrylic acid and a lyophilization protectant.

[0056] Among them, BSA stands for bovine serum albumin, which comes from Sigma Company, with product number V900933.

[0057] The hot-start DNA polymerase is a hot-start Taq DNA polymerase without glycerol, which is from Suzhou Cretaceous Biotechnology Co., Ltd. Jumbo Taq Hot-Start DNA Polymerase, Glycerol Free, Cat# CEN01641.

[0058] The molecular weight of polyacrylic acid is 450,000 and it comes from Aladdin Company, product number P104272.

[0059] The lyophilized protective agent is composed of cyclodextrin, mannitol, sucrose and polyvinyl alcohol. The concentrations of the components of the lyophilized protective agent in the qPCR amplification reaction composition are as follows: the concentration of cyclodextrin is 0.5% (w / V), the concentration of mannitol is 8% (w / V), the concentration of sucrose is 6% (w / V), and the concentration of polyvinyl alcohol is 0.05% (W / v).

[0060] The cyclodextrin is γ-cyclodextrin, which is from Sigma Company, product number C4892.

[0061] Wherein, mannitol is D-mannitol from Sigma Company, product number M4125.

[0062] Among them, sucrose comes from Aladdin Company, product number S112234.

[0063] The molecular weight of polyvinyl alcohol is 9,000-10,000 and is from Sigma Company, product number 360627.

[0064] Preparation Example 4

[0065] This preparation example provides a qPCR amplification reaction composition, which is provided in a 2X form and is also called 2X qPCR Mix, and is composed of: 60mM Tris-HCl, 25mM ammonium sulfate, 30mM KCl, 300μM dNTPs, 300μg / mL BSA, 2.5mM MgCl2, 0.15U / μL hot start DNA polymerase and 5mM polyacrylic acid and a lyophilization protectant.

[0066] The lyophilized protective agent is composed of cyclodextrin, mannitol, sucrose and polyvinyl alcohol. The concentrations of the components of the lyophilized protective agent in the qPCR amplification reaction composition are as follows: the concentration of cyclodextrin is 0.5% (w / V), the concentration of mannitol is 6% (w / v), the concentration of sucrose is 10% (w / V), and the concentration of polyvinyl alcohol is 0.1% (w / V).

[0067] The raw materials used in this preparation example are the same as those in Preparation Example 3.

[0068] Preparation Example 5

[0069] This preparation example provides a qPCR amplification reaction composition, which is substantially the same as that of Preparation Example 4, except that the qPCR amplification reaction composition of this preparation example does not contain BSA and polyacrylic acid.

[0070] Specifically, this preparation example provides a qPCR amplification reaction composition, which is provided in a 2X form, also called 2X qPCRMix, and is composed of: 60mM Tris-HCl, 25mM ammonium sulfate, 30mM KCl, 300μM dNTPs, 2.5mM MgCl2, 0.15U / μL hot start DNA polymerase and lyophilization protectant.

[0071] The lyophilized protective agent is composed of cyclodextrin, mannitol, sucrose and polyvinyl alcohol. The concentrations of the components of the lyophilized protective agent in the qPCR amplification reaction composition are as follows: the concentration of cyclodextrin is 0.5% (w / v), the concentration of mannitol is 6% (w / v), the concentration of sucrose is 10% (w / v), and the concentration of polyvinyl alcohol is 0.1% (w / v).

[0072] The raw materials used in this preparation example are the same as those in Preparation Example 3.

[0073] Example 1: Preparation of freeze-dried powder

[0074] 12.5ul of 2X qPCR Mix prepared in Examples 3, 4 and 5 was pipetted into 8-tube PCR tubes and lyophilized according to the procedure in Table 1 below.

[0075] Table 1. Vacuum freeze drying program

[0076] program temperature Temperature change rate Duration Vacuum degreePa Pre-freezing -50℃ 1.0℃ / min 180 / One-time drying -40℃ 0.5℃ / min 360 10 Secondary drying -20℃ 0.5℃ / min 360 10 Analytical drying 20℃ 0.5℃ / min 240 10

[0077] The inventors evaluated the morphology of the freeze-dried product, mainly judging whether the morphology was complete and not easy to break, and whether the bottom was shrunk. Figure 1-3 It can be seen from the results that the qPCR amplification reaction composition prepared by the present invention has good lyophilizability.

[0078] Example 2: Comparison of direct amplification and amplification after nucleic acid extraction of genomic DNA from oral swabs

[0079] Four subjects took oral swabs on the same day, which were named S1 to S4. The oral swabs were scraped with a flocked swab and dispersed in 1 mL of physiological saline. After vortexing for 1 minute, 200 μL was drawn into a centrifuge tube and DNA was extracted using QIAamp DNA MicroKit (Qiagen, Cat#56304) as Group A; 200 μL was drawn into a centrifuge tube again and an equal volume of the nucleic acid release agent of Preparation Example 1 was added as Group B. 200 μL was drawn into a centrifuge tube again and an equal volume of the nucleic acid release agent of Preparation Example 2 was added as Group C. The process of Group A is as follows: the nucleic acid extraction reagent is QIAamp DNA Micro Kit (Qiagen, Cat#56304), 200 μL of the sample is used for nucleic acid extraction, and finally 200 μL of Buffer AE is used for elution to obtain the DNA extraction product. The processes of Groups B and C are as follows: 200 μL is added with an equal volume of nucleic acid release agent, and vortexed at room temperature for 5 minutes to obtain a crude extract. The nucleic acid extraction products of group A and the crude extracts of groups B and C were tested using the freeze-dried powder (i.e., qPCR freeze-dried powder) obtained by freeze-drying the qPCR amplification reaction composition provided in preparation examples 3, 4, or 5 prepared in Example 1. The RNase P internal reference gene (a known and common housekeeping gene from the human body) was selected for amplification, and the ABI 7500Fast fluorescent quantitative PCR instrument (Applied Biosystems) was selected for quantification, and the sample loading amount was 5 μL template for testing.

[0080] The primers and probes used in this example are shown in Table 1 below.

[0081] Table 1

[0082]

[0083]

[0084] The qPCR reaction system of this example is shown in Table 2 below.

[0085] Table 2

[0086]

[0087] The amplification program used in this example is shown in Table 3 below.

[0088] Table 3

[0089]

[0090] The test scheme for each group is shown in Table 4 below.

[0091] Table 4

[0092]

[0093]

[0094] The test results (detection Ct values) of each group are shown in Table 5 below.

[0095] Table 5

[0096]

[0097] The results are shown in Table 5. As can be seen from Table 5, for Groups 1, 2 and 3, the results of the freeze-dried powder obtained after the nucleic acid extraction and the crude extraction were tested using the freeze-dried powder obtained by freeze-drying the qPCR amplification reaction composition provided in Preparation Example 3 prepared in Example 1, and the results were similar. The results of Groups 4, 5 and 6 were also the same. The data of Groups 7, 8 and 9 showed that the Ct values ​​of Groups 8 and 9 were delayed compared with the Ct value of Group 7, which proved that the freeze-dried powder obtained after freeze-drying the qPCR amplification reaction composition provided in Preparation Example 5 prepared in Example 1 could not tolerate the PCR inhibitors in the crude extraction product well.

[0098] Example 3: Sample tolerance of crude extract

[0099] The inventors further tested the tolerance of the crude extracted products. The inventors used the crude extract of sample S1 in Example 2 treated by group B, and tested the qPCR freeze-dried powder provided by Preparation Example 3 prepared in Example 1 and the qPCR freeze-dried powder provided by Preparation Example 5 prepared in Example 1. The sample template volumes were 5ul and 23.5ul, respectively. When 23.5ul of the template was added, no deionized water was needed.

[0100] like Figure 4 As described, when the qPCR lyophilized powder provided in Preparation Example 3 prepared in Example 1 was used, when the template was added as 5ul, the Ct value was 26.8, and when the template was added as 23.5ul, the Ct value was 25.0, and it was found from the amplification curve that the signal value did not decrease. When the qPCR lyophilized powder provided in Preparation Example 5 in Example 1 was used, when the template was added as 5ul, the Ct value was 27.2, and when the template was added as 23.5ul, there was no amplification signal, which indicates that when the crude lysis solution method (using the crude extraction product) is used, the qPCR lyophilized powder provided in Preparation Example 5 in Example 1 cannot tolerate the crude lysis method, which verifies that BSA and polyacrylic acid as PCR enhancers improve the direct amplification performance of the entire amplification system, which is conducive to the convenient application of molecular POCT.

[0101] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and modifications may be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. The various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A qPCR amplification reaction composition, characterized in that: The composition comprises: Tris-HCl, ammonium sulfate, KCl, dNTPs, BSA, MgCl2, hot start DNA polymerase, polyacrylic acid and lyophilization protectant, wherein the lyophilization protectant comprises cyclodextrin, mannitol, sucrose and polyvinyl alcohol.

2. The composition according to claim 1, characterized in that The composition comprises 20-100 mM Tris-HCl, 10-50 mM ammonium sulfate, 20-100 mM KCl, 100-500 μM dNTPs, 100-500 μg / mL BSA, 1-5 mM MgCl2, 0.1-0.2 U / μL hot start DNA polymerase, 0.1-10 mM polyacrylic acid, 0.25-0.5% (w / v) cyclodextrin, 6-10% (w / v) mannitol, 6-12% (w / v) sucrose and 0.025-0.4% (w / v) polyvinyl alcohol.

3. The composition according to claim 1 or 2, characterized in that The hot start DNA polymerase is a hot start Taq DNA polymerase; Preferably, the hot-start DNA polymerase is an antibody-modified, ligand-modified or chemically-modified hot-start Taq DNA polymerase; More preferably, the hot-start DNA polymerase is an antibody-modified hot-start Taq DNA polymerase.

4. The composition according to any one of claims 1 to 3, characterized in that: The molecular weight of the polyacrylic acid is 2,000 to 500,000; preferably, the molecular weight of the polyacrylic acid is 50,000 to 500,000.

5. The composition according to any one of claims 1 to 4, characterized in that: The cyclodextrin is γ-cyclodextrin; and / or The molecular weight of the polyvinyl alcohol is 5000-20000.

6. A nucleic acid releasing agent for use in conjunction with the composition according to any one of claims 1 to 5, characterized in that: The nucleic acid release agent comprises potassium hydroxide, dioxane, sodium dodecyl sulfate, Brij 58, lithium chloride and water.

7. The nucleic acid releasing agent according to claim 6, characterized in that: The nucleic acid release agent comprises 5-50 mM potassium hydroxide, 5-20% (w / v) dioxane, 0.01-0.5% (w / v) sodium dodecyl sulfate, 0.05-1% (w / v) Brij 58, 5-100 mM lithium chloride and the balance water; Preferably, the nucleic acid release agent comprises 10-25 mM potassium hydroxide, 5-10% (w / v) dioxane, 0.01-0.25% (w / v) sodium dodecyl sulfate, 0.1-0.5% (w / v) Brij 58, 10-25 mM lithium chloride and the balance water; More preferably, the nucleic acid releasing agent comprises 10 mM potassium hydroxide, 5% (w / v) dioxane, 0.05% (w / v) sodium dodecyl sulfate, 0.2% (w / v) Brij 58, 25 mM lithium chloride and the balance water.

8. A nucleic acid detection kit, characterized in that: The kit comprises a first reagent and / or a second reagent, wherein the first reagent is a reagent for preparing the composition according to any one of claims 1 to 5; and the second reagent is a reagent for preparing the nucleic acid releasing agent according to claim 6 or 7.

9. A method for quantitative or qualitative analysis of DNA in a biological sample, characterized in that: The method is performed using the composition according to any one of claims 1 to 5, the nucleic acid releasing agent according to claim 6 or 7, and / or the kit according to claim 8.

10. The method according to claim 9, characterized in that The method comprises the following steps: (1) adding a nucleic acid releasing agent to a biological sample to release nucleic acid from the biological sample, thereby preparing a crude sample; (2) adding a qPCR amplification reaction composition to the crude sample, and then adding primers and probes of corresponding detection targets to obtain a reaction working solution; (3) Using the reaction working solution to perform quantitative or qualitative analysis on the DNA in the biological sample through qPCR.

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