Sample nucleic acid release compositions, kits, and uses thereof

Abstract

The present application relates to the field of biological detection, and particularly relates to a sample nucleic acid releasing composition, a kit and application thereof. The present application provides a sample nucleic acid releasing composition, which comprises a lysing agent and a protein inhibitor; the lysing agent comprises a strong alkali, a non-ionic surfactant and an ionic surfactant. In application, the present application can provide that a swab sample is directly added into a sample releasing agent for lysis, and a lysis solution is directly reconstituted to freeze-dried reagents for amplification, without first soaking the swab into a sample storage solution, mixing the sample solution with the releasing agent according to a proportion, and then adding amplification reagents, so that the operation is more convenient.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to sample nucleic acid release compositions, kits, and their applications. Background Technology

[0002] Rapid nucleic acid diagnostic technology has become a key area of ​​development in nucleic acid testing. Common techniques for improving rapid nucleic acid diagnostics include combining rapid extraction and rapid amplification. A branch of rapid extraction technology is nucleic acid release agent technology. The basic principle is that the sample is lysed directly in chemical reagents without complex purification steps, releasing nucleic acids for downstream PCR or isothermal amplification. This release agent technology allows for rapid sample processing and nucleic acid release of simple samples, significantly reducing the sample processing time of nearly 20 minutes compared to existing magnetic bead or column extraction methods. Nucleic acid release agent technology can complete nucleic acid release in just 1-5 minutes. Furthermore, for simple swab samples with low inhibitor content, the sensitivity of this sample release method is almost equivalent to that of magnetic bead or column extraction methods.

[0003] Currently, there are two main types of nucleic acid release agents used in samples. One is alkaline lysis, which uses an alkaline solution to lyse the cell or viral protein coat. By adding certain proportions of surfactants and other components, the degradation effect of the alkaline solution on nucleic acids, especially RNA, is reduced. The other is lysis using a high concentration of surfactants, which uses a variety of non-ionic or ionic surfactant components to lyse the cell or viral protein coat. Guanidine salts are added to inhibit a large number of RNases in the sample, so as to avoid rapid RNA degradation.

[0004] The basic operating procedure for products currently on the market is to collect samples into a preservation solution, mix a portion of the collected preservation solution with a release agent in a certain proportion, and then add the mixture to a liquid amplification reagent for downstream reactions. The above operation requires several steps of tube transfer and sample addition, which is still inconvenient for clinical use.

[0005] For example, the first method uses an alkaline solution to rapidly release nucleic acids from the sample, exposing the RNA to the RNase environment. Although some patents include metal ion chelating agents such as EDTA, their effect is not significant, and they still cannot prevent RNA degradation by RNase. If the amount of alkali added is too small, it will not achieve sample lysis; if the amount of alkali added is too large, it will inhibit PCR. Therefore, in practice, the sample and the release agent should be mixed in a specific ratio for lysis to dilute the alkaline solution without affecting PCR amplification. The maximum volume ratio of sample to release agent is 1:1.2, which can be added to the downstream amplification reaction system without affecting subsequent amplification. This method requires mixing the sample and the release agent in a centrifuge tube to release nucleic acids first, and then adding part or all of it to the amplification reagent reaction tube for PCR reaction. At the same time, most amplification reagents are liquid reagents, requiring prior system construction, which also limits the sample volume, making the operation cumbersome.

[0006] The second method, using surfactants to lyse cell or viral protein coats, is relatively gentle but requires time, resulting in longer sample processing times. Furthermore, it requires high concentrations of surfactants for significant lysis, and the combination of high concentrations of guanidine salts to inhibit RNase leads to high concentrations of these two components entering the downstream amplification system, inhibiting normal PCR amplification and causing false negatives. Although some protocols incorporate enzyme protectants and stabilizers, complete non-inhibition is still impossible. The release agent application still requires mixing the sample and release agent in a specific ratio, or directly rinsing the collected swab sample into the release agent to release the sample nucleic acid. Part or all of the released nucleic acid is then added to liquid amplification reagents for downstream amplification. The amplification reagents require pre-construction of the system, which also limits the sample volume. This method is as cumbersome as the first method. Summary of the Invention

[0007] In view of this, the present invention provides a sample nucleic acid release composition, a kit, and its application. The technical solution of the present invention allows for the direct addition of a sample release agent to swab samples for lysis, and the direct reconstitution and lyophilization of the lysate with reagents for amplification. This eliminates the need to first rinse the swab into a sample preservation solution, mix the sample solution and release agent in a specific ratio, and then add the amplification reagent, making the operation much simpler.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a sample nucleic acid release composition comprising a lysing agent and a protein inhibitor;

[0010] The pyrolysis agent includes: a strong base, a nonionic surfactant, and an ionic surfactant.

[0011] In some embodiments of the present invention, the above-mentioned protein inhibitors include: erdosteine ​​at 0.4 mM, 8.02 mM, 8.42 mM, 10.02 mM or 16.04 mM; and / or TCEP at 0.2 mM, 3 mM or 12 mM.

[0012] In some embodiments of the present invention, the above-mentioned protein inhibitors include: 0.6 to 12.03 mM of erdosteine ​​and / or 0.5 to 10 mM of TCEP.

[0013] In some embodiments of the present invention, the above-mentioned protein inhibitors include: 8.02 mM erdosteine ​​and / or 3 mM TCEP.

[0014] In some embodiments of the present invention, the lysing agent in the above-mentioned sample nucleic acid release composition comprises 10-50 mM of a strong base, 0.1-6% (v / v) of a nonionic surfactant, and 0.002-0.6% (m / v) of an ionic surfactant.

[0015] In some embodiments of the present invention, the lysing agent in the above-mentioned sample nucleic acid release composition comprises 30-50 mM of a strong base, 0.2-5% (v / v) of a nonionic surfactant, and 0.005-0.5% (m / v) of an ionic surfactant.

[0016] In some embodiments of the present invention, the lysing agent in the above-mentioned sample nucleic acid release composition comprises a strong base of 30 mM, 40 mM, 44.6 mM or 45 mM.

[0017] In some embodiments of the present invention, the lysing agent in the above-mentioned sample nucleic acid release composition comprises 0.1% (v / v), 0.2% (v / v), 2% (v / v), 5% (v / v), or 6% (v / v) of a nonionic surfactant.

[0018] In some embodiments of the present invention, the lysis agent in the above-mentioned sample nucleic acid release composition includes 0.002% (m / v), 0.005% (m / v), 0.05% (m / v), or 0.06% (m / v) of an ionic surfactant.

[0019] In some embodiments of the present invention, in the above-mentioned sample nucleic acid release composition, the concentrations of the strong base, the nonionic surfactant, and the ionic surfactant in the lysis agent are all final concentrations.

[0020] In some embodiments of the present invention, the strong base in the above-mentioned sample nucleic acid release composition includes KOH and / or NaOH.

[0021] In some embodiments of the present invention, the final concentration of NaOH in the above-mentioned sample nucleic acid release composition is 30-45 mM.

[0022] In some embodiments of the present invention, the final concentration of NaOH in the above-mentioned sample nucleic acid release composition is 30mM, 40mM, 44.6mM or 45mM.

[0023] In some embodiments of the present invention, the nonionic surfactant in the above-mentioned sample nucleic acid release composition includes 35 and / or Triton X-100.

[0024] In some embodiments of the present invention, in the above-mentioned sample nucleic acid release composition, the... 35 is also called L23, or polyoxyethylene lauryl ether, is a nonionic surfactant that reduces the adsorption of non-specific proteins; Triton X-100, also known as polyethylene glycol octylphenyl ether, can penetrate cell membranes and nuclear membranes, non-specifically dissolving cell membranes and proteins. Other names include 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, and polyethylene glycol tert-octylphenyl ether. Its linear molecular formula is t-Oct-C6H4-(OCH2CH2)xOH, x = 9-10.

[0025] In some embodiments of the present invention, the ionic surfactant in the above-mentioned sample nucleic acid release composition includes sodium dodecyl sulfate.

[0026] In some embodiments of the present invention, the protein inhibitor in the above-described sample nucleic acid release composition includes erdosteine ​​and / or TCEP.

[0027] In some embodiments of the present invention, the erdosteine ​​in the above-mentioned sample nucleic acid release composition, also known as carboxymethylsteine, is a novel mucolytic agent that can significantly reduce the viscosity of sputum and significantly reduce the level of fucose (a marker of mucin glycoprotein) and the dry weight of macromolecules in sputum.

[0028] In some embodiments of the present invention, the TCEP in the above-mentioned sample nucleic acid release composition is named Tris(2-carboxyethyl)phosphine. TCEP can act as a strong reducing agent within a different pH range, reducing disulfide bonds in protein structures and inactivating the protein. The reaction time at room temperature does not exceed 5 minutes. Furthermore, TCEP has no unpleasant odor and is easily oxidized in air.

[0029] In some embodiments of the present invention, the above-mentioned sample nucleic acid release composition further includes: an RNase inhibitor, wherein the RNase inhibitor comprises 2-100 μg / mL sodium polyvinyl sulfonate and / or 0.5-3 mM metal ion chelating agent.

[0030] In some embodiments of the present invention, the RNase inhibitor in the above-mentioned sample nucleic acid release composition comprises: 5-80 μg / mL sodium polyvinyl sulfonate and / or 1.75-3 mM metal ion chelating agent.

[0031] In some embodiments of the present invention, the RNase inhibitor in the above-mentioned sample nucleic acid release composition includes sodium polyvinyl sulfonate at concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL.

[0032] In some embodiments of the present invention, the RNase inhibitor in the above-mentioned sample nucleic acid release composition includes: 1.75 mM, 2 mM or 3 mM of a metal ion chelating agent.

[0033] In some embodiments of the present invention, the metal ion chelating agent in the above-mentioned sample nucleic acid release composition includes EGTA and / or EDTA.

[0034] In some embodiments of the present invention, the RNase inhibitor in the above-mentioned sample nucleic acid release composition comprises: 5-80 μg / mL sodium polyvinyl sulfonate and 1.75-3 mM EGTA.

[0035] The present invention also provides the use of the above-described sample nucleic acid release composition in the preparation of kits for nucleic acid extraction and / or detection.

[0036] The present invention also provides a method for nucleic acid amplification, wherein the above-mentioned sample nucleic acid release composition is mixed with the sample to be tested, and a portion of the mixture is used as a nucleic acid template for nucleic acid amplification; the mixing does not include the step of mixing with a preservation solution; the nucleic acid amplification includes PCR or isothermal amplification.

[0037] In some embodiments of the present invention, the preservation solution in the above-described nucleic acid amplification method includes: cell preservation solution, TE buffer, physiological saline, UTM preservation solution, and VTM preservation solution.

[0038] The present invention also provides kits for nucleic acid extraction and / or detection, comprising the above-described sample nucleic acid release composition and acceptable adjuvants or carriers.

[0039] In some embodiments of the present invention, the acceptable adjuvants in the above-described kit include one or more of the following: amplifying agents, preservatives, and / or defoamers.

[0040] In some embodiments of the present invention, the above-mentioned kit further includes: buffer solution and / or 0.2 to 5% (v / v) Tween 20.

[0041] In some embodiments of the present invention, the amplification agent in the above-mentioned kit comprises: 0.5-5% (v / v) DMSO and 1-10% (m / v) mannitol.

[0042] In some embodiments of the present invention, the amplification agent in the above-mentioned kit comprises 0.5% (v / v), 2% (v / v), 4% (v / v), or 5% (v / v) DMSO.

[0043] In some embodiments of the present invention, the amplification agent in the above-mentioned kit comprises 1% (m / v), 4% (m / v), 6% (m / v), or 10% (m / v) mannitol.

[0044] In some embodiments of the present invention, the amplification agent in the above-described kit comprises 2% (v / v) DMSO and 4% (m / v) mannitol.

[0045] In some embodiments of the present invention, the amplification agent in the above-mentioned kit further includes: 1-5% (w / v) trehalose and 0.5-5 mg / mL BSA.

[0046] In some embodiments of the present invention, the amplification agent in the above-mentioned kit further includes: 4-4.7% (w / v) trehalose and 1.3-2.5 mg / mL BSA.

[0047] In some embodiments of the present invention, the amplification agent in the above-mentioned kit further includes 4% (w / v), 4.2% (w / v), 4.4% (w / v), 4.5% (w / v), or 4.7% (w / v) of trehalose.

[0048] In some embodiments of the present invention, the amplification agent in the above-mentioned kit further includes: 1.3 mg / mL, 1.5 mg / mL, 2.0 mg / mL or 2.5 mg / mL of BSA.

[0049] In some embodiments of the present invention, the preservative in the above-mentioned kit includes: 0.01 to 0.1% (v / v) NaN3.

[0050] In some embodiments of the present invention, the preservative in the above-mentioned kit includes: 0.05% (v / v) NaN3.

[0051] In some embodiments of the present invention, the defoamer in the above-mentioned kit comprises: 0.05 to 0.5% (v / v) SE-15.

[0052] In some embodiments of the present invention, the defoamer in the above-mentioned kit comprises: 0.3% (v / v) SE-15.

[0053] In some embodiments of the present invention, the buffer in the above-described kit comprises: 5-100 mM Tricine buffer and / or Hepes buffer.

[0054] The beneficial effects of this invention include:

[0055] (1) The sample release agent of the present invention contains a component that enhances the lysis ability, thereby improving the sample lysis ability;

[0056] (2) The sample release agent of the present invention incorporates the highly efficient RNase inhibitor PVSA to prevent the sample RNA from being rapidly degraded by RNase.

[0057] (3) In application, the technical solution of the present invention can provide that the swab sample can be directly added to the sample release agent for lysis, and the lysis solution can be directly reconstituted and lyophilized to amplify the reagent. There is no need to rinse the swab into the sample preservation solution first, mix the sample solution and the release agent in proportion and then add the amplification reagent, which is more convenient in operation. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0059] Figure 1 The diagram compares the existing technology flow in the market with the technology flow of the present invention; wherein: the left side shows the existing technology flow in the market; the right side shows the technology flow of the present invention. Detailed Implementation

[0060] This invention discloses a sample nucleic acid release composition, a kit, and its applications.

[0061] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0062] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0063] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0064] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0065] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0066] The sample release agent provided by this invention contains six components with different functions, namely:

[0067] (1) A 10-300mM alkaline solution with sample lysis function, 0.2%-5% Brij35, 0.2%-5% Triton X-100 nonionic surfactants to enhance the lysis function, and 0.005%-0.05% SDS ionic surfactants, wherein the alkaline solution can be KOH or NaOH.

[0068] (2) A component with RNase inhibitor function, one or more of 0.5mM~3mM EDTA / EGTA and 5μg / mL~80μg / mL sodium polyvinyl sulfonate (PVSA).

[0069] (3) Components with mucin-inhibiting function: 0.6-12.03mM erdosteine, 0.5mM-10mM TCEP.

[0070] (4) Components that reduce RNA degradation in alkaline environments: 0.2% to 5% Tween20.

[0071] (5) Components with buffering function: one of the 5mM to 100mM Tricine / Hepes buffer solutions.

[0072] (6) Components with enzyme protection, PCR or isothermal amplification enhancement: 0.5% to 5% DMSO, 1% to 10% mannitol, 1% to 8% glycerol, 1% to 5% trehalose, 0.5 mg / mL to 1.5 mg / mL BSA, 1 M to 5 M betaine.

[0073] (7) Contains preservative and defoamer components: preservative 0.01%~0.1% NaN3, defoamer 0.05%~0.5% SE-15.

[0074] Specific applications include:

[0075] The sample release agent of this invention allows for direct collection of swab samples for sample lysis and nucleic acid release. 20-50 μL of the released nucleic acid solution is then directly reconstituted with lyophilized reagent. The reconstituted lyophilized reagent, after centrifugation, can be directly used for PCR amplification. This lyophilized reagent can be a qPCR amplification reagent or an isothermal amplification reagent. Existing technologies require pre-collecting swabs in a preservation solution, then mixing a portion of the preservation solution with the release agent in a specific ratio for lysis. The release agent component in the lysed sample mixture has PCR inhibitory effects, making direct reconstitution with lyophilized reagent for further amplification impossible. Liquid amplification reagent needs to be added to reduce the final concentration of the release agent component in the system, thereby reducing amplification inhibition.

[0076] The technical solution of this invention is achieved through a combination of the following methods:

[0077] ① Reduce the concentration of the alkaline solution to a low concentration (10-50 mM) that provides a certain lysis effect without inhibiting downstream PCR. The alkaline solution can be KOH or NaOH. By adding surfactants to enhance lysis, the lysis effect is ensured. SDS can lyse the cell surface protein coat, but it is a strong PCR inhibitor. Triton X-100 reduces the inhibitory effect of SDS on PCR, achieving lysis of the cell protein coat without inhibiting PCR amplification. The concentrations of each component in the release agent are as above. Swabs contain a large amount of impurities and mucin. If the lysis and digestion are insufficient, a large amount of mucin will be introduced into the downstream amplification, leading to amplification inhibition. This technical solution allows for direct reconstitution of the lysed sample into the lysed reagent, i.e., adding more lysed sample. Mucin-inhibiting components, erdosteine ​​and TCEP, are added to the release agent. Erdosteine ​​is an expectorant used to treat patients with thick sputum and difficulty expectorating caused by chronic bronchitis, bronchial asthma, and other diseases. It is a mucolytic agent that digests mucoproteins, reduces mucus viscosity, and decreases the inhibition of amplification by mucus. The lysate is also easier to aspirate and less likely to clog pipette tips. The concentration of erdosteine ​​added to the system is 0.6–12.03 mM; the addition of >12.03 mM erdosteine ​​can also inhibit PCR amplification. TCEP is a reducing agent that can alter the rheological properties of mucus and reduce its viscosity by breaking disulfide bonds in mucoproteins. The concentration of TCEP added is 0.5–10 mM.

[0078] ② Swab samples also contain a large amount of RNase. When the nucleic acid in the sample is lysed and exposed, it is easily degraded by a large amount of RNase, reducing the nucleic acid detection rate. To improve the nucleic acid detection rate, one approach is to increase the amount of nucleic acid added. This technical solution directly reconstitutes the lysed sample nucleic acid into the lyophilized reagent to increase the amount added and improve detection sensitivity. Another approach is to add an RNase inhibitor to the release agent. This technical solution discovered a highly efficient and low-cost RNase inhibitor, PVSA (sodium polyvinyl sulfonate). However, PVSA is a strong inhibitor of PCR; 100 μg / mL is sufficient to completely inhibit PCR. The inventors unexpectedly discovered that even a very small amount of PVSA still has an inhibitory effect on RNase. When the amount added to the release agent is 0.5 μg / mL to 80 μg / mL, it meets the requirements for directly releasing the sample, reconstitutes the lyophilized reagent after lysation, and does not inhibit downstream amplification. Simultaneously, EGTA / EDTA is also added to the release agent, which can chelate metal ions, thereby inhibiting RNases containing metal ions.

[0079] ③ The combined effect of multiple PCR-inhibiting lysis components poses a significant challenge to amplification reagents. Therefore, in publicly available technical solutions, swab samples are collected into a preservation solution, mixed with a release agent in a specific ratio, and a portion or all of this mixture is added to the liquid amplification reagent. The purpose is to reduce the concentration of these PCR-inhibiting components in the amplification system. This technical solution incorporates a certain amount of PCR enhancer components to improve resistance to these inhibitory components. Glycerol and mannitol contain numerous polyhydroxy structures, which can competitively inhibit the destruction of enzyme and double-stranded DNA interstrand salt bridges by a large number of salt ions in the sample, thus enhancing the reagent's resistance to high-salt inhibitors. Trehalose can form a protective film on the surface of the amplification enzyme, reducing damage to the enzyme from inhibitors. DMSO can improve the amplification efficiency of the amplification enzyme, and betaine can provide an activator for isothermal amplification.

[0080] ④ Adding an antifoaming agent reduces the bubbles generated after the nucleic acid solution is reconstituted and lyophilized, thus reducing fluorescence interference.

[0081] This technical solution allows for the direct addition of swab samples to the lysing agent for lysis. The amount of swab sample added to the lysing agent can range from 400 μL to 5 mL. After lysis, the sample is immediately reconstituted with the lyophilized reagent before amplification, simplifying the operation, increasing the sample volume, and improving detection sensitivity. The sample volume for reconstituted lyophilized reagent is 20–50 μL. A volume <20 μL will result in insufficient sample in the amplification system and reduced sensitivity, while a volume >50 μL will result in a higher amount of inhibitor in the amplification system, leading to amplification inhibition.

[0082] The lyophilized reagent is obtained by lyophilizing a lyophilized liquid reagent. The volume of the lyophilized liquid reagent can be 5 μL to 80 μL. The procedure for directly adding the sample to the lyophilized reagent for amplification should be followed, and the ratio of sample volume to lyophilized reagent volume should be 1:4 to 10:1.

[0083] The lyophilization reagent formulation is as follows: raffinose 3%, β-cyclodextrin 1%, mannitol 2%, BSA 2%, PEG20000 1.5%.

[0084] In Examples 1 to 8 of this invention, all raw materials and reagents used can be purchased from the market.

[0085] The present invention will be further illustrated below with reference to the embodiments:

[0086] Example 1: The combination of SDS and Triton X-100 achieves the effect of lysing samples.

[0087] Mixing SDS at a certain concentration with Triton X100 can achieve lysis without inhibiting downstream amplification. Clinically positive and negative vaginal swab samples of Chlamydia trachomatis were used. A mixture of SDS and Triton X100 at a certain concentration was prepared with 1 mL of sterile water to obtain release agents 1–28 (see Table 1), with an aqueous solution as a control. A self-developed 15 μL lyophilized Chlamydia trachomatis PCR amplification reagent was used. The procedure was as follows: Clinically positive vaginal swab samples of Chlamydia trachomatis were rinsed into 3 mL of physiological saline and vortexed to obtain a positive sample dilution. 28 negative vaginal swab samples were rinsed into 1 mL of release agents 1–28 and vortexed. Then, 100 μL of the positive sample dilution was added to each release agent to obtain a simulated real clinically positive vaginal swab sample. After vortexing and standing for 1 minute, 25 μL of the solution was added to 15 μL of lyophilized amplification reagent, reconstituted, and directly amplified in a rapid amplification instrument.

[0088] The results are shown in Table 2. When the concentration of SDS in the release agent is between 0.005% and 0.05%, and the concentration of Triton X100 is between 0.2% and 5%, there are different degrees of sample lysis effect, and the inhibitory effect of SDS on PCR amplification can be weakened.

[0089] Table 1

[0090]

[0091]

[0092] Table 2. Detection of the effect of SDS and Triton X100 on sample lysis

[0093]

[0094]

[0095] Example 2: Comparison of pyrolysis effects between the combination of SDS and Triton X-114 and the combination of SDS and Triton X-100.

[0096] Clinically positive and negative vaginal swab samples of Chlamydia trachomatis were used. A mixture of SDS and Triton X114, and a mixture of SDS and Triton X100, were prepared using 1 mL of sterile water at specific concentrations. A self-developed 15 μL lyophilized Chlamydia trachomatis PCR amplification reagent was used. The procedure was as follows: a clinically positive vaginal swab sample was rinsed into 3 mL of physiological saline and vortexed to obtain a positive sample dilution. Six negative vaginal swab samples were rinsed into 1 mL of release agent and vortexed. Then, 100 μL of different positive sample dilutions were added to each release agent to obtain simulated real clinically positive vaginal swab samples 1-6. After vortexing and standing for 1 minute, 25 μL of the solution was added to 15 μL of lyophilized amplification reagent, reconstituted, and then directly amplified in a rapid amplification instrument.

[0097] The results are shown in Table 4. When SDS and Triton X114 were combined in the release agent, the lysis effect of the release agent on the sample was weakened, and the amplification Ct value was delayed by 2-3 compared with the combination of SDS and Triton X100, indicating that the combination of SDS and Triton X100 has a stronger sample lysis ability.

[0098] Table 3

[0099]

[0100]

[0101] Table 4 Comparison of lysis detection of SDS and Triton X100 samples with SDS and Triton X114 samples.

[0102]

[0103] Example 3: Detection of the ability of different concentrations of Brij35 to enhance the lysis of samples.

[0104] Using clinically positive and negative vaginal swab samples of Chlamydia trachomatis, 0.1-6% final concentration Brij35 was prepared with 1 mL of sterile water to obtain release agents 29-34, with an aqueous solution as a control. The reagent used was a self-developed 15 μL lyophilized Chlamydia trachomatis PCR amplification reagent. The procedure was as follows: Clinically positive vaginal swab samples of Chlamydia trachomatis were rinsed into 3 mL of physiological saline and vortexed to obtain a positive sample dilution. Six negative vaginal swab samples were rinsed into 1 mL of release agent 29-34 and vortexed. Then, 100 μL of the positive sample dilution was added to each release agent to obtain a simulated real clinically positive vaginal swab sample. After vortexing and standing for 1 minute, 20 μL of the sample was added to 15 μL of lyophilized amplification reagent, reconstituted, and directly amplified in a rapid amplification instrument.

[0105] The results are shown in Table 6. When the Brij35 concentration in the release agent was between 0.2% and 5%, the amplification Ct values ​​were all earlier than those of the control water samples, indicating that the sample lysis effect was different to varying degrees and could enhance the ability to release sample nucleic acids.

[0106] Table 5

[0107]

[0108] Table 6. Detection of the enhancement of sample lysis ability by different concentrations of Brij35.

[0109] water 0％ 36.9 36.8 36.5 36.7 Release agent 29 0.10％ 36.4 36.9 36.5 36.6 Release agent 30 0.20％ 35.4 35.2 35.8 35.5 Release agent 31 1.00％ 33.1 34.5 34.3 34.0 Release agent 32 3.00％ 32.5 33.3 33.6 33.1 Release agent 33 5.00％ 34.6 35.7 35.4 35.2 Release agent 34 6.00％ 35.9 36.6 36.3 36.3 Negative control / No Ct No Ct No Ct No Ct Positive quality control / 27.3 27.5 27.4 27.4

[0110] Example 4: Comparison of the effects of Brij35 and Brij58 on improving sample lysis ability.

[0111] Using clinically positive and negative vaginal swab samples of Chlamydia trachomatis, release reagents containing Brij35 and Brij58 were prepared in 1 mL of sterile water. The reagent used was a self-developed 15 μL lyophilized Chlamydia trachomatis PCR amplification reagent. The procedure was as follows: Clinically positive vaginal swab samples were rinsed into 3 mL of physiological saline and vortexed to obtain a positive sample dilution. Six negative vaginal swab samples were rinsed into 1 mL of each of the two release reagents and vortexed. Then, 100 μL of the different positive sample dilutions were added to each release reagent to obtain simulated real clinically positive vaginal swab samples 1–6. After vortexing and standing for 1 minute, 20 μL of the solution was added to 15 μL of lyophilized amplification reagent, reconstituted, and then directly amplified in a rapid amplification instrument.

[0112] The results are shown in Table 8. Adding Brij35 to the release agent resulted in earlier Ct values ​​compared to adding Brij58, indicating that Brij35 has a stronger ability to enhance sample lysis.

[0113] Table 7

[0114]

[0115]

[0116] Table 8. Comparison of Brij35 and Brij58 in improving sample lysis effect.

[0117]

[0118] Example 5: Comparison of RNase inhibition effects of different concentrations of PVSA.

[0119] The samples used were COVID-19 pharyngeal swab samples containing different concentrations of PVSA release agent 35–44 (see Table 9). The reagents used were a self-developed 15 μL lyophilized COVID-19 PCR amplification reagent. The procedure was as follows: COVID-19 positive pharyngeal swab samples were rinsed into 3 mL of physiological saline and vortexed to obtain a positive sample dilution. Eleven negative pharyngeal swab samples were rinsed into 1 mL of release agent 35–44 and vortexed. Then, 100 μL of the positive sample dilution was added to each release agent to obtain a simulated clinical positive pharyngeal swab sample. After vortexing and incubation at room temperature for 2 hours (with a 1-minute incubation period as a control), 20 μL of the sample was added to 15 μL of lyophilized amplification reagent, reconstituted, and directly amplified in a rapid amplification instrument.

[0120] The results are shown in Table 10. When no PVSA was added to the release agent, a large amount of RNase in the sample degraded RNA after standing for 2 hours. As the concentration of PVSA increased, RNase was inhibited, and the amplification Ct was close to that of the control after standing for 1 minute. When the amount of PVSA added was >80 μg / mL, it would lead to amplification inhibition. Therefore, a certain concentration of PVSA in the release agent has an RNase inhibitory effect, and the optimal concentration range is 5 μg / mL to 80 μg / mL.

[0121] Table 9

[0122]

[0123] Table 10 Comparison of RNase inhibitory effects of different concentrations of PVSA

[0124]

[0125] Example 6: Detection of the effect of erdosteine ​​and TCEP on the release agent to enhance tolerance in mucus samples.

[0126] The samples used were COVID-19 nasal swabs. Different concentrations of erdosteine ​​and TCEP were added to the release agent to obtain release agents ranging from 45 to 52. The reagents used were self-developed 10 μL lyophilized COVID-19 PCR amplification reagent. The procedure was as follows: COVID-19 positive nasal swab samples were collected and rinsed into 3 mL of physiological saline, then vortexed to obtain a positive sample dilution. Two 1 mL vials of release agent were taken and rinsed into one and two negative nasal swabs respectively, vortexed to mix, and then 100 μL of the positive sample dilution was added to each to obtain positive nasal swab samples simulating different clinical mucus concentrations. After vortexing and standing for 1 min, 20 μL of the solution was added to 10 μL of lyophilized amplification reagent, reconstituted, and then directly amplified in a rapid amplification instrument.

[0127] The results are shown in Table 12. The release agent containing 8.02 mM erdosteine ​​improved the reagent's tolerance to mucus samples, as did the release agent containing 3 mM TCEP. When added in combination to the release agent, the effect was the same as when added separately, and no stronger tolerance to mucus samples was observed.

[0128] Table 11

[0129]

[0130] Table 12 Detection of the effects of erdosteine ​​and TCEP on the enhancement of tolerance in mucus samples by release agents

[0131]

[0132]

[0133]

[0134] Example 7: Detection of the salt ion resistance of amplification reagents enhanced by DMSO and mannitol.

[0135] The samples used were nucleic acid solutions from vaginal swabs of Chlamydia trachomatis. Different concentrations of DMSO and mannitol were added to 25 μL of lyophilized amplification reagent. An additional 20 μL of NaCl solution with final concentrations of 30 mM, 60 mM, and 100 mM was added to the nucleic acid solution. 20 μL of the solution was added to 25 μL of lyophilized amplification reagent, and after reconstitution, the amplification reaction was carried out directly in a rapid amplification instrument.

[0136] The results are shown in Table 14. The results show that adding 0.5%–5% DMSO and 1%–10% mannitol to the amplification system can improve the tolerance of the amplification reagent to NaCl solution. When 2% DMSO and 4% mannitol are added to the solution at the same time, compared with the control without adding DMSO and mannitol, the tolerance of the amplification reagent to NaCl solution is improved, increasing the tolerance of the amplification reagent from 30mM NaCl solution to 60mM NaCl solution (detection of amplified Ct indicates tolerance, and non-detection indicates intolerance).

[0137] Table 13

[0138]

[0139]

[0140] Table 14. DMSO and mannitol enhance the salt ion tolerance of amplification reagents.

[0141]

[0142]

[0143] Example 8: Comparison of the processing methods and results of this technical solution for COVID-19 detection of pharyngeal swab samples with those of the commercially available Sansure Biotech COVID-19 test kit.

[0144] Six positive and two negative pharyngeal swab samples were used for the sample collection. Eight volunteers were recruited, and two pharyngeal swabs were collected from each volunteer. Two pharyngeal swabs were randomly rinsed into Sansure Biotech's preservation solution and 1 mL of the release agent from this protocol, respectively, resulting in eight collection solutions from the marketed kit's preservation solution and eight collection solutions from the protocol's release agent. 50 μL of the solution from the six positive pharyngeal swab samples and 50 μL of the solution from the two negative pharyngeal swab samples were added to each of the eight collection solutions. Subsequent testing was performed according to the respective manufacturer's instructions. A comparison of the operating steps between the marketed kit and this protocol is shown in Table 8. The concentrations of each component of the release agent 53 in this protocol are shown in Table 16. The sample volume and lyophilized reagent volume in this protocol are both 20 μL, resulting in a sample-to-reagent ratio of 1:1.

[0145] The results showed that the proposed method only involves three steps: lysing the pharyngeal swab sample with the release agent, reconstitute the lyophilized bulb reagent, and then amplify it. This reduces the need for commercially available kits to rinse the pharyngeal swab into the preservation solution and mix it with the release agent for lysis, as well as to prepare the amplification reagent solution, making the operation more convenient.

[0146] As shown in Table 17, the positive and negative concordance rate of this technical solution with the commercially available kits for COVID-19 sample detection is 100%. For median samples, the difference in amplified Ct values ​​between this technical solution and the commercially available kits is within 1 Ct. For low-value samples, this technical solution, due to its larger sample volume, achieves Ct values ​​more than 1 Ct earlier than the commercially available kits. In the case of positive samples, the commercially available kits showed some missed detections among replicates, while this solution detected all of them. This indicates that this solution has the advantages of simpler operation and higher detection sensitivity.

[0147] Table 15 Comparison of the operating procedures between commercially available reagent kits and this protocol.

[0148]

[0149] Table 16 Release Agent 53 Components

[0150] NaOH 44.6mM Brij35 4.33％ SDS 0.03％ Triton X100 5.00％ EGTA 1.75mM PVSA 43 μg / mL Edostein 11.23mM HEPES 35mM DMSO 4％ Trehalose 4.4％ BSA 1.3 mg / mL SE-15 0.30％ <![CDATA[NaN3]]> 0.05％

[0151] Table 17 Comparison of processing methods and results of this technical solution and commercially available reagent kits for COVID-19 testing of pharyngeal swab samples.

[0152]

[0153]

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for nucleic acid amplification for non-disease diagnostic purposes, characterized in that, After mixing the sample nucleic acid release composition with the sample to be tested, a portion of the mixture is used as a nucleic acid template for nucleic acid amplification. The mixing process does not include the step of mixing with the preservation solution; The nucleic acid amplification includes PCR or isothermal amplification; The sample nucleic acid release composition includes a lysing agent, a protein inhibitor of 0.2–16.04 mM, and an RNase inhibitor; The pyrolysis agent includes: a strong base, a nonionic surfactant, and an ionic surfactant; The pyrolysis agent comprises 10-50 mM of strong base, 0.2-5% (v / v) of nonionic surfactant, and 0.005-0.5% (w / v) of ionic surfactant; The strong base includes KOH and / or NaOH; The nonionic surfactants include Brij® 35 and / or Triton X-100; The ionic surfactant includes sodium dodecyl sulfate; The protein inhibitors include erdosteine ​​and / or TCEP; The RNase inhibitor comprises 5-80 μg / mL sodium polyvinyl sulfonate and / or 1.75-3 mM metal ion chelating agent.

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