Extraction-free rapid nucleic acid releasing agent and application thereof

Abstract

The invention provides an extraction-free rapid nucleic acid releasing agent and application thereof, and relates to the technical field of molecular biology, and the extraction-free rapid nucleic acid releasing agent comprises 10-100 mM of strong alkali, a surfactant with the mass-volume ratio of 0.1%-2%, 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 a PCR enhancer I. Cells or virus particles can be fully split, nucleic acid substances are released, and independent inactivation is not needed; the method has the advantages of strong cracking capability, short cracking time and simple steps, and can adapt to a rapid detection program.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to an extraction-free rapid nucleic acid release agent and application thereof. Background Art

[0002] In recent years, PCR technology has become one of the most widely used pathogen detection technologies, and has been widely used due to its advantages such as high sensitivity, strong specificity and short time consumption. Whether it is PCR technology or sequencing technology, the nucleic acid of the sample to be tested needs to be extracted before it can be used for subsequent testing.

[0003] There are many methods for nucleic acid extraction, mainly boiling method, centrifugal column method and magnetic bead method. These methods are time-consuming and cumbersome to operate. Therefore, the emergence of nucleic acid release agents is particularly important. Sample release agents have the advantages of low cost, convenient operation and short time.

[0004] With the development of molecular diagnostic POCT, rapid detection, simple operation, and high sensitivity are requirements and standards. Rapid acquisition of test results, ease of sample processing, and sensitivity of results are challenges and directions of molecular POCT. As for the products on the market, sample release agents are rapid sample pretreatment products, but their sensitivity is greatly affected due to the lack of purification steps. The sensitivity will be further affected when used with rapid detection procedures or equipment, and there are also problems such as limited pathogen targets.

[0005] Most nucleic acid releasers on the market do not have the inactivation function, and there is a certain risk of personal injury, and even require heating and other methods to treat some pathogenic microorganisms that are difficult to break through. Therefore, existing sample releasers still have problems such as long time consumption, many operation steps, and inability to adapt to rapid procedures. It is urgent to develop a nucleic acid releaser that is simple to operate, less time-consuming, highly safe, can treat multiple pathogens, and is compatible with rapid procedures or equipment.

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

[0007] One of the purposes of the present invention is to provide an extraction-free rapid nucleic acid releaser to solve the technical problems that the nucleic acid releasers in the prior art do not have an inactivation function, are time-consuming, have many operating steps, and cannot be adapted to rapid procedures.

[0008] The second object of the present invention is to provide the use of the above-mentioned extraction-free rapid nucleic acid releaser 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] A third object of the present invention is to provide a method for releasing nucleic acid.

[0010] A fourth object of the present invention is to provide a nucleic acid PCR amplification method.

[0011] A fifth object of the present invention is to provide a PCR amplification kit.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

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

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

[0015] Preferably, the lysis enhancer comprises at least one of guanidine isothiocyanate, guanidine hydrochloride or sodium lauroyl glutamate, preferably guanidine isothiocyanate;

[0016] Preferably, the PCR enhancer I comprises at least one of potassium chloride, sodium chloride or sucrose, preferably sucrose.

[0017] Further, when the surfactant is sodium dodecyl sulfate, the mass 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 0.5-2%;

[0020] Preferably, when the cleavage enhancer is guanidine thiocyanate, the concentration of the cleavage enhancer is 25 to 50 mM;

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

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

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

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

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

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

[0027] Preferably, it also includes grinding beads with a mass volume ratio of 0.1% to 0.5%, preferably grinding beads with a mass volume ratio of 0.2 to 0.4%;

[0028] Preferably, the composition further comprises a defoaming agent in a mass volume ratio of 0.05% to 1%.

[0029] Preferably, the defoaming agent comprises at least one of methyl silicone oil, foaming powder or n-butanol, preferably methyl silicone oil;

[0030] Preferably, the PCR enhancer II comprises polyvinyl pyrrolidone.

[0031] In a second aspect, the present invention provides the use of the above-mentioned extraction-free rapid nucleic acid release agent in any of the following items:

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

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

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

[0035] In a third aspect, the present invention provides a method for releasing nucleic acid, 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] Further, preferably, the shaking conditions include shaking at 2500-3500 rpm for 1-5 min;

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

[0038] Preferably, when the shaking speed is 3500 rpm, the shaking time is 1 to 3 min, preferably 1 min;

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

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

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

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

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

[0044] Preferably, a PCR reaction solution is also included.

[0045] The invention provides a kind of extraction-free rapid nucleic acid release agent, wherein the strong base can destroy the hydrogen bond and hydrophobic effect of the protein, causing protein denaturation, rapid lysis of pathogenic microorganisms, and contribute to the release of nucleic acid; the surfactant acts on the phospholipid bilayer of the cell membrane; the lysis enhancer can destroy the protein and cooperate with the surfactant to accelerate the lysis of the cell membrane. The metal ion chelator can inhibit the activity of the protease, protect the nucleic acid from being degraded, and ensure that a nucleic acid solution with sufficient activity is obtained; the buffer provides a certain buffer environment for the release of nucleic acid, which is convenient for the downstream PCR experiment; the use of PCR enhancer I can improve the compatibility of the buffer with the downstream PCR amplification system. The combination of specific concentrations of strong base, surfactant, buffer, metal ion chelator, lysis enhancer and PCR enhancer I can fully lyse cells or virus particles and release nucleic acid substances without separate inactivation; it has strong lysis ability, short lysis time, simple steps and can be adapted to the rapid detection program. DETAILED DESCRIPTION

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

[0047] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0048] On the one hand, the present invention provides an extraction-free rapid nucleic acid release agent, comprising 10-100 mM of a strong base, a surfactant with a mass volume ratio of 0.1% to 2%, 10-100 mM of a buffer solution, 0.1-2 mM of a metal ion chelator, 10-100 mM of a lysis enhancer and 50-100 mM of a PCR enhancer I.

[0049] Among them, strong alkali can destroy the hydrogen bonds and hydrophobic effects of proteins, causing protein denaturation, quickly lysing pathogenic microorganisms, and helping to release nucleic acids; surfactants act on the phospholipid bilayer of cell membranes; lysis enhancers can destroy proteins and cooperate with surfactants to accelerate the lysis of cell membranes. Metal ion chelators can inhibit protease activity, protect nucleic acids from degradation, and ensure that nucleic acid solutions with sufficient activity are obtained; buffers provide a certain buffer environment for nucleic acid release, which is convenient for downstream PCR experiments; the use of PCR enhancer I can improve the compatibility of buffers with downstream PCR amplification systems. The combination of specific concentrations of strong alkali, surfactants, buffers, metal ion chelators, lysis enhancers, and PCR enhancers I can fully lyse cells or virus particles and release nucleic acid substances without the need for separate inactivation; it has strong lysis ability, short lysis time, simple steps, and can be adapted to rapid detection procedures.

[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 lysis 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%; when the surfactant is Triton X1, the mass volume ratio of the surfactant is 0.5-2%.

[0054] In some specific embodiments, when the cleavage enhancer is guanidine isothiocyanate, the concentration of the cleavage enhancer is 25-50 mM; when the cleavage enhancer is guanidine hydrochloride, the concentration of the cleavage enhancer is 50-100 mM; when the cleavage enhancer is sodium lauroyl glutamate, the concentration of the cleavage 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; when PCR enhancer I is sucrose, the concentration of PCR enhancer I is 50-80 mM.

[0056] In some specific embodiments, a PCR enhancer II is included at a mass volume ratio of 1% to 2%, which cooperates with the PCR enhancer I to further improve the compatibility of the buffer with the downstream PCR amplification system.

[0057] The above-mentioned extraction-free rapid nucleic acid releaser can be used to lyse samples or sputum obtained by swab sampling. When the swab is collected, the pathogenic microorganisms in the sample will adhere to or inside the swab, and the sputum has a high viscosity, which is not conducive to the entry of pathogenic microorganisms into the extraction-free rapid nucleic acid releaser. In order to further increase the lysis effect of pathogenic microorganisms, in some specific embodiments, grinding beads with a mass volume ratio of 0.1% to 0.5% are also included, preferably grinding beads with a mass volume ratio of 0.2% to 0.4%. Grinding beads can provide external force to help some pathogenic microorganisms break the cell wall and release nucleic acids, so as to achieve the purpose of improving the swab elution efficiency.

[0058] In some specific embodiments, the composition further comprises a defoaming agent at a mass volume ratio of 0.05% to 1%. The defoaming agent is added to reduce the surface tension of the liquid, shorten the preparation time and enhance the interaction of molecules.

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

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

[0061] According to another aspect of the present invention, there is also provided the use of the above-mentioned extraction-free rapid nucleic acid release agent in any of the following items:

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

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

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

[0065] According to another aspect of the present invention, a nucleic acid release method is 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 no inactivation is required.

[0067] In some specific embodiments, the shaking conditions include shaking at 2500-3500 rpm for 1-5 min.

[0068] In some specific embodiments, when the shaking speed is 2500 rpm, the shaking time is 1 to 5 min, preferably 3 min.

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

[0070] In some specific embodiments, the shaking mixing further comprises standing for 1 to 10 minutes, preferably 5 minutes.

[0071] In some specific embodiments, the sample comprises 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 the nucleic acid solution prepared by the above nucleic acid release method as a template for PCR amplification.

[0073] In some specific embodiments, 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, there is also provided a PCR amplification kit, comprising the above-mentioned extraction-free rapid nucleic acid release agent;

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

[0076] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0077] 1. Extraction-free rapid nucleic acid release agent

[0078] The distribution ratios of the groups in the embodiment are shown in Table 1 and Table 2.

[0079] Table 1

[0080]

[0081] Example 8

[0082] The difference from Example 1 is that the strong base is replaced by potassium hydroxide instead of sodium hydroxide, and the rest is the same as Example 1.

[0083] Example 9

[0084] The difference from Example 1 is that the surfactant is replaced by Triton X100 instead of sodium dodecyl sulfate (SDS), and the rest is the same as Example 1.

[0085] Example 10

[0086] The difference from Example 1 is that the cleavage enhancer is replaced by guanidine hydrochloride instead of guanidine isothiocyanate, and the rest is the same as Example 1.

[0087] Embodiment 11

[0088] The difference from Example 1 is that the defoaming agent is replaced by foaming powder instead of methyl silicone oil, and the rest is the same as Example 1.

[0089] Example 12

[0090] The difference from Example 1 is that sucrose is replaced by sodium chloride in PCR enhancer I, and the rest is the same as Example 1.

[0091] Example 13

[0092] The difference from Example 1 is that the PVP material of PCR enhancer II is not added, and the rest is the same as Example 1.

[0093] Embodiment 14

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

[0095] Embodiment 15

[0096] The difference from Example 1 is that no defoaming agent 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 lysis enhancer is 10 mM.

[0107] Comparative Example 6

[0108] The difference from Example 1 is that the concentration of the lysis 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 solution: including 100 mM sodium hydroxide, 0.3% sodium dodecyl sulfate in a mass volume ratio, 100 mM Tris-HCl and 1 mM EDTA.

[0121] 2. Nucleic Acid Release Method

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

[0123] The sample may be a liquid sample or a swab sample. In order to further illustrate the effects of the nucleic acid releasers of Examples 1 to 15 and Comparative Examples 1 to 12, nucleic acid detection was performed on simulated samples of the same concentration using the nucleic acid releasers of the Examples and Comparative Examples.

[0124] For ease of implementation, the nucleic acid releasers used in the following examples were all placed in sample collection tubes, with each tube containing 2 mL of nucleic acid releaser.

[0125] Simulation sample:

[0126] Mixed pseudovirus solution samples containing influenza A virus (FluA), influenza B virus (FluB), adenovirus (ADV), respiratory syncytial virus (RSV), parainfluenza type 1 virus (PIV1), and parainfluenza type 3 virus (PIV3);

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

[0128] The operating method of Examples 1 to 15 and Comparative Examples 1 to 11 is as follows: take a collection tube containing a nucleic acid releaser, put in 200uL of pseudovirus solution and a throat swab from a healthy person respectively, shake at 2500rpm for 3min to mix, and then directly take 5uL of sample nucleic acid for detection on the machine.

[0129] The operation method of comparative example 12 is as follows: the sample needs to be placed in physiological saline first, and after oscillation and mixing, 10uL of the sample is taken and mixed with 10uL of nucleic acid release agent in a 1:1 equal volume, and then 10uL is taken for direct amplification.

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

[0131] Table 2

[0132] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0134] Table 3

[0135]

[0136] The simulated samples were tested and analyzed, and each sample was tested in duplicate. The test results are shown in Table 4.

[0137] Table 4

[0138]

[0139]

[0140]

[0141]

[0142] The lower the CT value, the more nucleic acid is detected, the less inhibition is, and the better the detection effect is. As shown in Table 4, Examples 1 to 15 are all better than Comparative Example 12. It can be seen that the extraction-free rapid nucleic acid releaser provided by the present invention is better than the prior art, can fully lyse cells or virus particles, release nucleic acid substances, and has a better detection effect in subsequent rapid nucleic acid detection, can be adapted to the rapid detection procedure, and is more convenient to operate. Among them, the CT value of Example 1 is the smallest.

[0143] Compared with Example 1, the CT values ​​of Examples 2 and 3 show an increasing trend. It can be seen that under the same strong base concentration, the surfactant, lysis enhancer and PCR enhancer I have a promoting effect on the release of nucleic acids, and can cooperate with the strong base to improve the overall effect. Although the content of the grinding beads in Examples 1 to 3 shows an increasing trend, it does not significantly improve the nucleic acid release effect. On the contrary, the CT value increases. It can be seen that the effect of the nucleic acid releaser does not continue to increase with the increase in the concentration of the strong base; the grinding beads can increase the elution of the swab and promote the release of nucleic acids, but the lysis ability of the nucleic acid releaser still needs to be considered. In a solution with strong lysis ability, excessive grinding beads will cause the sample to be over-acted, resulting in a stronger inhibitory effect, resulting in the inhibitory effect being greater than the release of nucleic acids, and ultimately causing the sample CT value to be too large.

[0144] Although the concentration of the strong base is further increased in Example 4, the CT value is not improved. It can be seen that when the concentration of each component material is fixed, the overall effect is not significantly different by adjusting the concentration of the strong base, which indicates that the effect of the strong base in the present invention can meet the requirements within a certain range.

[0145] Example 5 adjusted the contents of surfactant, buffer, lysis enhancer and grinding beads, but the CT value of Example 5 was greater than that of Example 4. It can be seen that each material has a synergistic effect and needs to be in a certain proportion to exert the best effect. The present invention screens the most suitable combination through combination optimization; and further adding surfactant and lysis enhancer does not bring further positive effects, indicating that the lysis ability of the current reagent has met the requirements of most samples.

[0146] Combined with Example 6, the CT value of Example 6 is close to Example 3 and greater than Example 1. Combined with Example 3 and Example 1, it is further proved that the current combination optimization and the continuous addition of individual materials cannot achieve the optimal result.

[0147] Combined with Example 7, the CT value of Example 7 is close to that of Example 6. This shows that the amount of each component material within a certain range can achieve the cracking effect, and the comprehensive cracking ability and the tolerance of the system need to be optimized to ensure the cracking ability and the tolerance of the system.

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

[0149] Compared with Example 1, Example 11 replaces the defoaming agent in Example 1. The CT value of Example 11 is close to that of Example 1 and lower than that of Comparative Example 13. There is no significant difference in the defoaming speed, and both can be used in nucleic acid release agents.

[0150] Compared with Example 1, Example 12 replaces the PCR enhancer in Example 1, and the CT value of Example 12 is greater than that of Example 1. The compatibility of the replaced material with the downstream PCR amplification system is not as good as that of the existing material. However, the CT value of Example 12 is less than that of Comparative Example 12, which shows that the effect of the replaced nucleic acid releaser is still better than that of the prior art.

[0151] Compared with Example 1, Example 13 eliminates the PVP in Example 1, and the CT value of Example 13 is greater than that of Example 1, indicating that PVP can enhance the lysis effect in cooperation with a strong base. At the same time, the addition of PVP can effectively bind and remove the inhibitory substance components, and can achieve a better effect. However, the CT value of Example 13 is less than that of Comparative Example 12. It can be seen that even without adding PVP, the effect of the nucleic acid releaser is still better than the prior art.

[0152] Compared with Example 1, Example 14 eliminates the grinding beads in Example 1. The CT value of Example 14 is greater than that of Example 1, indicating that the grinding beads can help the elution of the swab, release more nucleic acids, and improve sensitivity. However, the CT value of Example 13 is still smaller than that of Comparative Example 12. It can be seen that even without adding grinding beads, the effect of the nucleic acid releaser is still better than the prior art.

[0153] Compared with Example 1, Example 15 eliminates the methyl silicone oil in Example 1, and 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 pollution, and has no obvious effect on performance.

[0154] Compared with Example 1, the CT values ​​of Comparative Examples 1-12 are all greater than those of Example 1 and Comparative Example 12, and the effects are not as good as the existing technology, indicating that the amount used outside the material range or the absence of the addition of PCR enhancer I cannot take into account both the lysis ability and the tolerance of the system, and cannot meet the use requirements.

[0155] 3. Optimization of nucleic acid release conditions

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

[0157] Oropharyngeal swab samples mainly tested viruses, mycoplasmas and bacteria, including influenza A, Mycoplasma pneumoniae and Streptococcus pneumoniae; sputum samples mainly tested fungi and bacteria, including Aspergillus and Pseudomonas aeruginosa; vaginal swab samples mainly tested fungi, bacteria, parasites, mycoplasmas and viruses, including Candida, Prevotella, Trichomonas vaginalis, Ureaplasma urealyticum and HPV. The conditions of the nucleic acid release method are shown in Table 5.

[0158] Table 5

[0159]

[0160] The nucleic acid releaser prepared in Example 1 was used to perform nucleic acid detection on 10 throat swabs, 10 sputum samples and 10 vaginal swab samples, respectively. The operation steps were as follows: 10 throat swabs, 10 sputum samples and 10 vaginal swab samples were collected and added to the collection tube containing the nucleic acid releaser, respectively, and the mixture was mixed by inversion 10 times and then operated according to the oscillation frequency and time in Table 5. The control group was used before oscillation, and the experimental group was used after oscillation. Real-time fluorescence quantitative PCR amplification analysis was performed using a fluorescence quantitative PCR instrument, and the reaction system was shown in Table 6.

[0161] Table 6

[0162] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0164] Table 7

[0165]

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

[0167] Table 8 Oropharyngeal swab sample test data

[0168]

[0169]

[0170] Table 9 Sputum sample test data

[0171]

[0172] Table 10 Vaginal swab sample test data

[0173]

[0174]

[0175]

[0176]

[0177] As shown in the test results of Table 8, the CT values ​​of Examples 16 to 20 are significantly improved compared with the control group, indicating that under different shaking speeds and times, the nucleic acid releaser can lyse and obtain more nucleic acids. It can be seen from the test results of Examples 16 to 18 that when the shaking speed is 2500rpm, the CT value of Example 17 is less than that of Example 16, and the CT values ​​of Example 18 and Example 17 are similar. It can be seen from the test results of Examples 19 and 20 that when the shaking speed is 3500rpm, the CT values ​​of Examples 19 and 20 are similar, and the difference with the CT values ​​of Examples 17 and 18 is not much. The smaller the CT value, the more pathogenic microorganism nucleic acids are obtained in the sample under such operating conditions, and the higher the sensitivity. It can be seen. In the oropharyngeal swab sample type, when the shaking speed is 2500rpm, the effect of shaking for 1 minute is not sufficient to fully elute enough pathogens from the swab and obtain nucleic acid by maximal lysis; the effect of shaking for 3 minutes is equivalent to shaking for 5 minutes, and when the shaking speed is increased to 3500rpm, the effects of shaking for 1 minute and shaking for 3 minutes are equivalent.

[0178] As shown in the test results of Table 9, the CT values ​​of Examples 16 to 20 are significantly improved compared with the control group, indicating that the addition of glass beads also has a significant effect on sputum samples. It can be seen from the test results of Examples 16 to 18 that when the shaking speed is 2500rpm, the CT value of Example 17 is less than that of Example 16, and the CT values ​​of Example 18 and Example 17 are similar. It can be seen from the test results of Examples 19 and 20 that when the shaking speed is 3500rpm, the CT value of Example 19 is less than that of Example 20. It can be seen from the test results that the CT values ​​of Examples 17, 18 and 19 are similar. It can be seen that the components of the nucleic acid releaser in the present invention contain strong alkali, which can liquefy sputum samples and lyse pathogenic microorganisms, obtain nucleic acids of pathogenic microorganisms, and directly perform nucleic acid detection. In the sputum sample type, when the shaking speed is 2500rpm, the effect of shaking for 3 minutes is equivalent to shaking for 5 minutes, and when the shaking speed is increased to 3500rpm, the effect of shaking for 1 minute is also equivalent, but increasing the shaking time to 3 minutes, although it is still an improved effect, the effect will be slightly weaker than Examples 17 to 19. Sputum samples are relatively complex, and excessive shaking speed and time will not only increase the amount of nucleic acid of pathogenic microorganisms, but also bring more negative inhibitory effects.

[0179] As shown in the test results of Table 10, the CT values ​​of Examples 16 to 20 are significantly improved compared with the control group, indicating that at different shaking speeds and times, the nucleic acid release agent can lyse to obtain more nucleic acids. It can be seen from the test results of Examples 16 to 18 that when the shaking speed is 2500rpm, the CT value of Example 17 is less than that of Example 16, and the CT values ​​of Example 18 and Example 17 are similar. It can be seen from the test results of Examples 19 and 20 that when the shaking speed is 3500rpm, the CT values ​​of Examples 19 and 20 are similar, and the difference with the CT values ​​of Examples 17 and 18 is not significant. It can be seen that in the vaginal swab sample type, the effect of shaking for 1min is the best when the shaking speed is 3500rpm. When the shaking speed is 2500rpm, the effects of shaking for 3min and 5min are basically the same as the effect of shaking for 1min when the shaking speed is 3500rpm, but the average value of the overall improved CT value is within 0.1 CT, and the difference is not significant.

[0180] Combined with the test data of Table 9 and Table 10, considering the need for different sample types of detection effects, factors such as the oscillation speed and oscillation time of the glass beads need to be tested and verified. In the samples of oropharyngeal swabs and vaginal swabs, the results of Examples 16 to 18 show that the overall difference is not large. It is shown that the pathogenic microorganisms on the swab can be fully eluted and nucleic acids can be released at a shaking speed of 2500rpm for 3min. In sputum samples, the corresponding process of the extraction reagent of the prior art, whether manual or automatic, requires the liquefaction of the sample first, but the nucleic acid releaser provided by the present invention does not need to add an additional liquefaction step, and is directly added to the releaser to complete the liquefaction and nucleic acid extraction of the sample. From the results of Examples 17 to 19, it can be seen that for relatively complex samples such as sputum, extending the oscillation time may bring negative effects. This is because in the process of extending the oscillation time, not only more nucleic acids will be eluted and released, but the influence of inhibitors will also increase. From the test results, it can be seen that the oscillation speed and oscillation time need to be relatively balanced to ensure that the elution and release efficiency of pathogenic microorganisms is greater than the influence of inhibitors, so as to obtain higher amplification efficiency and smaller CT values.

[0181] Examples 16 to 18 are operated on conventional equipment and are more universal, and the operating time difference between Example 17 and Example 19 is not much different, so the applicability and cost of Example 17 are more preferred.

[0182] IV. Detection limit and precision analysis of novel coronavirus reference materials

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

[0184] The nucleic acid releaser prepared in Example 1 was used. 200uL of precision reference product 1, precision reference product 2 and detection limit reference product 1 were respectively added to the collection tube containing the nucleic acid releaser, and the mixture was shaken and mixed according to Example 17. 200uL of the mixed solution was extracted by magnetic bead method, and the real-time fluorescence quantitative PCR amplification analysis was directly performed using a fluorescence quantitative PCR instrument. The reaction system is shown in Table 11.

[0185] Table 11

[0186] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0188] Table 12

[0189]

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

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

[0192]

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

[0194] serial number Nucleic acid release agent Magnetic bead method serial number Nucleic acid release agent Magnetic bead method 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 Table 13 and Table 14, the CV% values ​​of the detection precision of the new coronavirus for reference material 1, the nucleic acid release agent and the magnetic bead method are 0.56% and 0.89% respectively; the CV% values ​​of the precision reference material 2, the nucleic acid release agent and the magnetic bead method are 0.78% and 0.50% respectively, both less than 5.0%, meeting the requirements. The detection limit of the new coronavirus for reference material is 2×10 3 copy / mL, because 200mL was added to 2mL of nucleic acid release agent, the detection limit was 200copy / mL, and both the nucleic acid release agent and the magnetic bead method could stably detect 20 times. The overall results showed that there was no significant difference in the extraction performance of the inactivated nucleic acid release agent treatment and the magnetic bead method for the new coronavirus pseudovirus (RNA virus).

[0196] 5. Validation of clinical samples of novel coronavirus

[0197] The nucleic acid releaser prepared in Example 1 was used to test 30 oropharyngeal swab samples for novel coronavirus nucleic acid. The specific operation was as follows: the collected dry swab samples were added to the collection tube containing the nucleic acid releaser, shaken and mixed, 200uL of the nucleic acid releaser was extracted using the magnetic bead method, and then the nucleic acid releaser containing the sample was operated according to Example 17 to release the nucleic acid into the nucleic acid releaser. Real-time fluorescent quantitative PCR amplification analysis was performed using a fluorescent quantitative PCR instrument, and the reaction system was shown in Table 15.

[0198] Table 15

[0199] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0201] Table 16

[0202]

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

[0204] Table 17

[0205]

[0206]

[0207] As shown in Table 17, all 30 oropharyngeal swab samples can be detected. The mean difference in CT values ​​between the magnetic bead method and the nucleic acid release agent sample detection was within 0.5 CT, and there was no significant difference in the test results between the two.

[0208] VI. Lactobacillus Clinical Sample Verification

[0209] The nucleic acid releaser prepared in Example 1 was used, and glass beads were pre-set to perform lactobacillus nucleic acid detection on 20 vaginal swab samples. The specific operation was as follows: the collected dry swab sample was added to a collection tube containing a nucleic acid releaser, shaken and mixed, 200uL of nucleic acid releaser was extracted using a magnetic bead method, and then the nucleic acid releaser containing the sample was operated according to Example 17 to release the nucleic acid in the nucleic acid releaser. Real-time fluorescent quantitative PCR amplification analysis was performed using a fluorescent quantitative PCR instrument, and the reaction system was shown in Table 18.

[0210] Table 18

[0211] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0213] Table 19

[0214]

[0215]

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

[0217] Table 20

[0218]

[0219] As shown in Table 20, all 20 vaginal swab samples can be detected. The mean difference in CT values ​​between the magnetic bead method and the nucleic acid release agent sample detection was within 0.5 CT, and there was no significant difference in the test results between the two.

[0220] VII. Validation of Aspergillus Clinical Samples

[0221] The nucleic acid releaser prepared in Example 1 was used, and glass beads were pre-placed to perform Aspergillus nucleic acid detection on 20 sputum samples. The specific operation was as follows: an appropriate amount of the collected sputum sample was added to the nucleic acid releaser, and 200uL of the nucleic acid releaser was taken for extraction using the magnetic bead method. Next, the nucleic acid releaser containing the sample was operated according to Example 17 to release the nucleic acid into the nucleic acid releaser. Real-time fluorescent quantitative PCR amplification analysis was performed using a fluorescent quantitative PCR instrument, and the reaction system was shown in Table 21:

[0222] Table 21

[0223] Components Reaction system 2X Buffer 10uL 20X Enzyme Mix 1uL Primer Probe Mix 2uL Nuclease-free water 2uL Nucleic acid release agent 5uL

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

[0225] Table 22

[0226]

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

[0228] Table 23

[0229]

[0230] As shown in the table, all 20 sputum samples can be detected. The mean difference of CT values ​​of samples detected by magnetic bead method and nucleic acid release agent is within CT, and there is no significant difference in the test results of 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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: The method comprises 10-100 mM of a strong base, a surfactant with a mass volume ratio of 0.1%-2%, a buffer solution of 10-100 mM, a metal ion chelating agent of 0.1-2 mM, a lysis enhancer of 10-100 mM and a PCR enhancer I of 50-100 mM.

2. The extraction-free rapid nucleic acid release agent according to claim 1, characterized in that: The surfactant comprises at least one of sodium dodecyl sulfate, Triton X1 or Tween 20, preferably sodium dodecyl sulfate; Preferably, the lysis enhancer comprises at least one of guanidine isothiocyanate, guanidine hydrochloride or sodium lauroyl glutamate, preferably guanidine isothiocyanate; Preferably, the PCR enhancer I comprises at least one of potassium chloride, sodium chloride or sucrose, preferably sucrose.

3. The extraction-free rapid nucleic acid release agent according to claim 2, characterized in that: When the surfactant is sodium dodecyl sulfate, the mass volume ratio of the surfactant is 0.1 to 0.5%; When the surfactant is Tween 20, the mass volume ratio of the surfactant is 0.1 to 1%; When the surfactant is Triton X1, the mass volume ratio of the surfactant is 0.5-2%; Preferably, when the cleavage enhancer is guanidine thiocyanate, the concentration of the cleavage enhancer is 25 to 50 mM; When the cleavage enhancer is guanidine hydrochloride, the concentration of the cleavage enhancer is 50 to 100 mM; When the cleavage enhancer is sodium lauroyl glutamate, the concentration of the cleavage enhancer is 10 to 30 mM; Preferably, when the PCR enhancer I is potassium chloride, the concentration of the PCR enhancer I is 70 to 100 mM; When the PCR enhancer I is sodium chloride, the concentration of the PCR enhancer I is 10 to 40 mM; When the PCR enhancer I is sucrose, the concentration of the PCR enhancer I is 50-80 mM.

4. The extraction-free rapid nucleic acid release agent according to any one of claims 1 to 3, characterized in that: Also included is a PCR enhancer II having a mass volume ratio of 1% to 2%; Preferably, it also includes grinding beads with a mass volume ratio of 0.1% to 0.5%, preferably grinding beads with a mass volume ratio of 0.2% to 0.4%; Preferably, it also includes a defoaming agent with a mass volume ratio of 0.05% to 1%; Preferably, the defoaming agent comprises at least one of methyl silicone oil, foaming powder or n-butanol, preferably methyl silicone oil; Preferably, the PCR enhancer II comprises polyvinyl pyrrolidone.

5. Use of the extraction-free rapid nucleic acid release agent according to any one of claims 1 to 4 in any of the following: A1. Application in nucleic acid release or in the preparation of products for nucleic acid release; A2. Application in nucleic acid amplification or in the preparation of products for nucleic acid amplification; A3. Application in the preparation of products for nucleic acid testing.

6. A method for releasing nucleic acid, characterized in that: The method comprises placing a sample in the extraction-free rapid nucleic acid release agent according to any one of claims 1 to 4, and mixing by shaking at room temperature to obtain a nucleic acid solution.

7. The method according to claim 6, characterized in that The shaking conditions include shaking at 2500-3500 rpm for 1-5 min; Preferably, when the shaking speed is 2500 rpm, the shaking time is 1 to 5 min, preferably 3 min; Preferably, when the shaking speed is 3500 rpm, the shaking time is 1 to 3 min, preferably 1 min; Preferably, after the shaking and mixing, the mixture is allowed to stand for 1 to 10 minutes, preferably 5 minutes; Preferably, the sample comprises a swab sample or a sputum sample.

8. A nucleic acid PCR amplification method, characterized in that: The method comprises using the nucleic acid solution prepared by the nucleic acid release method according to claim 6 as a template for PCR amplification.

9. The nucleic acid PCR amplification method according to claim 8, characterized in that: Taking a 20 μL system as an example, the amplification system includes 2 to 10 μL of nucleic acid solution, preferably 5 μL.

10. A PCR amplification kit, characterized in that: The invention comprises the extraction-free rapid nucleic acid release agent according to any one of claims 1 to 4; Preferably, a PCR reaction solution is also included.

Images