Cracking preservation solution, combined product, kit, preparation method of nucleic acid sample and detection method

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a lysis preservation solution, a combined product, a kit, a preparation method of a nucleic acid sample and a detection method. The preparation method comprises the following steps: fully adsorbing nucleic acid in a nucleic acid extracting solution of a biological sample to be detected by using cationic magnetic beads, collecting the cationic magnetic beads adsorbed with the nucleic acid, and preparing a nucleic acid sample. The nucleic acid sample prepared by adopting the preparation method disclosed by the invention can be directly subjected to PCR (Polymerase Chain Reaction) and other detections without cleaning, is simple to operate and is suitable for emergency treatment and other occasions.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering, and particularly relates to a lysis preservation solution, a combination product, a kit, a preparation method of a nucleic acid sample, and a detection method. Background Art

[0002] With the continuous advancement of molecular biology technology, the detection and typing of pathogens has become an important branch in the field of molecular diagnosis. In this process, efficient and simple extraction and purification of target nucleic acids from biological samples is a key step in achieving molecular detection.

[0003] At present, the purification technology of viral nucleic acid is mainly divided into three methods: traditional method, column membrane method, magnetic bead method and nucleic acid releaser. Traditional nucleic acid purification technology usually involves cell lysis to release nucleic acid, followed by the use of organic solvents such as phenol and chloroform to remove impurities such as protein, and the collection of nucleic acid by alcohol precipitation. Finally, the nucleic acid needs to be dissolved in water or buffer. The column membrane method and magnetic bead method adsorb and purify nucleic acid through silica membrane or magnetic beads. These methods first use lysis solution to lyse cells to release nucleic acid, and then use column membrane or magnetic beads to adsorb nucleic acid under specific conditions, and then use washing solution to remove impurities such as protein, and finally elute nucleic acid from column membrane or magnetic beads through elution solution. Like the traditional method, their operation steps are also cumbersome and time-consuming, and it is difficult to meet the needs of rapid detection in emergency and other scenarios. Among them, the magnetic bead method nucleic acid extraction uses silicon hydroxy magnetic beads, which bind nucleic acid under low pH, high salt and alcohol conditions, and elute nucleic acid under high pH and low salt conditions. Nucleic acid releasers release nucleic acids by lysing samples, and then add the lysed products to the amplification system. The operation is convenient. However, since the releasers dilute the samples and the amount of nucleic acids added to the amplification system is small, the detection sensitivity is low, which limits its application, especially the release of nucleic acids in large-volume samples.

[0004] Traditional nucleic acid extraction methods have problems such as cumbersome operations and are difficult to meet the needs of occasions such as emergency situations.

[0005] In view of this, this application is hereby filed. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a lysis preservation solution, a combination product, a kit, a preparation method of a nucleic acid sample, and a detection method. The technical solutions include the following:

[0007] One or more embodiments of the present application provide a method for preparing a nucleic acid sample, the preparation method comprising the following steps:

[0008] The nucleic acid in the nucleic acid extract of the biological sample to be tested is fully adsorbed by cationic magnetic beads, and the cationic magnetic beads adsorbed with the nucleic acid are collected to prepare a nucleic acid sample.

[0009] In some embodiments of the present application, the cationic magnetic beads include amino magnetic beads.

[0010] In some embodiments of the present application, the amount of the cationic magnetic beads per 1 mL of the nucleic acid extract is 1 μg-200 μg;

[0011] Optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 1 μg-100 μg;

[0012] Optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 5 μg-50 μg.

[0013] In some embodiments of the present application, the collecting method includes magnetic attraction.

[0014] In some embodiments of the present application, the step of preparing the nucleic acid extract comprises: mixing the biological sample to be tested with a lysis preservation solution to prepare the nucleic acid extract.

[0015] In some embodiments of the present application, the lysis preservation solution comprises:

[0016] pH 7.5-10 buffer,

[0017] 0.5mM-5mM EDTA,

[0018] 0.25%-2% (v / v) NP-40 (Nonidet(R) P-40, Chinese name: ethylphenyl polyethylene glycol),

[0019] 0.25%-2% (v / v) Tween-20,

[0020] 10mM-100mM guanidine isothiocyanate (GITC),

[0021] 20mM-250mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol,

[0022] 0.01%-0.5% (v / v) Proclin 300, and

[0023] 1%-5% (v / v) glycerol;

[0024] Optionally, the buffer comprises one or more of Tris-HCl buffer, citrate buffer and HEPES buffer;

[0025] Optionally, the buffer comprises a Tris-HCl buffer of pH 8.2-9.5, 5mM-50mM;

[0026] Optionally, the lysis preservation solution comprises:

[0027] pH 8.2-9.5, 10mM-20mM Tris-HCl buffer,

[0028] 0.5mM-3mM EDTA,

[0029] 0.25%-1% (v / v) NP-40,

[0030] 0.25%-1% (v / v) Tween-20,

[0031] 10mM-30mM guanidine thiocyanate,

[0032] 50mM-250mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol,

[0033] 0.1%-0.25% (v / v) Proclin 300, and

[0034] 1%-3% (v / v) glycerol.

[0035] In some embodiments of the present application, the biological sample to be tested includes a swab sample.

[0036] In some embodiments of the present application, the biological sample to be tested includes one or more of a serum sample, a plasma sample, a urine sample, a whole blood sample, and an environmental sample;

[0037] Optionally, the environmental sample comprises a water sample.

[0038] One or more embodiments of the present application provide a method for detecting a target nucleic acid in a biological sample to be tested, the detection method comprising the following steps:

[0039] Prepare a nucleic acid sample according to the preparation method; and

[0040] The nucleic acid sample is tested.

[0041] In some embodiments of the present application, the detection method includes PCR method;

[0042] Optionally, the PCR method comprises QPCR.

[0043] In some embodiments of the present application, the amplification system includes 15 μL-25 μL of the nucleic acid sample.

[0044] One or more embodiments of the present application provide a combination product, the combination product comprising:

[0045] Cationic magnetic beads as defined above; and

[0046] The lysis storage medium defined above.

[0047] One or more embodiments of the present application provide a lysis preservation solution, which includes the lysis preservation solution defined above.

[0048] One or more embodiments of the present application provide a kit, comprising:

[0049] The combination product; or

[0050] The lysis preservation solution.

[0051] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0052] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0054] the term

[0055] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0056] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0057] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0058] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0059] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0060] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0061] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0062] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0063] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0064] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0065] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0066] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0067] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0068] In this application, m / v means mass-to-volume ratio, and % (m / v) means the mass percentage contained in a certain volume of a mixed system. For example, if the concentration of substance A in a mixed system is 5% (m / v), it means that 5 grams of substance A is contained in every 100 ml of the mixed system.

[0069] In this application, mmol / L and mM both represent millimoles per liter and can be used interchangeably. mol / L and M both represent moles per liter and can be used interchangeably.

[0070] In the present application, the concentration or dosage of each component in the preservation lysate, unless otherwise specified, refers to the final concentration or final dosage in the preservation lysate, which is equivalent to the final concentration or final dosage in the reagent constituting the preservation lysate.

[0071] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0072] Current rapid nucleic acid purification technologies, such as the technical solutions described in CN105420403B, CN108315325A, and CN110607297B, are more convenient and efficient in operation than traditional nucleic acid purification technologies, but there is still room for improvement. The present application has developed a whole-sample nucleic acid enrichment and extraction scheme, which uses cationic magnetic beads to capture nucleic acids by electrostatic binding, and can be amplified with magnetic beads without washing after capture. The present application is simple to operate, low in cost, and can quickly extract and enrich nucleic acids in samples, significantly improving detection sensitivity, and is particularly suitable for rapid detection in emergency and other occasions. The present application can meet the needs for sensitivity and convenience.

[0073] In a first aspect of an embodiment of the present application, a method for preparing a nucleic acid sample is provided, the method comprising the following steps:

[0074] The nucleic acid in the nucleic acid extract of the biological sample to be tested is fully adsorbed by cationic magnetic beads, and the cationic magnetic beads adsorbed with the nucleic acid are collected to prepare a nucleic acid sample.

[0075] The present application does not specifically limit the type of cationic magnetic beads, as long as they can capture nucleic acids by electrostatic binding. In some embodiments of the present application, the cationic magnetic beads include amino magnetic beads, such as Bio MA0308N or Merck Chemical 39572001. The magnetic beads can effectively adsorb and enrich nucleic acids in the sample through electrostatic action, significantly improving the sensitivity of detection.

[0076] In some embodiments of the present application, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 1 μg-200 μg (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 , 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 μg); optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 1 μg-100 μg; optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 5 μg-50 μg; optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 15 μg-25 μg; optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 18 μg-22 μg.

[0077] In the present application, the cationic magnetic beads after adsorbing nucleic acid can be collected by centrifugation, magnetic absorption, etc. In some embodiments of the present application, the collection method is magnetic absorption, which can achieve efficient collection of cationic magnetic beads.

[0078] In some embodiments of the present application, the step of preparing the nucleic acid extract comprises: mixing the biological sample to be tested with a lysis preservation solution to prepare the nucleic acid extract.

[0079] The present application does not specifically limit the formulation of the lysis preservation solution, as long as it can lyse cells, release nucleic acids and keep nucleic acids stable. In some embodiments of the present application, the lysis preservation solution includes:

[0080] (1) a buffer having a pH of 7.5-10, wherein the pH of the buffer is, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0; the buffer is mainly used to prevent changes in the pH value of the reagent;

[0081] (2) 0.5 mM-5 mM EDTA, the concentration of EDTA is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mM; in the present application, EDTA is used to inhibit the activity of nucleases and maintain the stability of nucleic acids;

[0082] (3) 0.25%-2% (v / v) NP-40, the concentration of NP-40 is, for example, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60% , 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.8 1%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.00%, 1.01%, 1. 02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, 1.41%, 1.42%, 1.43% , 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.63%, 1.6 4%, 1.65%, 1.66%, 1.67%, 1.68%, 1.69%, 1.70%, 1.71%, 1.72%, 1.73%, 1.74%, 1.75%, 1.76%, 1.77%, 1.78%, 1.79%, 1.80%, 1.81%, 1.82%, 1.83%, 1.84%, 1.85％、1.86％、1.87％、1.88％、1.89％、1.90％、1.91％、1.92％、1.93％、1.94％、1.95％、1.96％、1.97％、1.98％、1.99％、2.00％；.

[0083] (4) 0.25%-2% (v / v) Tween-20, the concentration of Tween-20 is, for example, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59% , 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8 0%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.00%, 1. 01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, 1.41%, 1.42% , 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.6 3%, 1.64%, 1.65%, 1.66%, 1.67%, 1.68%, 1.69%, 1.70%, 1.71%, 1.72%, 1.73%, 1.74%, 1.75%, 1.76%, 1.77%, 1.78%, 1.79%, 1.80%, 1.81%, 1.82%, 1.83%, 1.84%, 1.85%, 1.86%, 1.87%, 1.88%, 1.89%, 1.90%, 1.91%, 1.92%, 1.93%, 1.94%, 1.95%, 1.96%, 1.97%, 1.98%, 1.99%, 2.00%; NP-40 and Tween-20 in this application are used to lyse cells and release nucleic acids, can destroy the connection between protein-lipid and lipid-lipid, and are often used as components in PCR reaction systems to improve the activity of Taq enzymes;.

[0084] (5) 10 mM to 100 mM guanidine thiocyanate, wherein the concentration of guanidine thiocyanate is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, GITC can promote sample lysis and inhibit the activity of nucleases.

[0085] (6) 20 mM-250 mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol; in the present application, sorbitol may be contained alone, mannitol may be contained alone, or sorbitol and mannitol may be added simultaneously, or polyethylene glycol (including but not limited to polyethylene glycol 8000) may be added; wherein the concentration of sorbitol and / or mannitol is, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 mM and the concentration of polyethylene glycol is, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%.

[0086] (7) 0.01%-0.5% (v / v) Proclin 300, the concentration of Proclin 300 is, for example, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%; and

[0087] (8) 1%-5% (v / v) glycerol, the concentration of glycerol is, for example, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%.

[0088] In the present application, glycerol, sorbitol, mannitol, and (PEG) polyethylene glycol mainly play the role of maintaining the stability of nucleic acids, etc. Proclin300 mainly plays the role of preservation and antibacterial.

[0089] In the case of being able to maintain a specific pH, the present application does not particularly limit the type of buffer. In some embodiments of the present application, the buffer includes one or more of Tris-HCl buffer, citrate buffer and HEPES buffer; optionally, the buffer includes Tris-HCl buffer with a pH of 8.2-9.5 and 5mM-50mM.

[0090] Optionally, the lysis preservation solution comprises:

[0091] pH 8.2-9.5, 10mM-20mM Tris-HCl buffer,

[0092] 0.5mM-3mM EDTA,

[0093] 0.25%-1% (v / v) NP-40,

[0094] 0.25%-1% (v / v) Tween-20,

[0095] 10mM-30mM guanidine thiocyanate,

[0096] 50mM-250mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol,

[0097] 0.1%-0.25% (v / v) Proclin 300, and

[0098] 1%-3% (v / v) glycerol.

[0099] The use of the lysis preservation solution of the present application for nucleic acid extraction is simple and easy to operate, does not require the use of organic reagents, can effectively release the nucleic acids in the sample, and can also be used in conjunction with cationic magnetic beads to enrich the nucleic acids (about 5 minutes, short operation time, and high extraction and enrichment efficiency). After enrichment, PCR detection can be directly performed without washing. The operation is convenient, the sensitivity can be significantly improved, and large-volume samples can be extracted and enriched, which has a broader application prospect.

[0100] The lysis preservation solution of the present application can have multiple functions, for example, it can also be used as a diluent, and the nucleic acid extract can be a product diluted with the diluent.

[0101] The present application does not specifically limit the type and sampling form of the biological sample to be tested. In some embodiments of the present application, the biological sample to be tested includes but is not limited to a swab sample.

[0102] In this application, the terms "biological sample", "sample" and the like refer to animal samples; tissues or organs, tissue lysates, which may be from animals (preferably at least including mammals, such as primates, including humans); cells (in a subject, directly taken from a subject, or maintained in culture or from a cultured cell line), cell lysates (or lysate portions) or cell extracts; solutions containing one or more molecules derived from cells, cellular material or viral material (such as polypeptides or nucleic acids); or solutions containing naturally or non-naturally occurring nucleic acids, which are or can be assayed as described herein.

[0103] The sample contains nucleic acid. The sample may also be any body fluid or excreta containing one or more cells, cell components or nucleic acids, including but not limited to cellular, nuclear or cell-free nucleic acids. In particular, in some embodiments of the present application, the biological sample to be tested includes but is not limited to one or more of a serum sample, a plasma sample, a urine sample, a whole blood sample and an environmental sample; the environmental sample includes but is not limited to a water sample.

[0104] In some embodiments of the present application, the nucleic acid includes DNA and / or RNA, and its form is various, and can be in the form of short oligonucleotides, long oligonucleotides or polynucleotides. It can also be double-stranded DNA and single-stranded DNA and double-stranded RNA and single-stranded RNA. In some specific embodiments, the nucleic acid can be a gene, cDNA molecule, mRNA, tRNA, rRNA, non-coding RNA molecule, etc., and a fragment of the above-mentioned nucleic acid form, such as an oligonucleotide.

[0105] In a second aspect of the embodiment of the present application, a method for detecting a target nucleic acid in a biological sample to be tested is provided, the detection method comprising the following steps:

[0106] Prepare a nucleic acid sample according to the preparation method; and

[0107] The nucleic acid sample is tested.

[0108] The detection method of nucleic acid may or may not rely on amplification of nucleic acid. As used herein, the term "detection method of nucleic acid" refers to any method for determining the nucleotide composition of a target nucleic acid, including but not limited to DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays, (Hologic, Inc.) and described in, e.g., U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17 :292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009 / 0253142, each of which is incorporated herein by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, which are incorporated herein by reference in their entirety); polymerase chain reaction (PCR) as described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5, 849,481, 5,710,264, 5,124,246 and 5,624,802, which are incorporated herein by reference in their entireties); rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, which are incorporated herein by reference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); E-transfer sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entireties); circular probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entireties); Dade Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,8 6 7 and 5,7 9 2, 6 1 4, which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety).

[0109] The term "amplification" or "amplification" refers to the production of multiple copies of polynucleotides or parts of polynucleotides when they occur together in the context of the term "nucleic acid", usually starting from a small amount of polynucleotides (e.g., as few as a single polynucleotide molecule), wherein the amplification product or amplicon is usually detectable. The amplification of polynucleotides includes a variety of chemical and enzymatic methods. The production of multiple DNA copies from one or several copies of a target DNA or template DNA molecule during polymerase chain reaction (PCR), rolling circle amplification (RCA) or ligase chain reaction (LCR) is a form of amplification. Amplification is not limited to the strict replication of the starting molecule. For example, the use of reverse transcription RT-PCR to produce multiple cDNA molecules from a limited amount of RNA in a sample is a form of amplification. In addition, the production of multiple RNA molecules from a single DNA molecule during the transcription process is also a form of amplification.

[0110] In some embodiments of the present application, the detection method includes a PCR method; optionally, the PCR method includes QPCR.

[0111] In some embodiments of the present application, the detection method of the present application may be for non-diagnostic purposes.

[0112] In some embodiments of the present application, the detection method of the present application may be for diagnostic purposes.

[0113] In some embodiments of the present application, the amplification system includes 15 μL-25 μL (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 μL) of the nucleic acid sample.

[0114] A third aspect of the embodiments of the present application provides a combination product, the combination product comprising:

[0115] Cationic magnetic beads as defined above; and

[0116] The lysis storage medium defined above.

[0117] In the present application, "combination product" means that the components thereof are not necessarily mixed together, and one or more components thereof may be separately packaged.

[0118] According to a fourth aspect of the embodiments of the present application, a lysis preservation solution is provided, wherein the lysis preservation solution includes the lysis preservation solution defined above.

[0119] In a fifth aspect of the embodiments of the present application, a kit is provided, the kit comprising:

[0120] The combination product; or

[0121] The lysis preservation solution.

[0122] In some embodiments of the present application, the kit further contains one or more of an extracting agent, an amplifying agent, and a detecting agent for nucleic acid.

[0123] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0124] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0125] Cationic magnetic beads: Merck Chemicals 39572001.

[0126] Silicon hydroxy magnetic beads: MS02H from Du Biotechnology.

[0127] Example 1. Optimization of the components of the lysis storage solution

[0128] 1.1 Materials and methods

[0129] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0130] (2) Amplification instrument: Slan96P.

[0131] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0132] Table 1. Nucleic acid lysis storage solution

[0133]

[0134]

[0135] 1.2 Sample preparation

[0136] The reference S1 in the six respiratory pathogen nucleic acid reference plates was diluted 10 times with each lysis storage solution to 0.2 TCID of INF A virus culture. 50 / mL, INF B virus culture 0.2TCID 50 / mL, RSV artificially constructed vector 50 copies / mL.

[0137] 1.3 Experimental procedures

[0138] (1) Take 2 mL of sample diluted with various lysis storage solutions, add 20 μg of cationic magnetic beads, shake and mix, and let stand for 3-5 minutes;

[0139] (2) The dropper was then placed on a magnetic stand and magnetically attracted for 30 seconds to 1 minute until the magnetic beads were completely attracted and gathered at the mouth of the dropper. Then, 20 μL of the nucleic acid enrichment extract containing magnetic beads was dropped into the QPCR amplification reagent for QPCR amplification detection. Each group was repeated 3 times.

[0140] 1.4 Amplification Procedure

[0141] Table 2. Amplification procedures

[0142]

[0143] 1.5 Experimental Results

[0144] Table 3. Comparison of enrichment effects of different lysis storage solutions

[0145]

[0146]

[0147] By comparison, it was found that the lysis preservation solution 2 had the highest enrichment efficiency and was used as the lysis preservation solution component for verification in subsequent examples.

[0148] Example 2: Comparison of the amount of magnetic beads used

[0149] 2.1 Materials and methods

[0150] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0151] (2) Amplification instrument: Slan96P.

[0152] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0153] 2.2 Sample preparation

[0154] (1) Dilute the reference sample S1 in the six respiratory pathogen nucleic acid reference plates with lysis storage buffer in a 10-fold concentration gradient to 2 TCID of INF A virus culture 50 / mL, INF B virus culture 2TCID 50 / mL, RSV artificially constructed vector 500 copies / mL;

[0155] (2) The diluted product was further diluted with lysis storage solution to 0.4 TCID of INF A virus culture 50 / mL, INF B virus culture 0.4TCID 50 / mL, RSV artificially constructed vector 100 copies / mL;

[0156] (3) Further dilute with lysis storage solution to 0.1 TCID of INF A virus culture 50 / mL, INF B virus culture 0.1TCID 50 / mL, RSV artificially constructed vector 25 copies / mL.

[0157] The lysis preservation solution in this example is the lysis preservation solution 2 in Table 1.

[0158] 2.3 Experimental procedures

[0159] (1) Take 2 mL of the diluted sample of each concentration gradient and add it to a dropper, add 10 μg, 20 μg, 30 μg, 40 μg and 50 μg of cationic magnetic beads respectively, mix well and let stand at room temperature for 3 min;

[0160] (2) The dropper was then placed on a magnetic stand and magnetically attracted for 30 seconds to 1 minute until the magnetic beads were completely attracted and gathered at the mouth of the dropper. Then, 20 μL of the nucleic acid enrichment extract containing magnetic beads was dropped into the QPCR amplification reagent for QPCR amplification detection. Each group was repeated 3 times.

[0161] 2.4 Amplification Procedure

[0162] Table 4. Amplification procedures

[0163]

[0164] 2.5 Experimental results:

[0165] Table 5. Comparison of enrichment effects of different magnetic beads in high concentration samples

[0166]

[0167] Table 6. Comparison of enrichment effects of different magnetic beads in medium concentration samples;

[0168]

[0169] Table 7. Comparison of enrichment effects of different magnetic beads in low concentration samples

[0170]

[0171] By comparing samples with three concentration gradients of high, medium and low, 10μg, 20μg, 30μg, 40μg and 50μg of cationic magnetic beads were added to 2mL of diluted samples. The amplification results showed that the average Ct value of the detection was advanced and the detection rate was the highest when 20μg of magnetic beads were added. Low concentration of magnetic beads may affect the enrichment of low-concentration samples, and too high concentration of magnetic beads may inhibit the amplification system or affect fluorescence collection.

[0172] Example 3: Comparison of pH values ​​of lysis storage solutions

[0173] 3.1 Materials and methods

[0174] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0175] (2) Amplification instrument: Slan96P.

[0176] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0177] (4) Lysis buffer storage solution:

[0178] A: Adjust the pH of the lysis storage solution to 7.5;

[0179] B: Adjust the pH of the lysis storage solution to 8.0;

[0180] C: Adjust the pH value of the lysis storage solution to 10;

[0181] D: Original lysis storage solution (pH 8.5).

[0182] The pH value of the lysis preservation solution of this example is adjusted based on the lysis preservation solution 2 in Table 1.

[0183] 3.2 Sample preparation

[0184] The reference S1 in the six respiratory pathogen nucleic acid reference plates was diluted 10 times with each lysis storage solution to 0.2 TCID of INF A virus culture. 50 / mL, INF B virus culture 0.2TCID 50 / mL, RSV artificially constructed vector 50 copies / mL.

[0185] 3.3 Experimental procedures

[0186] (1) Take 2 mL of sample diluted with various lysis storage solutions, add 20 μg of cationic magnetic beads, shake and mix, and let stand for 3-5 minutes;

[0187] (2) The dropper was then placed on a magnetic stand and magnetically attracted for 30 seconds to 1 minute until the magnetic beads were completely attracted and gathered at the mouth of the dropper. Then, 20 μL of the nucleic acid enrichment extract containing magnetic beads was dropped into the QPCR amplification reagent for QPCR amplification detection. Each group was repeated 3 times.

[0188] 3.4 Amplification Procedure

[0189] Table 8. Amplification procedures

[0190]

[0191] 3.5 Experimental Results

[0192] Table 9. Comparison of enrichment effects of lysis storage solutions with different pH values

[0193]

[0194] By comparing gradients of different pH values ​​(7.5-10), the lysis and enrichment effect at pH 8.5 was the best, while the Ct values ​​of other pH gradient tests were basically the same. Finally, the lysis preservation solution of pH 8.5 was selected.

[0195] Example 4: Comparison of extraction and enrichment efficiency with silanol magnetic beads

[0196] 4.1 Materials and methods

[0197] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0198] (2) Amplification instrument: Slan96P.

[0199] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0200] 4.2 Sample preparation

[0201] (1) Dilute the reference sample S1 in the six respiratory pathogen nucleic acid reference plates with lysis storage buffer in a 10-fold concentration gradient to 2 TCID of INF A virus culture 50 / mL, INF B virus culture 2TCID 50 / mL, RSV artificially constructed vector 500 copies / mL.

[0202] (2) The diluted product was further diluted with lysis storage solution to 0.2 TCID of INF A virus culture 50 / mL, INF B virus culture 0.2TCID 50 / mL, RSV artificially constructed vector 50 copies / mL.

[0203] The lysis preservation solution in this example is the lysis preservation solution 2 in Table 1.

[0204] 4.3 Experimental procedures

[0205] (1) Dilute each to 2TCID 50 / mL of INF A virus culture, 2TCID 50 20 μg of cationic magnetic beads, silanol magnetic beads and H2O were added to 2 mL of INF B virus culture at 500 copies / mL and 2 mL of RSV artificial vector at 500 copies / mL, respectively. The mixture was allowed to stand at room temperature for 3 min. The dropper was then placed on a magnetic stand and magnetically attracted for 30 s to 1 min until the magnetic beads were completely attracted and gathered to the dropper mouth. Finally, 20 μL of nucleic acid enriched extract containing magnetic beads was dropped into the QPCR amplification reagent for QPCR amplification detection. Each group was repeated 3 times.

[0206] (2) Dilute each to 0.2 TCID 50 / mL of INF A virus culture, 0.2TCID 50 / mL INF B virus culture, 50 copies / mL RSV artificial vector 2mL, add 20μg of cationic magnetic beads and silicon hydroxyl magnetic beads respectively, a total of 6 groups, mix and stand at room temperature for 3min, of which 3 groups are used as controls, place the dropper on the magnetic stand, magnetically absorb for 30s-1min until the magnetic beads are completely magnetically absorbed and gathered at the dropper mouth, and then drop 20μL of nucleic acid enriched extract containing magnetic beads into the QPCR amplification reagent, and perform QPCR amplification detection, with 3 repetitions for each group. The other 3 groups are placed on the magnetic stand for 1min adsorption until the magnetic beads are completely adsorbed, the supernatant is discarded, and then 20μL of lysis preservation solution is added to mix by blowing, and 20μL of nucleic acid enriched extract containing magnetic beads is added to the prepared amplification solution for QPCR amplification detection.

[0207] The amplification procedures used for the detection are shown in the following table.

[0208] Table 10. Amplification Program

[0209]

[0210]

[0211] 4.4 Experimental Results

[0212] Table 11. Comparison of nucleic acid enrichment effects of silicon hydroxy magnetic beads and cationic magnetic beads under medium concentration samples

[0213]

[0214] Table 12. Comparison of nucleic acid enrichment effects of silicon hydroxy magnetic beads and cationic magnetic beads after washing in low concentration samples

[0215]

[0216] For samples with medium concentration (IFA: 2TCID 50 / mL, IFB: 2TCID 50 / mL, RSV: 500 copies / mL), the Ct values ​​of direct release and adding silicon hydroxy magnetic beads were basically the same, while the samples with the addition of cationic magnetic beads were about 3 Cts earlier, and the sensitivity was increased by about 10 times (Table 11); while the samples with low concentration (IFA: 0.2TCID 50 / mL, IFB: 0.2TCID 50 / mL, RSV:RSV:50copies / mL) after one wash, the Ct value of the sample enriched by the silicon hydroxy magnetic beads showed a significant decline (about 3 Ct), while the Ct values ​​of the samples of the cationic magnetic beads were basically the same regardless of whether they were washed (Table 12). The results of this example show that the enrichment effect of the silicon hydroxy magnetic beads in the nucleic acid releaser is very poor. It is speculated that the silicon hydroxy magnetic beads need high salt and alcohol to adsorb nucleic acids, while the cationic magnetic beads have a significant nucleic acid enrichment effect in the nucleic acid releaser.

[0217] Example 5. Comparison of different extraction methods

[0218] 5.1 Materials and methods

[0219] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0220] (2) Amplification instrument: Slan96P.

[0221] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0222] 5.2 Sample preparation

[0223] (1) Six respiratory pathogen nucleic acid reference plate S1, first diluted 50 times to 4 TCID of INF A virus culture 50 / mL, INF B virus culture 4TCID 50 / mL, RSV artificially constructed vector 1×10 3 copies / mL;

[0224] (2) The diluted product was then diluted with lysis storage solution in a gradient dilution to 0.4 TCID of INF A virus culture. 50 / mL, INF B virus culture 0.4TCID 50 / mL, RSV artificially constructed vector 100 copies / mL;

[0225] (3) The diluted product was then diluted with lysis storage solution in a gradient dilution to 0.2 TCID of INF A virus culture. 50 / mL, INF B virus culture 0.2TCID 50 / mL, RSV artificially constructed vector 50 copies / mL;

[0226] (4) The diluted product was diluted with the preservation solution in a gradient dilution to 0.1 TCID of INF A virus culture. 50 / mL, INF B virus culture 0.1TCID 50 / mL, RSV artificially constructed vector 25 copies / mL.

[0227] The lysis preservation solution in this example is the lysis preservation solution 2 in Table 1.

[0228] 5.3 Experimental procedures

[0229] (1) Nucleic acid direct amplification

[0230] Take 20 μL of the diluted samples at each concentration gradient and add them to the prepared amplification reagent for amplification.

[0231] (2) Magnetic bead-based nucleic acid extraction

[0232] Take 400 μL of the diluted samples of each concentration gradient, extract nucleic acid according to the instructions of the magnetic bead method nucleic acid extraction reagent (nucleic acid extraction or purification reagent S20025, Shengxiang Bio), and then take the nucleic acid extraction product for nucleic acid amplification.

[0233] (3) Magnetic beads enrichment

[0234] Each concentration gradient was diluted to 2 mL, and 20 μg of enriched cationic magnetic beads were added to each sample. The mixture was mixed and allowed to stand for 3-5 min. Then the dropper was placed on the magnetic rack and magnetically attracted for 30 s to 1 min until the cationic magnetic beads were completely attracted to the dropper mouth. Then 20 μL of nucleic acid enriched extract containing cationic magnetic beads was dropped into the QPCR amplification reagent for QPCR amplification detection. Each group was repeated 3 times.

[0235] 5.4 Amplification Procedure

[0236] Table 13. Amplification procedures

[0237]

[0238] 5.5 Experimental Results

[0239] Table 14. Comparison of various extraction methods for medium concentration samples

[0240]

[0241]

[0242] Table 15. Comparison of various extraction methods for low concentration samples

[0243]

[0244] Table 16. Comparison of various extraction methods for extremely low concentration samples

[0245]

[0246] In the samples of INF A: 0.4TCID50 / mL, INF B: 0.4TCID50 / mL, and RSV 100Copies / mL, the Ct value of cationic magnetic bead enrichment was earlier than that of direct amplification and magnetic bead nucleic acid extraction, with a detection rate of 4 / 4. In the samples of low concentration (INF A: 0.2TCID50 / mL; INF B: 0.2TCID50 / mL; RSV: 50Copies / mL) and extremely low concentration (INF A: 0.1TCID50 / mL; INF B: 0.1TCID50 / mL; RSV: 25Copies / mL), the effect of cationic magnetic bead enrichment was significantly better than the other two methods. The speculated reason may be that the enrichment efficiency is high and the amount of enriched sample is large, which can significantly improve the sensitivity of detection.

[0247] Example 6. Comparison of oropharyngeal swab samples

[0248] 6.1 Materials and methods

[0249] (1) Sample: oropharyngeal swab (Fuzhou Children's Hospital).

[0250] (2) Amplification instrument: Slan96P.

[0251] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0252] 6.2 Sample preparation

[0253] Place the positive human rhinovirus oropharyngeal swab in the lysis preservation solution and shake to mix as the sample.

[0254] 6.3 Experimental procedures

[0255] (1) Nucleic acid direct amplification

[0256] Take 20 μL of the prepared sample and add it to the prepared amplification reagent for amplification.

[0257] (2) Magnetic bead-based nucleic acid extraction

[0258] Take 400 μL of the prepared sample, extract nucleic acid according to the instructions of the magnetic bead nucleic acid extraction reagent (nucleic acid extraction or purification reagent S20025, Shengxiang Bio), and then take the nucleic acid extraction product for nucleic acid amplification.

[0259] (3) Magnetic beads enrichment

[0260] Take 2 mL of the prepared sample, add 20 μg of cationic magnetic beads to each sample, mix well and let stand for 3-5 minutes, then place the dropper on the magnetic stand and magnetically absorb for 30 seconds to 1 minute until the cationic magnetic beads are completely magnetically attracted to the dropper mouth, then drop 20 μL of nucleic acid enrichment extract containing cationic magnetic beads into the QPCR amplification reagent for QPCR amplification detection.

[0261] 6.4 Amplification Procedure

[0262] Table 17. Amplification Program

[0263]

[0264] 6.5 Experimental Results

[0265] Table 18. Comparison of positive human rhinovirus oropharyngeal swab sample tests

[0266] sample Magnetic bead extraction direct expansion Magnetic beads enrichment 1 27.34 32.11 27.06 2 28.72 27.44 25.04 3 22.01 21.11 19.1 4 25.39 24.05 22.44 5 40 36.18 33.11 6 36.94 40 33.61

[0267] Verification results in clinical samples showed that the Ct value detected after cationic magnetic bead enrichment was significantly earlier than that of magnetic bead method and direct amplification extraction. On the one hand, cationic magnetic beads can effectively enrich nucleic acids in samples. On the other hand, cationic magnetic bead enrichment is not limited by sample size and is suitable for nucleic acid enrichment and extraction of large-volume samples.

[0268] Example 7: Comparison of direct amplification and enrichment direct amplification of sewage samples

[0269] 7.1 Materials and methods

[0270] (1) Sample: Six respiratory pathogen nucleic acid reference plates (Changsha Sokoya Biotechnology Co., Ltd., catalog number: SKY-230023).

[0271] (2) Amplification instrument: Slan96P.

[0272] (3) Amplification kit: Six-item respiratory pathogen nucleic acid detection kit (PCR-fluorescent probe method), Shengxiang Bio, batch number: 24022.

[0273] 7.2 Sample preparation

[0274] (1) The sewage collected by Changsha Center for Disease Control and Prevention was centrifuged at 2000 g for 3 min, and the supernatant was used as the matrix.

[0275] (2) Using treated sewage as the matrix, dilute the S1 reference sample in the reference plate in a 10-fold gradient to 2 TCID of INF A virus culture. 50 / mL, INF B virus culture 2TCID 50 / mL, RSV artificially constructed vector 500 copies / mL.

[0276] 7.3 Experimental procedures

[0277] (1) Magnetic beads enrichment

[0278] Take 2 mL of diluted sample, add 2 mL of lysis preservation solution 2 and 20 μg of cationic magnetic beads, mix well and let stand for 3-5 minutes, then place the dropper on the magnetic stand and magnetically absorb for 30 seconds to 1 minute until the cationic magnetic beads are completely magnetically attracted to the dropper mouth, then drop 20 μL of nucleic acid enrichment extract containing cationic magnetic beads into the QPCR amplification reagent for QPCR amplification detection, and perform 3 replicates for each group.

[0279] (2) Direct expansion

[0280] Take 2 mL of diluted sample and add 2 mL of lysis preservation solution 2, mix well and let stand for 3-5 minutes, take 20 μL of released product for QPCR amplification detection, and repeat 3 times for each group.

[0281] 7.4 Amplification Procedure

[0282] Table 19. Amplification Program

[0283]

[0284] 7.5 Experimental Results

[0285] Table 20. Comparison of direct amplification and cationic magnetic bead enrichment in sewage samples

[0286]

[0287] The results showed that the detection results after cationic magnetic bead enrichment were significantly improved by about 3-4 Ct compared with the direct amplification samples. The sensitivity of detection can be significantly improved after the sewage samples are lysed by nucleic acid release agents and enriched with cationic magnetic beads. It also proved that the cationic magnetic bead method is suitable for the detection of large volume samples.

[0288] On the whole, the combination of the lysis preservation solution and cationic magnetic beads of the present application can quickly lyse pathogens to release nucleic acids while the cationic magnetic beads capture nucleic acids. Through magnetic bead amplification, it is suitable for various scenarios such as outpatient and emergency departments. At the same time, it significantly improves the sensitivity of detection and reduces the risk of missed detection. It is especially suitable for nucleic acid extraction and enrichment of large-volume liquid samples.

[0289] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0290] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for preparing a nucleic acid sample, characterized in that: The preparation method comprises the following steps: The nucleic acid in the nucleic acid extract of the biological sample to be tested is fully adsorbed by cationic magnetic beads, and the cationic magnetic beads adsorbed with the nucleic acid are collected to prepare a nucleic acid sample.

2. The method for preparing a nucleic acid sample according to claim 1, characterized in that: The cationic magnetic beads include amino magnetic beads.

3. The method for preparing a nucleic acid sample according to claim 1, characterized in that: The amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 1 μg-200 μg; Optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 1 μg-100 μg; Optionally, the amount of the cationic magnetic beads corresponding to each 1 mL of the nucleic acid extract is 5 μg-50 μg.

4. The method for preparing a nucleic acid sample according to any one of claims 1 to 3, characterized in that: The collection method includes magnetic attraction.

5. The method for preparing a nucleic acid sample according to any one of claims 1 to 3, characterized in that: The preparation step of the nucleic acid extract comprises: mixing the biological sample to be tested with a lysis preservation solution to prepare the nucleic acid extract.

6. The method for preparing a nucleic acid sample according to claim 5, characterized in that: The lysis preservation solution comprises: pH 7.5-10 buffer, 0.5mM-5mM EDTA, 0.25%-2% (v / v) NP-40, 0.25%-2% (v / v) Tween-20, 10mM-100mM guanidine thiocyanate, 20mM-250mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol, 0.01%-0.5% (v / v) Proclin 300, and 1%-5% (v / v) glycerol; Optionally, the buffer comprises one or more of Tris-HCl buffer, citrate buffer and HEPES buffer; Optionally, the buffer comprises a Tris-HCl buffer of pH 8.2-9.5, 5mM-50mM; Optionally, the lysis preservation solution comprises: pH 8.2-9.5, 10mM-20mM Tris-HCl buffer, 0.5mM-3mM EDTA, 0.25%-1% (v / v) NP-40, 0.25%-1% (v / v) Tween-20, 10mM-30mM guanidine thiocyanate, 50mM-250mM sorbitol and / or mannitol or 0.5%-3% (w / v) polyethylene glycol, 0.1%-0.25% (v / v) Proclin 300, and 1%-3% (v / v) glycerol.

7. The method for preparing a nucleic acid sample according to claim 5, characterized in that: The biological sample to be tested includes a swab sample.

8. The method for preparing a nucleic acid sample according to claim 5, characterized in that: The biological sample to be tested includes one or more of a serum sample, a plasma sample, a urine sample, a whole blood sample and an environmental sample; Optionally, the environmental sample comprises a water sample.

9. A method for detecting a target nucleic acid in a biological sample to be tested, characterized in that: The detection method comprises the following steps: Preparing a nucleic acid sample according to the preparation method of any one of claims 1 to 8; and The nucleic acid sample is tested.

10. The method for detecting a target nucleic acid in a biological sample to be tested according to claim 9, characterized in that: Detection methods include PCR; Optionally, the PCR method comprises QPCR.

11. The method for detecting a target nucleic acid in a biological sample to be tested according to claim 10, characterized in that: The amplification system includes 15 μL-25 μL of the nucleic acid sample.

12. A combination product, characterized in that The combination product comprises: Cationic magnetic beads as defined in any one of claims 1 to 8; and A lysis preservation solution as defined in any one of claims 5 to 8.

13. A lysis preservation solution, characterized in that: The lysis preservation solution comprises the lysis preservation solution defined in any one of claims 5 to 8.

14. A kit, characterized in that The kit comprises: The combination product as claimed in claim 12; or The lysis preservation solution as claimed in claim 13.

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