A quick nucleic acid releasing agent without extraction and its application

Abstract

The application provides a non-extraction fast nucleic acid releasing agent and application thereof, and relates to the technical field of molecules, and comprises 10-100 mM of strong alkali, 0.1-2% of surfactant in mass / volume ratio, 10-100 mM of buffer, 0.1-2 mM of metal ion chelating agent, 10-100 mM of lysis enhancer and 50-100 mM of PCR enhancer I. The application can sufficiently lyse cells or virus particles, release nucleic acid substances, does not need to be inactivated separately, has strong lysis capacity, short lysis time, simple steps and can be adapted to fast detection procedures.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to an extraction-free rapid nucleic acid release agent and its application. Background Technology

[0002] In recent years, PCR technology has become one of the most widely used pathogen detection techniques, due to its advantages such as high sensitivity, high specificity, and short processing time. Both PCR and sequencing technologies require the extraction of nucleic acids from the sample before subsequent detection can be performed.

[0003] Nucleic acid extraction methods are diverse, mainly including boiling, centrifugation, and magnetic bead extraction. However, these methods suffer from drawbacks such as excessive time consumption and cumbersome operation. Therefore, the emergence of nucleic acid release agents is particularly important, as they offer advantages such as low cost, ease of operation, and short extraction time.

[0004] With the development of molecular diagnostic point-of-care testing (POCT), rapid detection, ease of operation, and high sensitivity are not only requirements but also standards. Rapidly obtaining test results, simplifying sample processing, and ensuring high sensitivity are both challenges and directions for molecular POCT. However, commercially available products, such as sample release agents, which are rapid sample pretreatment products, suffer from significantly reduced sensitivity due to the lack of a purification step. Furthermore, pairing them with rapid detection programs or devices further impacts sensitivity, and there are also issues such as limited pathogen target targeting.

[0005] Most commercially available nucleic acid release agents lack inactivation capabilities, posing certain risks to human health, and some even require heating or other methods to treat difficult-to-break pathogens. Therefore, existing sample release agents still suffer from problems such as long processing times, numerous steps, and incompatibility with rapid procedures. There is an urgent need to develop a nucleic acid release agent that is simple to operate, quick to use, highly safe, capable of handling multiple pathogens, and compatible with rapid procedures or equipment.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide an extraction-free rapid nucleic acid release agent to solve the technical problems of existing nucleic acid release agents that do not have inactivation function, and that are time-consuming, have many operation steps, and cannot be adapted to rapid procedures.

[0008] The second objective of this invention is to provide the above-mentioned extraction-free rapid nucleic acid release agent for use in nucleic acid release or preparation of products for nucleic acid release, nucleic acid amplification or preparation of products for nucleic acid amplification, or preparation of products for nucleic acid detection.

[0009] The third objective of this invention is to provide a method for releasing nucleic acids.

[0010] The fourth objective of this invention is to provide a nucleic acid PCR amplification method.

[0011] The fifth objective of this invention is to provide a PCR amplification kit.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0013] In a first aspect, the present invention provides an extraction-free rapid nucleic acid release agent, comprising 10-100 mM of a strong base, 0.1%-2% of a surfactant by mass / volume, 10-100 mM of a buffer solution, 0.1-2 mM of a metal ion chelating agent, 10-100 mM of a lysis enhancer, and 50-100 mM of PCR enhancer I.

[0014] Furthermore, the surfactant includes at least one of sodium dodecyl sulfate, Triton X1, or Tween 20, preferably sodium dodecyl sulfate;

[0015] Preferably, the pyrolysis enhancer includes at least one of guanidine isothiocyanate, guanidine hydrochloride, or sodium lauroyl glutamate, and is preferably guanidine isothiocyanate;

[0016] Preferably, the PCR enhancer I includes at least one of potassium chloride, sodium chloride, or sucrose, with sucrose being the most preferred.

[0017] Furthermore, when the surfactant is sodium dodecyl sulfate, the mass-to-volume ratio of the surfactant is 0.1% to 0.5%.

[0018] When the surfactant is Tween 20, the mass-volume ratio of the surfactant is 0.1% to 1%.

[0019] When the surfactant is Triton X1, the mass-volume ratio of the surfactant is 0.5% to 2%.

[0020] Preferably, when the pyrolysis enhancer is guanidine isothiocyanate, the concentration of the pyrolysis enhancer is 25-50 mM;

[0021] When the pyrolysis enhancer is guanidine hydrochloride, the concentration of the pyrolysis enhancer is 50–100 mM;

[0022] When the pyrolysis enhancer is sodium lauroyl glutamate, the concentration of the pyrolysis enhancer is 10–30 mM;

[0023] Preferably, when PCR enhancer I is potassium chloride, the concentration of PCR enhancer I is 70–100 mM;

[0024] When PCR enhancer I is sodium chloride, the concentration of PCR enhancer I is 10–40 mM;

[0025] When PCR enhancer I is sucrose, the concentration of PCR enhancer I is 50–80 mM.

[0026] Furthermore, it also includes PCR enhancer II at a mass-to-volume ratio of 1% to 2%;

[0027] Preferably, it further includes grinding beads with a mass-to-volume ratio of 0.1% to 0.5%, and more preferably grinding beads with a mass-to-volume ratio of 0.2% to 0.4%.

[0028] Preferably, it further includes a defoamer at a mass-volume ratio of 0.05% to 1%.

[0029] Preferably, the defoamer includes at least one of methyl silicone oil, defoaming powder, or n-butanol, with methyl silicone oil being the most preferred.

[0030] Preferably, the PCR enhancer II comprises polyvinylpyrrolidone.

[0031] Secondly, the present invention provides the application of the above-mentioned extraction-free rapid nucleic acid release agent in any of the following:

[0032] A1. Applications in nucleic acid release or in the preparation of products for nucleic acid release;

[0033] A2. Applications in nucleic acid amplification or the preparation of products for nucleic acid amplification;

[0034] A3. Application in the preparation of products for nucleic acid detection.

[0035] Thirdly, the present invention provides a nucleic acid release method, comprising placing a sample in the above-mentioned extraction-free rapid nucleic acid release agent, and mixing by shaking at room temperature to obtain a nucleic acid solution.

[0036] Furthermore, preferably, the oscillation conditions include oscillation at 2500-3500 rpm for 1-5 minutes;

[0037] Preferably, when the oscillation speed is 2500 rpm, the oscillation time is 1 to 5 minutes, preferably 3 minutes;

[0038] Preferably, when the oscillation speed is 3500 rpm, the oscillation time is 1 to 3 minutes, preferably 1 minute;

[0039] Preferably, after shaking and mixing, the mixture is allowed to stand for 1 to 10 minutes, more preferably 5 minutes;

[0040] Preferably, the sample includes a swab sample or a sputum sample.

[0041] Fourthly, the present invention provides a nucleic acid PCR amplification method, comprising using a nucleic acid solution prepared by the above-mentioned nucleic acid release method as a template for PCR amplification.

[0042] Furthermore, taking a 20 μL system as an example, the amplification system includes 2–10 μL of nucleic acid solution, preferably 5 μL.

[0043] Fifthly, the present invention provides a PCR amplification kit, comprising the above-mentioned extraction-free rapid nucleic acid release agent;

[0044] Preferably, it also includes PCR reaction solution.

[0045] This invention provides an extraction-free rapid nucleic acid release agent. A strong base disrupts the hydrogen bonds and hydrophobic interactions of proteins, leading to protein denaturation and rapid lysis of pathogenic microorganisms, thus facilitating nucleic acid release. A surfactant acts on the phospholipid bilayer of the cell membrane. A lysis enhancer disrupts proteins and, in conjunction with the surfactant, accelerates cell membrane lysis. A metal ion chelating agent inhibits protease activity, protecting nucleic acids from degradation and ensuring a sufficiently active nucleic acid solution. A buffer provides a buffering environment for nucleic acid release, facilitating downstream PCR experiments. The use of PCR enhancer I improves the compatibility of the buffer with downstream PCR amplification systems. The specific concentration combination of a strong base, surfactant, buffer, metal ion chelating agent, lysis enhancer, and PCR enhancer I effectively lyses cells or virus particles, releasing nucleic acid without the need for separate inactivation. It exhibits strong lysis capability, short lysis time, simple steps, and compatibility with rapid detection programs. Detailed Implementation

[0046] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0047] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0048] One aspect of this invention provides an extraction-free rapid nucleic acid release agent, comprising 10–100 mM of a strong base, 0.1%–2% of a surfactant by mass / volume, 10–100 mM of a buffer solution, 0.1–2 mM of a metal ion chelating agent, 10–100 mM of a lysis enhancer, and 50–100 mM of PCR enhancer I.

[0049] The strong base disrupts the hydrogen bonds and hydrophobic interactions of proteins, leading to protein denaturation and rapid lysis of pathogens, thus facilitating nucleic acid release. Surfactants act on the phospholipid bilayer of the cell membrane. Lysis enhancers can break down proteins and, in conjunction with surfactants, accelerate cell membrane lysis. Metal ion chelators inhibit protease activity, protecting nucleic acids from degradation and ensuring a sufficiently active nucleic acid solution. Buffer solutions provide a buffering environment for nucleic acid release, facilitating downstream PCR experiments. The use of PCR enhancer I improves the compatibility of the buffer solution with downstream PCR amplification systems. This specific combination of strong base, surfactant, buffer solution, metal ion chelators, lysis enhancers, and PCR enhancer I effectively lyses cells or virus particles, releasing nucleic acid without the need for separate inactivation. It exhibits strong lysis capabilities, short lysis time, simple procedures, and compatibility with rapid detection programs.

[0050] In some specific embodiments, the surfactant includes at least one of sodium dodecyl sulfate, Triton X1, or Tween 20, preferably sodium dodecyl sulfate.

[0051] In some specific embodiments, the pyrolysis enhancer includes at least one of guanidine isothiocyanate, guanidine hydrochloride, or sodium lauroyl glutamate, preferably guanidine isothiocyanate.

[0052] In some specific embodiments, the PCR enhancer I includes at least one of potassium chloride, sodium chloride, or sucrose, preferably sucrose.

[0053] In some specific embodiments, when the surfactant is sodium dodecyl sulfate, the mass-volume ratio of the surfactant is 0.1-0.5%; when the surfactant is Tween 20, the mass-volume ratio of the surfactant is 0.1-1%; and when the surfactant is Triton X1, the mass-volume ratio of the surfactant is 0.5-2%.

[0054] In some specific embodiments, when the pyrolysis enhancer is guanidine isothiocyanate, the concentration of the pyrolysis enhancer is 25-50 mM; when the pyrolysis enhancer is guanidine hydrochloride, the concentration of the pyrolysis enhancer is 50-100 mM; and when the pyrolysis enhancer is sodium lauroyl glutamate, the concentration of the pyrolysis enhancer is 10-30 mM.

[0055] In some specific embodiments, when PCR enhancer I is potassium chloride, the concentration of PCR enhancer I is 70-100 mM; when PCR enhancer I is sodium chloride, the concentration of PCR enhancer I is 10-40 mM; and when PCR enhancer I is sucrose, the concentration of PCR enhancer I is 50-80 mM.

[0056] In some specific implementations, PCR enhancer II, with a mass-to-volume ratio of 1% to 2%, is also included to work in conjunction with PCR enhancer I to further improve the compatibility of the buffer with the downstream PCR amplification system.

[0057] The aforementioned extraction-free rapid nucleic acid release agent can be used to lyse samples or sputum obtained through swab sampling. During swab collection, pathogenic microorganisms in the sample adhere to or are inside the swab. Sputum has a high viscosity, which hinders the entry of pathogenic microorganisms into the extraction-free rapid nucleic acid release agent. To further enhance the lysis effect on pathogenic microorganisms, some specific embodiments also include grinding beads at a mass-to-volume ratio of 0.1% to 0.5%, preferably 0.2% to 0.4%. The grinding beads provide external force to help break down the cell walls of some pathogenic microorganisms, releasing nucleic acids and thus improving swab elution efficiency.

[0058] In some specific embodiments, a defoamer at a mass-volume ratio of 0.05% to 1% is also included. Adding a defoamer reduces the surface tension of the liquid, shortens preparation time, and enhances molecular interactions.

[0059] In some specific embodiments, the defoamer includes at least one of methyl silicone oil, defoaming powder, or n-butanol, preferably methyl silicone oil.

[0060] In some specific embodiments, the PCR enhancer II comprises polyvinylpyrrolidone.

[0061] According to another aspect of the present invention, the use of the above-described extraction-free rapid nucleic acid release agent in any of the following:

[0062] A1. Applications in nucleic acid release or in the preparation of products for nucleic acid release;

[0063] A2. Applications in nucleic acid amplification or the preparation of products for nucleic acid amplification;

[0064] A3. Application in the preparation of products for nucleic acid detection.

[0065] According to another aspect of the present invention, a method for releasing nucleic acid is also provided, comprising placing a sample in the above-mentioned extraction-free rapid nucleic acid release agent, and mixing by shaking at room temperature to obtain a nucleic acid solution.

[0066] The nucleic acid solution prepared by this method can be directly used for PCR amplification. The operation method is simple and does not require inactivation.

[0067] In some specific implementations, the oscillation conditions include oscillation at 2500–3500 rpm for 1–5 minutes.

[0068] In some specific implementations, when the oscillation speed is 2500 rpm, the oscillation time is 1 to 5 minutes, preferably 3 minutes.

[0069] In some specific implementations, when the oscillation speed is 3500 rpm, the oscillation time is 1 to 3 minutes, preferably 1 minute.

[0070] In some specific implementations, the mixture is further allowed to stand for 1 to 10 minutes after shaking and mixing, preferably 5 minutes.

[0071] In some specific implementations, the sample includes a swab sample or a sputum sample.

[0072] According to another aspect of the present invention, a nucleic acid PCR amplification method is also provided, comprising using a nucleic acid solution prepared by the above-described nucleic acid release method as a template for PCR amplification.

[0073] In some specific implementations, taking a 20 μL system as an example, the amplification system includes 2 to 10 μL of nucleic acid solution, preferably 5 μL.

[0074] According to another aspect of the present invention, a PCR amplification kit is also provided, comprising the above-described extraction-free rapid nucleic acid release agent;

[0075] In some specific implementations, a PCR reaction solution is also included.

[0076] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0077] I. Extraction-free rapid nucleic acid release agent

[0078] The group assignments in the embodiments are shown in Tables 1 and 2.

[0079] Table 1

[0080]

[0081] Example 8

[0082] The difference from Example 1 is that the strong alkali is replaced by potassium hydroxide instead of sodium hydroxide, otherwise it is the same as Example 1.

[0083] Example 9

[0084] The difference from Example 1 is that the surfactant was replaced with Triton X100 instead of sodium dodecyl sulfate (SDS), otherwise it is the same as Example 1.

[0085] Example 10

[0086] The difference from Example 1 is that the pyrolysis enhancer is replaced by guanidine isothiocyanate with guanidine hydrochloride, otherwise it is the same as Example 1.

[0087] Example 11

[0088] The difference from Example 1 is that the defoamer is replaced by defoaming powder instead of methyl silicone oil, otherwise it is the same as Example 1.

[0089] Example 12

[0090] The difference from Example 1 is that PCR enhancer I is replaced with sodium chloride instead of sucrose, otherwise it is the same as Example 1.

[0091] Example 13

[0092] The difference from Example 1 is that the PVP material without PCR enhancer II is not added; otherwise, it is the same as Example 1.

[0093] Example 14

[0094] The difference from Example 1 is that no grinding beads are added.

[0095] Example 15

[0096] The difference from Example 1 is that no defoamer is added.

[0097] Comparative Example 1

[0098] The difference from Example 1 is that the concentration of the strong base is 5 mM.

[0099] Comparative Example 2

[0100] The difference from Example 1 is that the concentration of the strong base is 150 mM.

[0101] Comparative Example 3

[0102] The difference from Example 1 is that the concentration of the surfactant is 0.05%.

[0103] Comparative Example 4

[0104] The difference from Example 1 is that the concentration of the surfactant is 1%.

[0105] Comparative Example 5

[0106] The difference from Example 1 is that the concentration of the pyrolysis enhancer is 10 mM.

[0107] Comparative Example 6

[0108] The difference from Example 1 is that the concentration of the pyrolysis enhancer is 80 mM.

[0109] Comparative Example 7

[0110] The difference from Example 1 is that the concentration of PCR enhancer I is 30 mM.

[0111] Comparative Example 8

[0112] The difference from Example 1 is that the concentration of PCR enhancer I is 100 mM.

[0113] Comparative Example 9

[0114] The difference from Example 1 is that the concentration of PCR enhancer II is 0.5%.

[0115] Comparative Example 10

[0116] The difference from Example 1 is that the concentration of PCR enhancer II is 3%.

[0117] Comparative Example 11

[0118] The difference from Example 1 is that PCR enhancer I is not added.

[0119] Comparative Example 12

[0120] Nucleic acid lysis buffer: includes 100 mM sodium hydroxide, 0.3% sodium dodecyl sulfate by mass / volume, 100 mM Tris-HCl and 1 mM EDTA.

[0121] II. Nucleic Acid Release Methods

[0122] The specific method includes: thoroughly mixing and shaking the sample with the nucleic acid release agent to obtain a nucleic acid solution.

[0123] The samples can be liquid or swab samples. To further illustrate the effectiveness of the nucleic acid release agents in Examples 1-15 and Comparative Examples 1-12, nucleic acid detection was performed on simulated samples of the same concentration using the nucleic acid release agents from each example and comparative example.

[0124] For ease of implementation, the nucleic acid release agent used in the following examples is placed in the sample collection tube, with each tube containing 2 mL of nucleic acid release agent.

[0125] Simulated sample:

[0126] A mixed pseudovirus solution sample containing influenza A virus (FluA), influenza B virus (FluB), adenovirus (ADV), respiratory syncytial virus (RSV), parainfluenza type I virus (PIV1), and parainfluenza type III virus (PIV3);

[0127] Pharyngeal swab samples from healthy individuals that do not contain influenza A virus, influenza B virus, adenovirus, respiratory syncytial virus, parainfluenza type I virus, or parainfluenza type III virus.

[0128] The operating methods of Examples 1-15 and Comparative Examples 1-11 are as follows: Take one collection tube containing nucleic acid release agent, add 200uL of pseudovirus solution and one pharyngeal swab from a healthy person respectively, shake at 2500rpm for 3 minutes to mix, and then directly take 5uL of sample nucleic acid for testing.

[0129] The procedure for Comparative Example 12 is as follows: The sample needs to be placed in physiological saline first, shaken and mixed, and then 10uL of sample and 10uL of nucleic acid release agent are mixed thoroughly in a 1:1 ratio. Then, 10uL of sample is taken for direct amplification.

[0130] Real-time quantitative PCR amplification analysis was performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 2.

[0131] Table 2

[0132] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0133] The real-time quantitative PCR amplification procedure is shown in Table 3.

[0134] Table 3

[0135]

[0136] The simulated samples were tested and analyzed. Each sample underwent repeated testing, and the results are shown in Table 4.

[0137] Table 4

[0138]

[0139]

[0140]

[0141]

[0142] A lower CT value indicates a higher amount of detected nucleic acid, less inhibition, and better detection results. Table 4 shows that Examples 1-15 are all superior to Comparative Example 12, demonstrating that the extraction-free rapid nucleic acid release agent provided by this invention is superior to existing technologies. It can fully lyse cells or virus particles, releasing nucleic acid substances, resulting in better detection performance in subsequent rapid nucleic acid testing. It is compatible with rapid testing procedures and is more convenient to operate. Among them, Example 1 has the lowest CT value.

[0143] Compared with Example 1, the CT values ​​of Examples 2 and 3 showed an increasing trend. This indicates that at the same strong alkali concentration, surfactants, lysis enhancers, and PCR enhancer I have a promoting effect on nucleic acid release and can work together with strong alkali to improve the overall effect. Although the content of grinding beads in Examples 1-3 showed an increasing trend, it did not significantly improve the nucleic acid release effect; on the contrary, the CT value increased. This shows that the effect of nucleic acid release agents does not continuously improve with the increase of strong alkali concentration. Grinding beads can increase swab elution and promote nucleic acid release, but the lysis ability of nucleic acid release agents still needs to be considered. In solutions with strong lysis ability, excessive grinding beads can lead to excessive sample action, resulting in a stronger inhibitory effect. This inhibitory effect is greater than the nucleic acid release effect, ultimately leading to a higher CT value of the sample.

[0144] Although the concentration of strong alkali was further increased in Example 4, the CT value did not increase. This shows that when the concentrations of each component are fixed, adjusting the concentration of strong alkali does not result in a significant difference in the overall effect. This indicates that the strong alkali in this invention can meet the requirements within a certain range.

[0145] Example 5 adjusted the contents of surfactant, buffer solution, lysis enhancer, and grinding beads, but the CT value of Example 5 was greater than that of Example 4. This indicates that the materials work synergistically and require specific proportions to achieve optimal results. This invention optimizes the combination to select the most suitable combination. Furthermore, adding more surfactant and lysis enhancer does not produce any further positive effects, indicating that the lysis capacity of the current reagent already meets the requirements of most samples.

[0146] In conjunction with Example 6, the CT value of Example 6 is close to that of Example 3 and greater than that of Example 1. Combining Examples 3 and 1 further demonstrates that the current approach is a combination optimization; continuously increasing the dosage of individual materials does not achieve the optimal result.

[0147] In conjunction with Example 7, the CT value of Example 7 is close to that of Example 6. This indicates that within a certain range, the dosage of each component material can achieve the pyrolysis effect. Considering both the pyrolysis capability and the system's tolerance, it is necessary to optimize the optimal combination to ensure both pyrolysis capability and system tolerance.

[0148] Compared with Example 1, Examples 8-10 replaced the strong alkali, surfactant and lysis enhancer in Example 1 respectively. The CT values ​​of Examples 8-10 were similar and all greater than those of Example 1, but still lower than those of Comparative Example 13. That is, the extraction-free rapid nucleic acid release agent after replacing the new materials is still superior to the prior art.

[0149] Compared with Example 1, Example 11 replaces the defoamer in Example 1. The CT value of Example 11 is close to that of Example 1 and lower than that of Comparative Example 13. The defoaming speed is not significantly different. Both can be used in nucleic acid release agents.

[0150] Compared to Example 1, Example 12 replaced the PCR enhancer in Example 1. The CT value of Example 12 was greater than that of Example 1, indicating that the compatibility of the replaced material with the downstream PCR amplification system was not as good as that of the currently used material. However, the CT value of Example 12 was less than that of Comparative Example 12, indicating that the replaced nucleic acid release agent was still superior to the existing technology.

[0151] Compared with Example 1, Example 13 removed PVP from Example 1. The CT value of Example 13 was greater than that of Example 1, indicating that PVP and strong base can enhance the lysis effect. At the same time, the addition of PVP can effectively bind and remove inhibitory substances, achieving better results. However, the CT value of Example 13 was less than that of Comparative Example 12, indicating that even without the addition of PVP, the nucleic acid release agent is still more effective than the prior art.

[0152] Compared with Example 1, Example 14 removed the grinding beads from Example 1. The CT value of Example 14 was greater than that of Example 1, indicating that the grinding beads could help the swabs to wash out more nucleic acids and improve sensitivity. However, the CT value of Example 13 was still less than that of Comparative Example 12. It can be seen that even without the addition of grinding beads, the nucleic acid release agent is still more effective than the prior art.

[0153] Compared with Example 1, Example 15 removes the methyl silicone oil from Example 1. The CT value of Example 15 is close to that of Example 1. The addition of methyl silicone oil is mainly to assist in defoaming during production and actual use, reduce the risk of contamination, and has no significant impact on performance.

[0154] Compared with Example 1, the CT values ​​of Comparative Examples 1-12 were all greater than those of Example 1 and Comparative Example 12, and the effects were not as good as the existing technology. This indicates that the amount of material outside the range or the absence of PCR enhancer I cannot balance the lysis ability and the system's tolerance, and cannot meet the usage requirements.

[0155] III. Optimization of Nucleic Acid Release Methods and Conditions

[0156] The combined use of grinding beads and nucleic acid release agents is crucial for the processing of different sample types and the lysis of various pathogenic microorganisms. Therefore, considering the versatility of nucleic acid release agents, we selected different sample types to process several representative pathogenic microorganisms, including fungi, viruses, and bacteria.

[0157] Oropharyngeal swab samples primarily detect viruses, mycoplasma, and bacteria, including influenza A, Mycoplasma pneumoniae, and Streptococcus pneumoniae; sputum samples primarily detect fungi and bacteria, including Aspergillus and Pseudomonas aeruginosa; vaginal swab samples primarily detect fungi, bacteria, parasites, mycoplasma, and viruses, including Candida spp., Prevotella, Trichomonas vaginalis, Ureaplasma urealyticum, and HPV. Nucleic acid release methods and conditions are shown in Table 5.

[0158] Table 5

[0159]

[0160] The nucleic acid release agent prepared in Example 1 was used to perform nucleic acid testing on 10 pharyngeal swabs, 10 sputum samples, and 10 vaginal swabs. The operation steps are as follows: 10 pharyngeal swabs, 10 sputum samples, and 10 vaginal swabs were collected and added to collection tubes containing the nucleic acid release agent. After inverting and mixing 10 times, the shaking frequency and time were performed according to Table 5. The control group was before shaking, and the experimental group was after shaking. Real-time quantitative PCR amplification and analysis were performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 6.

[0161] Table 6

[0162] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0163] The real-time quantitative PCR amplification procedure is shown in Table 7.

[0164] Table 7

[0165]

[0166] The real samples were tested and analyzed, and the results are shown in Tables 8 to 10.

[0167] Table 8. Test data of oropharyngeal swab samples

[0168]

[0169]

[0170] Table 9. Sputum Sample Test Data

[0171]

[0172] Table 10 Test data of vaginal swab samples

[0173]

[0174]

[0175]

[0176]

[0177] As shown in Table 8, the CT values ​​of Examples 16-20 were significantly higher than those of the control group, indicating that the nucleic acid releasing agent could lyse more nucleic acids under different vibration velocities and times. The detection results of Examples 16-18 show that when the vibration velocity was 2500 rpm, the CT value of Example 17 was lower than that of Example 16, while the CT values ​​of Examples 18 and 17 were similar. The detection results of Examples 19 and 20 show that when the vibration velocity was 3500 rpm, the CT values ​​of Examples 19 and 20 were similar and not significantly different from those of Examples 17 and 18. A smaller CT value indicates that under these operating conditions, more pathogenic microbial nucleic acids were obtained from the sample, and the sensitivity was higher. In oropharyngeal swab samples, at a shaking speed of 2500 rpm, shaking for 1 minute is not sufficient to wash away enough pathogens from the swab and obtain nucleic acids to the maximum extent. Shaking for 3 minutes is equivalent to shaking for 5 minutes, and when the shaking speed is increased to 3500 rpm, shaking for 1 minute and shaking for 3 minutes are equivalent.

[0178] As shown in Table 9, the CT values ​​of Examples 16-20 were significantly higher than those of the control group, indicating that the addition of glass beads also has a significant effect on sputum samples. The test results of Examples 16-18 show that when the vibration velocity is 2500 rpm, the CT value of Example 17 is lower than that of Example 16, while the CT values ​​of Examples 18 and 17 are similar. The test results of Examples 19 and 20 show that when the vibration velocity is 3500 rpm, the CT value of Example 19 is lower than that of Example 20. The test results indicate that the CT values ​​of Examples 17, 18, and 19 are similar. It can be seen that the nucleic acid releasing agent in this invention contains a strong alkali, which can liquefy and lyse pathogenic microorganisms in sputum samples to obtain the nucleic acids of the pathogenic microorganisms for direct nucleic acid detection. In sputum sample types, at a shaking speed of 2500 rpm, shaking for 3 minutes is equivalent to shaking for 5 minutes. Increasing the shaking speed to 3500 rpm also yields the same effect after 1 minute of shaking. However, increasing the shaking time to 3 minutes, while still improving the effect, is slightly weaker than in Examples 17-19. Sputum samples are relatively complex; excessively high shaking speeds and times, besides leading to a positive increase in the amount of pathogenic microbial nucleic acids, can also result in more negative inhibitory effects.

[0179] As shown in Table 10, the CT values ​​of Examples 16-20 were significantly higher than those of the control group, indicating that the nucleic acid releasing agent could lyse more nucleic acids at different vibration velocities and times. The detection results of Examples 16-18 show that when the vibration velocity was 2500 rpm, the CT value of Example 17 was lower than that of Example 16, while the CT values ​​of Examples 18 and 17 were similar. The detection results of Examples 19 and 20 show that when the vibration velocity was 3500 rpm, the CT values ​​of Examples 19 and 20 were similar and not significantly different from those of Examples 17 and 18. It can be concluded that, for vaginal swab sample types, vibration at 3500 rpm for 1 minute is the most effective. Vibration at 2500 rpm for 3 minutes and 5 minutes is essentially the same as vibration at 3500 rpm for 1 minute, but the average increase in CT value is less than 0.1 CT, indicating no significant difference.

[0180] Based on the detection data in Tables 9 and 10, considering the need to test and verify the detection effects on different sample types, factors such as the shaking speed and shaking time of the glass beads need to be tested and verified. In oropharyngeal swabs and vaginal swabs, the results of Examples 16-18 show little overall difference. This indicates that shaking at 2500 rpm for 3 minutes is sufficient to fully wash away pathogenic microorganisms and release nucleic acids from the swabs. In sputum samples, the existing extraction reagents, whether manual or automated, require sample liquefaction first. However, the nucleic acid release agent provided by this invention does not require an additional liquefaction step; it can be directly added to the release agent to complete sample liquefaction and nucleic acid extraction. The results of Examples 17-19 show that for relatively complex samples like sputum, extending the shaking time may have a negative effect. This is because during extended shaking time, not only will more nucleic acids be eluted and released, but the influence of inhibitors will also increase. The test results show that the shaking speed and shaking time need to be relatively balanced to ensure that the elution and release efficiency of pathogenic microorganisms is greater than the influence of inhibitors in order to obtain higher amplification efficiency and lower CT values.

[0181] Examples 16-18 are operated on conventional equipment and are more universal, while the operation time of Examples 17 and 19 is not much different. Therefore, Example 17 is more suitable and cost-effective.

[0182] IV. Detection Limit and Precision Analysis of Novel Coronavirus Reference Materials

[0183] The inactivated novel coronavirus reference sample was diluted to 10 using TE buffer. 5 copy / mL, 10 4 copy / mL and 2×103 copy / mL. 10 5 104 copies / mL was used as precision reference 1, and 2×10⁴ copies / mL was used as precision reference 2. 3 The copy / mL value was used as the detection limit reference. The precision reference was tested 10 times, and the detection limit reference was tested 20 times.

[0184] The nucleic acid release agent prepared in Example 1 was used. 200 μL of each of the precision reference standard 1, precision reference standard 2, and detection limit reference standard 1 were added to a collection tube containing the nucleic acid release agent. The mixture was shaken and mixed according to Example 17. 200 μL of the mixture was extracted using the magnetic bead method, and then directly analyzed by real-time quantitative PCR using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 11.

[0185] Table 11

[0186] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0187] The real-time quantitative PCR amplification procedure is shown in Table 12.

[0188] Table 12

[0189]

[0190] The novel coronavirus pseudovirus reference sample was tested and analyzed, and the test results are shown in Tables 13-14.

[0191] Table 13 CT values ​​of the novel coronavirus precision reference material

[0192]

[0193] Table 14 CT values ​​of reference materials for novel coronavirus detection limits

[0194] 1 36.36 35.79 11 36.19 35.80 2 36.44 36.05 12 35.94 35.94 3 35.98 35.77 13 36.56 35.96 4 36.27 36.12 14 36.57 35.78 5 36.39 36.10 15 36.05 35.51 6 36.50 35.59 16 36.46 35.54 7 36.00 35.91 17 36.11 36.18 8 36.26 35.88 18 36.12 35.94 9 36.33 35.78 19 35.93 35.91 10 36.56 35.93 20 35.94 36.21

[0195] As shown in Tables 13 and 14, the CV% values ​​for the precision reference standard 1, nucleic acid release agent, and magnetic bead method for detecting the novel coronavirus are 0.56% and 0.89%, respectively; the CV% values ​​for the precision reference standard 2, nucleic acid release agent, and magnetic bead method are 0.78% and 0.50%, respectively, both less than 5.0%, meeting the requirements. The detection limit reference standard for the novel coronavirus is 2 × 10⁻⁶. 3 The detection limit was 200 copies / mL, as 200 mL was added to 2 mL of nucleic acid release agent. Both the nucleic acid release agent and the magnetic bead method showed stable detection for 20 tests. Overall, the results showed no significant difference in extraction performance between the inactivated nucleic acid release agent treatment and the magnetic bead method against novel coronavirus pseudoviruses (RNA viruses).

[0196] V. Validation of Clinical Samples of Novel Coronavirus

[0197] The nucleic acid release agent prepared in Example 1 was used to detect novel coronavirus nucleic acid in 30 oropharyngeal swab samples. The specific operation was as follows: the collected dry swab samples were added to a collection tube containing the nucleic acid release agent, vortexed and mixed, and 200 μL of the nucleic acid release agent was extracted using the magnetic bead method. Next, the nucleic acid release agent containing the sample was processed according to Example 17 to release the nucleic acid into the nucleic acid release agent. Real-time quantitative PCR amplification and analysis were performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 15.

[0198] Table 15

[0199] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0200] The real-time quantitative PCR amplification procedure is shown in Table 16.

[0201] Table 16

[0202]

[0203] The real samples were tested and analyzed, and the results are shown in Table 17.

[0204] Table 17

[0205]

[0206]

[0207] As shown in Table 17, all 30 oropharyngeal swab samples were detectable. The mean difference in CT values ​​between the magnetic bead method and the nucleic acid release agent method was within 0.5 CT, indicating no significant difference in the detection results between the two methods.

[0208] VI. Validation of Clinical Samples of Lactobacillus

[0209] Using the nucleic acid release agent prepared in Example 1 and pre-filled with glass beads, lactobacillus nucleic acid was detected in 20 vaginal swab samples. The specific procedure was as follows: The collected dry swab samples were added to a collection tube containing the nucleic acid release agent, vortexed to mix, and 200 μL of the nucleic acid release agent was extracted using the magnetic bead method. Next, the nucleic acid release agent containing the sample was processed according to Example 17 to release the nucleic acid into the nucleic acid release agent. Real-time quantitative PCR amplification and analysis were performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 18.

[0210] Table 18

[0211] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0212] The real-time quantitative PCR amplification procedure is shown in Table 19.

[0213] Table 19

[0214]

[0215]

[0216] The real samples were tested and analyzed, and the results are shown in Table 20.

[0217] Table 20

[0218]

[0219] As shown in Table 20, all 20 vaginal swab samples were detectable. The mean difference in CT values ​​between the magnetic bead method and the nucleic acid release agent method was within 0.5 CT, indicating no significant difference in the detection results between the two methods.

[0220] VII. Validation of Aspergillus spp. clinical samples

[0221] The nucleic acid release agent prepared in Example 1, with pre-placed glass beads, was used to detect Aspergillus nucleic acid in 20 sputum samples. The specific operation was as follows: An appropriate amount of the collected sputum sample was added to the nucleic acid release agent, and the mixture was shaken and mixed. 200 μL of the nucleic acid release agent was then extracted using the magnetic bead method. Next, the nucleic acid release agent containing the sample was processed according to Example 17 to release the nucleic acid into the nucleic acid release agent. Real-time quantitative PCR amplification and analysis were performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 21.

[0222] Table 21

[0223] 2X Buffer 10uL 20X enzyme mixture 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

[0224] The real-time quantitative PCR amplification procedure is shown in Table 22.

[0225] Table 22

[0226]

[0227] The real samples were tested and analyzed, and the results are shown in Table 23.

[0228] Table 23

[0229]

[0230] As shown in the table, all 20 sputum samples were detectable. The mean difference in CT values ​​between the magnetic bead method and the nucleic acid release agent method was within one CT value, indicating no significant difference in the detection results between the two methods.

[0231] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid nucleic acid release agent without extraction, characterized in that, It includes a strong base of 50-100mM, a surfactant of 0.3% by volume, a buffer of 10-100mM, a metal ion chelating agent of 1mM, a lysis enhancer of 25mM, PCR enhancer I of 80mM, and PCR enhancer II of 1% by volume. The strong base is sodium hydroxide; The surfactant is sodium dodecyl sulfate; The buffer solution is Tris-HCl; The metal ion chelating agent is EDTA; The pyrolysis enhancer is guanidine isothiocyanate; The PCR enhancer I is sucrose; The PCR enhancer II is polyvinylpyrrolidone.

2. The extraction-free rapid nucleic acid release agent according to claim 1, characterized in that, It also includes grinding beads with a mass-to-volume ratio of 0.1% to 0.5%.

3. The extraction-free rapid nucleic acid release agent according to claim 1, characterized in that, It also includes defoamers with a mass-volume ratio of 0.5% to 1%; The defoamer is at least one of methyl silicone oil and defoaming powder.

4. The use of the extraction-free rapid nucleic acid release agent according to any one of claims 1 to 3 in any of the following: A1. Applications in nucleic acid release or in the preparation of products for nucleic acid release; A2. Applications in nucleic acid amplification for non-diagnostic purposes or in the preparation of products for nucleic acid amplification; A3. Application in the preparation of products for nucleic acid detection.

5. A method for releasing nucleic acids, characterized in that, The method includes placing the sample in the extraction-free rapid nucleic acid release agent according to any one of claims 1 to 3, and mixing by shaking at room temperature to obtain a nucleic acid solution.

6. The method according to claim 5, characterized in that, The oscillation conditions include oscillation at 2500~3500 rpm for 1~5 minutes.

7. The method according to claim 6, characterized in that, When the oscillation speed is 2500 rpm, the oscillation time is 1~5 min.

8. The method according to claim 6, characterized in that, When the oscillation speed is 3500 rpm, the oscillation time is 1~3 minutes.

9. The method according to claim 5, characterized in that, After shaking and mixing, the mixture should be left to stand for 1 to 10 minutes.

10. The method according to claim 5, characterized in that, The samples include swab samples or sputum samples.

11. A nucleic acid PCR amplification method for non-diagnostic purposes, characterized in that, This includes using the nucleic acid solution prepared by the nucleic acid release method described in claim 5 as a template for PCR amplification.

12. The non-diagnostic nucleic acid PCR amplification method according to claim 11, characterized in that, A 20 μL amplification system includes 2–10 μL of nucleic acid solution.

13. A PCR amplification kit, characterized in that, Includes the extraction-free rapid nucleic acid release agent as described in any one of claims 1 to 3.

14. The PCR amplification kit according to claim 13, characterized in that, It also includes PCR reaction solution.

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