Nucleic acid detection method

Abstract

The present disclosure belongs to the field of molecular biological detection, and specifically relates to a nucleic acid detection method, more specifically relating to a method for rapid nucleic acid extraction and detection. The present disclosure provides a nucleic acid detection method, comprising the following steps: S1, mixing a sample to be tested with a direct amplification preservation solution and ultrasonicating, to obtain a sample nucleic acid solution; S2, adding the sample nucleic acid solution obtained in S1 to an amplification reaction solution, to obtain a mixed solution; S3, using the mixed solution to perform an amplification reaction and detect nucleic acid. The direct amplification preservation solution comprises: a protective agent, a nuclease inhibitor, a surfactant, a buffer system and an antibacterial agent. Using the method of the present disclosure, the entire detection process is simple, the process is less susceptible to influence, and the detection time is short.

Description

A nucleic acid detection method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202311674732.4, application date December 7, 2023, invention name “A nucleic acid detection method”, and application number 202311677300.9, application date December 7, 2023, invention name “A sample preservation composition”, and claims the priority of these Chinese patent applications. The entire contents of these Chinese patent applications are hereby incorporated into this application by introduction. Technical Field

[0003] The present disclosure belongs to the field of molecular biological detection, and specifically relates to a nucleic acid detection method and a sample preservation composition used therein, and more specifically to a method for rapid extraction and detection of nucleic acids and a sample preservation composition for direct amplification without extraction used therein. Background Art

[0004] Detecting pathogens or disease markers through molecular diagnostic means is one of the important auxiliary methods for clinical diagnosis. At present, molecular diagnosis usually includes three parts: sample collection, storage and transportation, sample extraction, and nucleic acid testing. However, molecular diagnosis has certain requirements for the purity and integrity of nucleic acid samples. When the quality of nucleic acid cannot meet the requirements, it will affect the accuracy and detection limit of subsequent test results. The sample collection and transportation process, extraction method, and efficiency will all have an impact on nucleic acid. In addition, general nucleic acid extraction has relatively high requirements for instruments and personnel, and the entire extraction process takes a long time. From sample collection to test results, the entire process is cumbersome and time-consuming, taking more than 4 hours. Existing nucleic acid tests have problems such as complex entire detection process, easy contamination during operation, and long detection time, which does not meet the increasing demand for accurate and rapid detection.

[0005] Currently, the commonly used sample preservation methods mainly use Hank's solution or phosphate buffered saline for preservation. Their disadvantages are: 1. A subsequent nucleic acid extraction step is required, which is time-consuming and prone to contamination; 2. The preservation effect of various preservation solutions is of poor quality, and RNA is extremely easy to degrade; 3. After sampling, the samples need to be stored at low temperatures, and the storage time is short.

[0006] Conventional extraction-free sample preservation solutions contain a high concentration of lysing components after sampling, which have a strong effect and significantly damage the structure of nucleic acids. After a short storage period, some nucleic acids degrade, reducing detection effectiveness and leading to the risk of false negatives. Furthermore, after crude extraction using conventional extraction-free sample preservation solutions, the absence of a purification stage leads to higher levels of inhibitors, significantly impacting subsequent nucleic acid testing.

[0007] Therefore, there is an urgent need to develop a solution that can rapidly inactivate infectious viruses, eliminate the need for nucleic acid extraction from subsequent samples, reduce steps in the overall process, and not affect subsequent nucleic acid, especially RNA, detection. It also allows for long-term storage at room temperature. A cell preservation solution that can be directly tested, as well as a nucleic acid detection method, can shorten overall detection time.

[0008] Summary of the Invention

[0009] In view of this, in a first aspect, the present disclosure provides a nucleic acid detection method, comprising the following steps:

[0010] S1. Mix the sample to be tested with the direct amplification storage solution and sonicate to obtain a sample nucleic acid solution;

[0011] S2, adding the sample nucleic acid solution obtained in S1 to the nucleic acid amplification reaction solution to obtain a mixed solution;

[0012] S3, using the mixed solution to perform an amplification reaction and detect nucleic acid;

[0013] The direct amplification preservation solution includes: a protective agent, a nuclease inhibitor, a surfactant, a buffer system, and an antibacterial agent.

[0014] The term "direct amplification" as used herein refers to the release and amplification of nucleic acids from a sample without the need for sample extraction or purification. This refers to direct nucleic acid amplification testing of a sample without the need for sample extraction or purification.

[0015] The direct amplification preservation fluid used in this application has the functions of sample preservation, sample inactivation and nucleic acid release. Combined with ultrasonic treatment, it accelerates the sample extraction process and simultaneously fully mixes it, which can shorten the sample nucleic acid release and extraction time to within 3 minutes; the sample nucleic acid solution after ultrasonic treatment is directly added to the amplification reaction solution for amplification detection, which greatly shortens the detection time. The entire process can be completed within 1 hour, significantly improving the detection efficiency and meeting the needs of rapid nucleic acid detection.

[0016] In some specific embodiments, in step S1, the sample to be tested is mixed with the direct amplification storage solution in a container with pipetting capabilities; the sample nucleic acid solution is then added directly to the amplification reaction premix via the container. This avoids the traditional method of pipetting, which requires pipettes of varying sizes and tips, simplifying the process and preventing contamination during aspiration, pipetting, and pipetting, thus saving costs.

[0017] Furthermore, the direct amplification storage solution is loaded into a container with a pipetting function, and the sample to be tested is added to the direct amplification storage solution for mixing and ultrasonication. For example, the container can be a pipette, a centrifuge tube, etc.

[0018] In some specific embodiments, the pipette is a vesicle pipette. Furthermore, the vesicle pipette has a quantitative pipetting function.

[0019] In a specific embodiment, as shown in FIG1 , the vesicle pipette includes a tube body, a dripper, a cover body and an extrusion body, the dripper is detachably arranged at the first end of the tube body, the cover body is detachably mounted on the outer peripheral side of the dripper and is used to seal the liquid outlet on the dripper, the extrusion body is arranged at the second end of the tube body and is formed with a flexible vesicle portion, and the interior of the flexible vesicle portion is formed with an extrusion cavity for communicating with the accommodating cavity. Each time the flexible vesicle portion is pressed to deform it into a planar state, the space inside the reagent tube can only be reduced by a preset size each time, and accordingly, the liquid inside the reagent tube can only be squeezed out of a preset volume. The pipette has a simple structure, and when using it for pipetting, it is not easy to produce pipetting deviation due to fatigue operation, and can better ensure the accuracy of quantitative pipetting and improve the accuracy of pipetting.

[0020] In this way, the vesicle pipette can be used to load direct amplification storage solution, add the sample to be tested for ultrasonic treatment, and achieve quantitative pipetting, while avoiding sample contamination caused by the use of additional pipettes during sample transfer.

[0021] In some specific embodiments, the protective agent includes sucrose, choline chloride, mannitol, polyvinyl pyrrolidone, and glycerol.

[0022] In some specific embodiments, in the direct expansion preservation solution, the sucrose concentration is 0.15% to 1.5% (w / v), the choline chloride concentration is 1 mM to 10 mM, the polyvinyl pyrrolidone concentration is 1 mM to 10 mM, the mannitol concentration is 0.045% to 0.45% (v / v), and the glycerol concentration is 0.05% to 0.5% (v / v).

[0023] In the definitions of the present disclosure, the term "w / v" represents mass volume concentration, and the term "v / v" represents volume fraction, wherein w is measured in g and v is measured in mL. In some specific embodiments, the concentration of the nuclease inhibitor is 0.01% to 0.1% (v / v), for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0024] The nuclease inhibitor is selected from at least one of RNasin, diethyl pyrophosphate, or guanidine isothiocyanate. In a specific embodiment, the nuclease inhibitor is RNasin.

[0025] In some specific embodiments, the concentration of the surfactant is 0.01% to 0.5% (v / v), such as 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The surfactant is selected from at least one of Triton X-100, Tween 20, sodium lauryl ether sulfate, or sodium lauryl sulfate. In a specific embodiment, the surfactant is Triton X-100 or Tween 20.

[0026] In some specific embodiments, the concentration of the antibacterial agent is 0.01% to 0.1% (v / v), and the concentration of the buffer system is 80mM to 120mM. The antibacterial agent is Proclin 300 and / or potassium sorbate. In a specific embodiment, the antibacterial agent is Proclin 300.

[0027] In some specific embodiments, the buffer system is selected from at least one of Tris-HCl buffer, phosphate buffer, and TE buffer. In a specific embodiment, the buffer system is Tris-HCl buffer. Furthermore, the concentration of the buffer system is 80 mM to 120 mM.

[0028] In some specific embodiments, the pH value of the direct expansion preservation solution is 11 to 13. Preferably, the pH value of the direct expansion preservation solution is about 12.

[0029] The use of the direct amplification preservation solution can improve the detection effect after the direct amplification method of the present disclosure.

[0030] In some specific embodiments, the ultrasound frequency in step S1 is 30kHz to 80kHz, and the duration is 30s to 150s. Preferably, the ultrasound frequency is 40kHz, and the duration is 120s.

[0031] In some specific embodiments, the ultrasonic treatment comprises applying ultrasound to the side or bottom of the container. Preferably, ultrasound is applied to the side of the container.

[0032] In some specific embodiments, the amplification reaction solution in step S2 is a PCR reaction solution, which includes primers, probes, Mg 2+ , dNTPs, Taq enzyme, MMLV enzyme (reverse transcriptase), or one or more of PCR buffer.

[0033] Furthermore, the PCR reaction solution includes Mg 2+4mM-8mM, dNTPs 0.2mM-0.6mM, Taq enzyme 5U-30U, primers 200nM-600nM, probe 100nM-500nM, RNasin 1U-20U. In some embodiments, the PCR reaction solution also includes reverse transcriptase 5U-30U.

[0034] In some specific embodiments, the PCR reaction solution is a fully premixed reaction solution, and the pH value of the PCR reaction solution is 8-9.

[0035] Preferably, the PCR reaction solution includes Mg 2+ 4mM, dNTPs 0.4mM, MMLV 5U, Taq enzyme 15U, primers 200nM, probe 100nM, RNasin 5U. This PCR reaction solution provides the best storage results.

[0036] In some specific embodiments, the detection in step S3 is PCR, and the PCR time does not exceed 60 minutes. Furthermore, the PCR time is 30-60 minutes.

[0037] Furthermore, the PCR procedure is as follows:

[0038] The method disclosed herein can quickly and fully process samples, release nucleic acids, easily and accurately transfer sample nucleic acids, and efficiently and quickly perform amplification detection, thereby simplifying the entire detection process, reducing contamination, shortening detection time, and ensuring the accuracy of detection results.

[0039] In yet another aspect, the present disclosure provides a sample preservation composition, comprising: a protective agent, wherein the protective agent comprises sucrose, choline chloride, mannitol, polyvinyl pyrrolidone, and glycerol.

[0040] The applicant creatively discovered that the use of the sample preservation composition can enable the sample to be stored at room temperature for a long time, and the simple composition does not affect its performance, and its detection sensitivity and stability can be well maintained.

[0041] Furthermore, the concentrations of the sucrose, choline chloride, mannitol, polyvinyl pyrrolidone, and glycerol in the protective agent are: sucrose concentration is 15% (w / v), choline chloride concentration is 0.1M, polyvinyl pyrrolidone concentration is 0.1M, mannitol concentration is 4.5% (v / v), and glycerol concentration is 5% (v / v).

[0042] After the protective agent is prepared into a sample preservation composition, in the sample preservation composition, the concentration of sucrose is 0.15% to 1.5% (w / v), the concentration of choline chloride is 1 mM to 10 mM, the concentration of polyvinyl pyrrolidone is 1 mM to 10 mM, the concentration of mannitol is 0.045% to 0.45% (v / v), and the concentration of glycerol is 0.05% to 0.5% (v / v).

[0043] In the definitions of the present disclosure, the term “w / v” represents mass volume concentration, and the term “v / v” represents volume fraction, wherein w is measured in g and v is measured in mL.

[0044] Furthermore, the sample preservation composition further comprises one or more of a nuclease inhibitor, a surfactant, a buffer system, and an antibacterial agent.

[0045] In a specific embodiment, the sample preservation composition includes a protective agent, a nuclease inhibitor, a surfactant, a buffer system, and an antibacterial agent.

[0046] In some specific embodiments, the concentration of the protective agent is 1% to 10% (v / v), for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. Preferably, the concentration of the protective agent is 1% to 5% (v / v), more preferably, the concentration of the protective agent is 1%.

[0047] In some specific embodiments, the concentration of the nuclease inhibitor is 0.01% to 0.1% (v / v), for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. The nuclease inhibitor can be one or more of RNasin, diethyl pyrophosphate, and guanidine isothiocyanate. In a specific embodiment, the nuclease inhibitor is RNasin.

[0048] In some specific embodiments, the concentration of the surfactant is 0.01% to 0.5% (v / v), for example, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The surfactant can be one or more of Triton X-100, Tween 20, sodium lauryl ether sulfate, and sodium lauryl sulfate. In a specific embodiment, the surfactant is Triton X-100 or Tween 20.

[0049] In some specific embodiments, the concentration of the antibacterial agent is 0.01% to 0.1% (v / v), for example, 0.01%, 0.02%, 0.05%, 0.07%, 0.08%, 0.09%, or 0.1%. The antibacterial agent can be one or more of Proclin 300, potassium sorbate, and the like. In a specific embodiment, the antibacterial agent is Proclin 300.

[0050] In some specific embodiments, the buffer system can be one or more of Tris-HCl buffer, phosphate buffer, TE, etc. In a specific embodiment, the buffer system is a Tris-HCl buffer with a pH of 8.0 to 8.5. Furthermore, the concentration of the buffer system is 80 mM to 120 mM.

[0051] The sample preservation composition disclosed herein can be used for detection without the need for lysis and nucleic acid extraction steps, and can enable samples to be stored at room temperature for a long time. The simple composition does not affect its performance, and its detection sensitivity and stability can be well maintained.

[0052] In yet another aspect, the present disclosure provides a PCR reaction composition comprising the sample preservation composition as described above.

[0053] Furthermore, the PCR reaction composition also includes a sample and a PCR reaction reagent.

[0054] Furthermore, the sample is a biological sample containing nucleic acid, preferably a biological sample containing RNA, such as a virus whose genetic material is RNA.

[0055] Furthermore, the PCR reaction reagents also include primers and probes for PCR.

[0056] Furthermore, the PCR reaction reagents also include one or more of dNTP, reverse transcriptase, DNA polymerase, and PCR buffer.

[0057] In the definition of the present disclosure, the term "PCR reaction composition" refers to a mixture capable of detecting nucleic acids using PCR.

[0058] In a specific embodiment, the PCR reaction composition includes the sample preservation composition as described above, primers and probes, dNTPs, reverse transcriptase, DNA polymerase, and PCR buffer.

[0059] In another aspect, the present disclosure provides an extraction-free nucleic acid detection kit, which includes the sample preservation composition described above. Furthermore, the nucleic acid detection kit is a PCR detection kit, which also includes the PCR reaction composition described above.

[0060] In another aspect, the present disclosure provides a use of the above composition in preparing an extraction-free nucleic acid detection kit. Furthermore, the nucleic acid detection kit is a PCR detection kit.

[0061] In yet another aspect, the present disclosure provides a method for extraction-free PCR detection, comprising the steps of mixing a sample with the sample preservation composition and the PCR reaction reagent.

[0062] The PCR reaction reagents include, for example, dNTPs, reverse transcriptase, DNA polymerase, and PCR buffer, and may further include primers and probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a schematic diagram of a vesicle pipette;

[0064] Figure 2 shows the amplification curves of samples treated with different compositions and methods (AG is magnetic bead method, direct amplification storage solution 1 to 6, in that order);

[0065] FIG3 is a graph showing the test results of sample 1 treated with different treatment methods;

[0066] FIG4 is a graph showing the test results of sample 2 treated with different treatment methods;

[0067] FIG5 is a graph showing the results of detecting influenza A virus by treating samples with the disclosed method and the magnetic bead method, respectively;

[0068] FIG6 is a graph showing the results of detecting influenza B virus by treating samples with the disclosed method and the magnetic bead method, respectively;

[0069] FIG7 is a graph showing the results of detecting the novel coronavirus using the disclosed method and the magnetic bead method, respectively, for treating samples;

[0070] FIG8 is a graph showing the results of detecting Mycoplasma pneumoniae using the disclosed method and the magnetic bead method to process samples, respectively. DETAILED DESCRIPTION

[0071] The present disclosure will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present disclosure will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present disclosure, rather than to limit the present disclosure.

[0072] Currently, the main methods used for nucleic acid extraction from respiratory pathogens include centrifugal column nucleic acid extraction, magnetic bead method, and alkaline lysis method. Among them, the centrifugal column nucleic acid extraction method requires specific adsorption and repeated washing of impurities, so it takes up to 1 to 2 hours. The magnetic bead method for nucleic acid extraction has become the mainstream method in the industry due to its high degree of automation, large sample throughput, good nucleic acid concentration and purity, and high safety. However, the extraction process is easily contaminated and takes 30 minutes to 2 hours. The alkaline lysis method is a relatively simple extraction method that uses a strong base to lyse the sample. The nucleic acid after sample lysis in this method is extremely easy to degrade, has a very short storage time, and cannot be retested. It takes about 10 minutes. Traditional PCR reaction reagents are usually separately packaged as primer probes, DNA polymerase, RNA polymerase, PCR buffer and other components. They need to be prepared before use, which is complicated to operate. At the same time, the traditional PCR amplification procedure is: 50℃, 30min; 95℃, 5min; (95℃, 1min; 60℃, 30sec; 72℃, 30sec) 45 cycles, and the PCR detection time takes about 2h.

[0073] In order to meet the needs of rapid clinical testing, the inventors of this application have developed a new nucleic acid detection method. Through the detection scheme of direct expansion ultrasound + pipette + premixing + rapid amplification, the detection time can be greatly shortened, the detection process can be shortened to within 1 hour, the contamination caused by sample nucleic acid transfer can be reduced, the detection efficiency can be improved, and the accuracy and reliability of the test results can be guaranteed.

[0074] Example 1

[0075] Prepare direct amplification preservation solution

[0076] Step 1: Prepare the protective agent.

[0077] Protective agent 1: In 50 mL of sterile water, add 15 g of sucrose, 1.3963 g of choline chloride, and 1.1114 g of polyvinylpyrrolidone. After dissolution, add 4.5 mL of mannitol and 5 mL of glycerol, and dilute to 100 mL with sterile water.

[0078] Protective agent 2: In 50 mL of sterile water, add 15 g of sucrose, 1.3963 g of choline chloride, and 3.423 g of trehalose. After dissolution, add 4.5 mL of mannitol and 5 mL of glycerol, and dilute to 100 mL with sterile water.

[0079] Protective agent 3: In 50 mL of sterile water, add 15 g of sucrose and 1.1114 g of polyvinylpyrrolidone. After dissolution, add 4.5 mL of mannitol, 5 mL of glycerol, and 1 mL of dimethyl sulfoxide. Dose the mixture up to 100 mL with sterile water.

[0080] Protective agent 4: Add 15 g of sucrose to 50 mL of sterile water. After dissolution, add 4.5 mL of mannitol and 5 mL of glycerol, and dilute to 100 mL with sterile water.

[0081] Step 2: Prepare direct expansion preservation solution.

[0082] Use protective agent 1 to prepare direct amplification preservation solution 1: add 1 mL of protective agent 1, 5 μL of Triton X-100, 5 μL of Tween 20, 10 μL of RNasin, 10 μL of Proclin 300, and 1.2608 g of Tris-HCl solid to 50 mL of sterile water, adjust the pH, and make up to 100 mL with sterile water to prepare direct amplification preservation solution 1.

[0083] Use protective agent 1 to prepare direct amplification preservation solution 2: add 5 mL of protective agent 1, 50 μL of Triton X-100, 50 μL of Tween 20, 50 μL of RNasin, 50 μL of Proclin 300, and 1.576 g of Tris-HCl solid to 50 mL of sterile water, adjust the pH, and make up to 100 mL with sterile water to prepare direct amplification preservation solution 2.

[0084] Use protective agent 1 to prepare direct amplification preservation solution 3: add 10 mL of protective agent 1, 250 μL of Triton X-100, 250 μL of Tween 20, 100 μL of RNasin and 100 μL of Proclin 300, and 1.8912 g of Tris-HCl solid to 50 mL of sterile water, adjust the pH, and make up to 100 mL with sterile water to prepare direct amplification preservation solution 2.

[0085] Use protective agent 2-4 to prepare direct amplification preservation solution 4-6: add 1 mL of protective agent 2-4 to each of three 50 mL sterile water, then add 5 μL of Triton X-100, 5 μL of Tween 20, 10 μL of RNasin and 10 μL of Proclin 300, and 1.2608 g of Tris-HCl solid, adjust the pH, and make up to 100 mL with sterile water to prepare direct amplification preservation solution 4-6.

[0086] Use the above-prepared 6 direct expansion preservation solutions 1 to 6 to 1*10 5 The influenza A virus sample with 10 copies / mL was chemically treated and diluted to 1*10 4 copies / mL, followed by sonication (40 kHz, 120 s).

[0087] In addition, nucleic acid extraction and purification reagent S10015 from Shengxiang Biotechnology Co., Ltd. was used to extract nucleic acid from influenza A virus samples using magnetic beads.

[0088] Testing was performed using the Influenza A Virus Universal Nucleic Acid Detection Kit from Shengxiang Biotechnology Co., Ltd. The reaction system consisted of 20 μL of treated sample and 30 μL of PCR reaction solution. Testing was performed on a SLAN-96P fully automated medical PCR analysis system. The PCR protocol is shown in Table 1 below:

[0089] Table 1

[0090] The results are shown in FIG2 , where A is the amplification result of the sample extracted by the magnetic bead method, and BG are the amplification results of the samples treated with sample treatment solutions 1-6, respectively.

[0091] As shown in Figure 2, all six sample treatment solutions can be directly amplified without the need for additional nucleic acid extraction, meeting testing requirements. However, relatively speaking, sample treatment solutions 1-3 exhibited higher Ct values, greater fluorescence increments, and improved nucleic acid release, with comparable results compared to the magnetic bead extraction control group, demonstrating consistent results with magnetic bead extraction.

[0092] Example 2

[0093] (1) Use a pharyngeal swab to gently scrape epidermal mucosal cells from the surface of the tongue or buccal mucosa (cheeks on both sides of the mouth), and soak the swab in a vesicle dropper (as shown in Figure 1) containing 2 mL of direct expansion preservation solution 1 in Example 1. Break off the swab head, cover the tube tightly, and number them 1-13 respectively.

[0094] (2) Dilute the influenza A virus simulation sample solution to 1*10 5 copies / mL (sample 1), and added to the treatment solutions No. 1-13 in (1) above to obtain samples No. 1-13.

[0095] (3) Samples 1-13 in (2) were placed in an ultrasonic instrument for ultrasonic treatment. The ultrasonic treatment conditions are shown in Table 2. After ultrasonic treatment, the samples were placed on ice for cooling for 1 minute.

[0096] Table 2

[0097] (4) Samples 1-13 were tested using the universal nucleic acid detection kit for influenza A virus produced by Shengxiang Biotechnology Co., Ltd. A dropper (20 μL) of sample was placed into a PCR tube containing 30 μL of PCR premixed reaction solution after ultrasonication. The tube cap was tightly closed and the test was performed on a SLAN-96P fully automatic medical PCR analysis system. The test procedure is shown in Table 2. The test results are shown in Table 3.

[0098] In addition, the simulated samples of the novel coronavirus (sample 2) and Mycoplasma pneumoniae (sample 3) were processed respectively with reference to the above (1) to (4), and the novel coronavirus nucleic acid detection kit and Mycoplasma pneumoniae nucleic acid detection kit of Shengxiang Biotechnology Co., Ltd. were used for detection. The test results are shown in Table 3.

[0099] Table 3

[0100] As shown in Table 3, excellent detection results were achieved when the ultrasonic treatment conditions ranged from 30kHz to 80kHz and lasted from 30s to 150s. Compared to sample 7 without ultrasonic treatment, its Ct value was up to six points higher, and its detection results were also superior to those of the control samples using the remaining ultrasonic treatment conditions.

[0101] Furthermore, a comparison was performed on Samples 1 and 2 using ultrasound alone, without using Direct Amplification Preservation Solution 1. The results are shown in Table 4 and Figures 3-4. As can be seen from Table 4, the detection effect of using both methods to treat the samples was superior to that of ultrasound alone.

[0102] Table 4

[0103] Example 3

[0104] Referring to the method of Example 2, the influenza A virus sample was chemically treated using the direct amplification preservation solution 1 of Example 1, followed by ultrasonic treatment (40 kHz, 120 s). The treated samples were then mixed with the PCR reaction solutions shown in Table 5 below (after storage at -20°C for 6 months), stored at -20°C for the treatment time shown in Table 6, and finally detected on a SLAN-96P fully automatic medical PCR analysis system.

[0105] Table 5

[0106] The results are shown in Table 6. As can be seen from Table 6, PCR reaction solution 1 has the best preservation effect.

[0107] Table 6

[0108] Example 4: Detection of different pathogens using the disclosed method and magnetic bead method

[0109] The nucleic acid detection method disclosed in the present invention was used to detect samples (influenza A virus, influenza B virus, new coronavirus, and Mycoplasma pneumoniae) respectively using the magnetic bead method. The test results are shown in Figures 5 to 8. The results show that the effect of the disclosed method is significant and is equivalent to the effect of nucleic acid detection after nucleic acid extraction using the magnetic bead method. The detection reagents are from the new coronavirus 2019-nCoV, influenza A virus, influenza B virus nucleic acid detection kit, Mycoplasma pneumoniae kit, etc. produced by Shengxiang Biotechnology Co., Ltd.

[0110] Furthermore, the detection time of the two methods was compared, and the results are shown in Table 6 (taking influenza A virus samples as an example). Using the method disclosed herein, the detection process can be shortened by at least 1 to 2 hours, making the entire detection process more rapid.

[0111] Table 6

[0112] Example 5: Temperature stability (preservation) effect of the sample preservation composition disclosed herein

[0113] (1) Prepare sample preservation solutions No. 1, 2, and 3 according to the protective agent 1 of Example 1. Prepare control solutions No. 4, 5, and 6 using protective agents 2 to 4 of Example 1.

[0114] Use a throat swab to gently scrape the epidermal mucosal cells from the human tongue or buccal mucosa (cheeks on both sides of the mouth), soak the swab in 2 mL of sample preservation solution 1 to 6 respectively, close the tube cap tightly, mix upside down for 90 seconds, and then centrifuge instantly.

[0115] (2) Add 100 μL of 1*10 5 Influenza A virus at a concentration of 100 copies / mL was added to the six virus preservation solutions of (1) and divided into three groups, respectively, and stored at 37°C, 4°C, and -80°C, respectively. Samples were taken at set days and ultrasonicated for 120 seconds at a frequency of 40 kHz, followed by testing. The specific test arrangements are shown in Table 7 below.

[0116] Table 7. Temperature setting table for each group

[0117] (4) Saving effect detection:

[0118] According to the experimental arrangement in the table above, samples were collected at different time periods for testing. Testing was performed according to the instructions for the Universal Nucleic Acid Detection Kit for Influenza A Virus from Shengxiang Biotechnology Co., Ltd. The test results at -80°C are shown in Table 8. As can be seen in Table 2, kits 1 to 3 performed the best. After 14 days of storage at -80°C, their detection performance remained the same as on day 0. Kits 4 and 5 performed second best, with their CT values ​​fluctuating around 1, while kit 6 performed the worst.

[0119] Table 8. Comparison of Ct values ​​of samples stored at -80℃ with different preservation solutions

[0120] The results of the 4°C test are shown in Table 3. Table 9 shows that samples 1, 2, and 3 performed best, maintaining the same performance after 7 days of storage at 4°C as on day 0. Samples 4 and 5 performed second best, with a lag of more than 3 Ct values ​​after 7 days. Sample 6 performed the worst, becoming undetectable after 7 days, indicating poor storage.

[0121] Table 9. Comparison of Ct values ​​of samples stored at low temperature (4°C) using different preservation solutions

[0122] The test results at 37°C are shown in Table 4. As can be seen from Table 10, samples 1 to 3 performed best, with their performance remaining the same after 7 days of storage at 37°C compared to day 0. Samples 4 and 5 performed poorly, with their Ct values ​​falling behind by more than two units after 3 days of storage. Sample 6 performed the worst, becoming undetectable after 3 days, indicating poor storage.

[0123] Table 10. Comparison of Ct values ​​of samples stored at 37°C in different preservation solutions

Claims

1. A nucleic acid detection method comprising the following steps: S1. Mix the sample to be tested with the direct amplification storage solution and perform ultrasound to obtain a sample nucleic acid solution; S2, adding the sample nucleic acid solution obtained in S1 to the nucleic acid amplification reaction solution to obtain a mixed solution; S3, using the mixed solution to perform an amplification reaction and detect nucleic acid; The direct expansion preservation solution comprises: a protective agent, a nuclease inhibitor, a surfactant, a buffer system, and an antibacterial agent.

2. The nucleic acid detection method according to claim 1, wherein: The protective agent includes sucrose, choline chloride, mannitol, polyvinyl pyrrolidone and glycerol.

3. The nucleic acid detection method according to claim 2, wherein: In the direct expansion preservation solution, the mass / volume concentration of sucrose is 0.15% to 1.5%, the concentration of choline chloride is 1mM to 10mM, the concentration of polyvinyl pyrrolidone is 1mM to 10mM, the volume / volume concentration of mannitol is 0.045% to 0.45%, and the volume / volume concentration of glycerol is 0.05% to 0.5%.

4. The nucleic acid detection method according to claim 1, wherein: The volume / volume concentration of the nuclease inhibitor is 0.01% to 0.1%, the volume / volume concentration of the surfactant is 0.01% to 0.5%, the volume / volume concentration of the antibacterial agent is 0.01% to 0.1%, and the concentration of the buffer system is 80mM to 120mM.

5. The nucleic acid detection method according to claim 1, wherein: The frequency of the ultrasound is 30kHz to 80kHz, and the time is 30S to 150S.

6. The nucleic acid detection method according to claim 1, wherein: In the step S1, the sample to be tested is mixed with a direct amplification storage solution in a container with a pipetting function; The sample nucleic acid solution is directly added to the amplification reaction premix through the container.

7. The nucleic acid detection method according to claim 1, wherein: The amplification reaction solution in step S2 is a PCR reaction solution, which includes primers, probes, Mg 2+ , dNTPs, Taq enzyme, reverse transcriptase, or one or more of PCR buffer.

8. The nucleic acid detection method according to claim 6, wherein: The PCR reaction solution includes Mg 2+ 4mM-8mM, dNTPs 0.2mM-0.6mM, Taq enzyme 5U-30U, primers 200nM-600nM, probe 100nM-500nM, RNasin 1U-20U.

9. The nucleic acid detection method according to claim 1, wherein: The detection in step S3 is PCR, wherein the time of the PCR does not exceed 60 minutes.

10. The nucleic acid detection method according to claim 9, wherein: The PCR procedure is as follows:

11. A sample preservation composition, wherein: The sample preservation composition comprises a protective agent, and the protective agent comprises sucrose, choline chloride, mannitol, polyvinyl pyrrolidone and glycerol.

12. The sample preservation composition according to claim 11, wherein In the sample preservation composition, the mass / volume concentration of sucrose is 0.15% to 1.5%, the concentration of choline chloride is 1mM to 10mM, the concentration of polyvinyl pyrrolidone is 1mM to 10mM, the volume / volume concentration of mannitol is 0.045% to 0.45%, and the volume / volume concentration of glycerol is 0.05% to 0.5%.

13. The sample preservation composition according to claim 12, wherein The sample preservation composition further comprises one or more of a nuclease inhibitor, a surfactant, a buffer system, or an antibacterial agent.

14. The sample preservation composition according to claim 13, wherein The nuclease inhibitor is selected from at least one of RNasin, diethyl pyrophosphate, or guanidine isothiocyanate; the surfactant is selected from at least one of Triton X-100, Tween 20, sodium dodecyl alcohol ether sulfate, or sodium dodecyl sulfate; the buffer system is selected from at least one of Tris-HCl buffer, phosphate buffer, or TE buffer; the antibacterial agent is selected from at least one of Proclin300 or potassium sorbate.

15. The sample preservation composition according to claim 14, wherein The nuclease inhibitor is RNasin; the surfactant is Triton X-100 and Tween 20; the buffer system is Tris-HCL buffer; and the antibacterial agent is Proclin 300.

16. The sample preservation composition according to any one of claims 13 to 15, wherein The volume / volume concentration of the nuclease inhibitor is 0.01% to 0.1%, the volume / volume concentration of the surfactant is 0.01% to 0.5%, the volume / volume concentration of the antibacterial agent is 0.01% to 0.1%, and the concentration of the buffer system is 80mM to 120mM.

17. An extraction-free nucleic acid detection kit, comprising the sample preservation composition according to any one of claims 11 to 16.

18. The extraction-free nucleic acid detection kit according to claim 17, wherein: The detection kit is a PCR detection kit.

19. Use of the sample preservation composition according to any one of claims 11 to 16 in preparing an extraction-free nucleic acid detection kit.

20. The use according to claim 19, wherein The extraction-free nucleic acid detection kit is an extraction-free PCR detection kit.

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