CN115976170A - Chimeric primer-mediated nucleic acid detection method and detection kit - Google Patents

Abstract

The present application relates to a nucleic acid detection method and detection kit mediated by chimeric primers. The detection method provided by the present invention is mainly to introduce the chimeric primer into the constant temperature amplification system, and highlight the RNA part binding site in the chimeric primer by degrading and amplifying the RNA part in the hybrid DNA double strand formed by ribonuclease, In the follow-up, relying on ribonuclease to degrade the prominent gap generated by RNA, under the action of strong strand displacement activity of strand displacement DNA polymerase, the exponential amplification of target gene can be realized. During the whole amplification process, there is no need for high temperature denaturation of nucleic acid, Simple operation and low cost.

Description

Chimeric primer-mediated nucleic acid detection method and detection kit

technical field

The invention relates to the technical field of molecular biology, and specifically provides a chimeric primer-mediated nucleic acid detection method and a detection kit.

Background technique

Polymerase chain reaction (PCR, polymerase chain reaction) was founded in 1983 by Kary Mullis of Cetus Company in the United States, and has a history of more than 30 years. Due to its advantages in sensitivity and specificity, PCR technology is rapidly applied to various aspects of scientific research and clinical research. This PCR technology, which is similar to the natural replication process of DNA, relies on the specificity of oligonucleotide primers complementary to both ends of the target sequence, and can amplify the number of target DNA fragments by more than one million times. Under the catalysis of polymerase, the mother strand DNA is used as the template, and the specific primer is used as the starting point of extension, and the daughter strand DNA complementary to the mother strand template DNA is replicated in vitro through steps such as denaturation, annealing, and extension. The PCR process is specifically divided into three steps: (1) Denaturation: use high temperature to denature and separate the DNA double strands. The hydrogen bond between the DNA double strands is broken at high temperature (93-98°C); (2) Annealing (Annealing): After the DNA double strands are separated, the temperature is lowered so that the primer can bind to the single-stranded DNA; (3 ) Extension (Extension): The DNA polymerase synthesizes a complementary strand along the DNA strand from the primer bound when the temperature is lowered. After the extension is completed, a cycle is completed, and the number of DNA fragments is doubled. Repeat these three steps 25-35 times, and the number of DNA fragments will increase exponentially.

After decades of development, PCR technology has been extended from the original common PCR, such as landing PCR, hot start PCR, long fragment amplification PCR, nested PCR, multiplex PCR, fluorescent quantitative PCR and other technologies. Although these techniques have been used in microbial research, disease diagnosis, drug screening, etc., there are some limitations in the application of this common PCR-based technology: (1) PCR technology requires expensive amplification instruments in application, and at the same time The instrument must have a fine temperature control program and a heating template, so that it can realize the denaturation of the DNA template strand at high temperature and the primer annealing and template extension at a lower temperature, so that the exponential amplification of the template amount can be realized after dozens of cycles of repeated temperature changes . Since each cycle time is short, the instrument must be able to quickly and accurately raise and lower the temperature. Therefore, this requires high requirements for the heating module of the instrument. (2) The DNA polymerase used in PCR amplification has the ability to withstand high temperature, otherwise new enzymes must be added in each cycle to achieve the amplification of the next cycle, which is very easy to cause pollution and increase the cost . (3) In the PCR cycle, the annealing time of the primers in each cycle is very short (several seconds to tens of seconds), which requires the primers to quickly find the homologously matched segment on the template to achieve extension. This requires that the PCR system must have an excess of PCR primers. Excess primers can mismatch with the template, misprime amplification, primer dimers, etc., which in turn inhibit PCR amplification, especially when the amount of template is low, which can exacerbate this situation. (4) In practical applications, common PCR technology or technology based on its extension requires a long amplification reaction time, usually 1.5 h or more, which is not conducive to the application of rapid amplification.

Due to the limitations of traditional PCR technology, a variety of in vitro isothermal nucleic acid amplification technologies have been developed, such as: CPA technology (cross-primed nucleic acid amplification technology), LAMP (loop-mediated isothermal amplification technology), NASBA technology (nucleic acid sequence-dependent Amplification technology) SPIA (single primer isothermal nucleic acid amplification technology), RPA technology (recombinase polymerase isothermal nucleic acid amplification technology), etc., these technologies can achieve high efficiency at a constant temperature (37°C to 65°C). Nucleic acid amplification does not require the use of a PCR machine that precisely controls the temperature. However, technologies such as CPA technology and LAMP technology require higher temperatures and still require temperature control equipment, which is not conducive to the operation in the field, customs, border defense and other laboratory environments; while NABSA, RPA, SPIA and other technology amplification systems It is complex, requires many types of enzymes, and is expensive. These factors limit the application of constant temperature amplification technology.

Contents of the invention

Based on this, the purpose of the embodiments of the present application includes providing a nucleic acid detection method mediated by chimeric primers, which combines chimeric primers with reverse transcriptase, ribonuclease, and strand-displacing DNA polymerase, etc., and the reaction system It is simple and can achieve nucleic acid amplification only at a single temperature.

The purpose of the embodiment of the present application can be achieved through the following technical solutions:

In the first aspect of the present application, a chimeric primer-mediated nucleic acid detection method is provided, the detection method comprising the following steps:

Provide nucleic acid fragments to be tested;

A chimeric primer pair is designed for the target fragment in the nucleic acid fragment; in the chimeric primer pair, one chimeric primer specifically binds to the target fragment, and the other chimeric primer binds to the complementary DNA of the target fragment Fragment-specific binding, each chimeric primer contains an RNA fragment at the 5' end and a DNA fragment at the 3' end, and the length of the chimeric primer is 30nt-40nt;

Mix the nucleic acid fragment, the pair of chimeric primers, reverse transcriptase, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer, amplify at a constant temperature, and detect.

In the present application, reverse transcriptase is the DNA polymerase that depends on RNA, has the DNA polymerase activity of 5'-3' terminal RNA guidance and the polymerase activity of DNA guidance, has no RNase H activity; Strand displacement DNA polymerase is to have strong Strand displacement activity and 5'→3' DNA polymerase activity, but no 5'→3' exonuclease activity and 3'→5' exonuclease activity.

In some embodiments of the present application, in the chimeric primer, the length of the RNA fragment at the 5' end is 10nt-20nt, and the length of the DNA fragment at the 3' end is 15nt-20nt.

In some embodiments of the present application, the reverse transcriptase is one or more of AMV reverse transcriptase and M-MLV reverse transcriptase.

In some embodiments of the present application, the ribonuclease is one or more of the nucleases RNase A, RNase T1 and RNase H.

DNA聚合酶和Bsu DNA聚合酶I中的一种或者多种。In some embodiments of the present application, the strand-displacing DNA polymerase is Bst DNA polymerase I,

One or more of DNA polymerase and Bsu DNA polymerase I.

In some embodiments of the present application, the amplification reaction buffer comprises KCl 10mM-100mM, MgCl ₂ 2mM-20mM, dNTPs 0.1mM-1mM, dithiothreitol 1mM-10mM and BSA 50ng/μL-200ng/ μL, and Tris-HCl buffer solution with a pH value of 8.0-8.5, 10mM-200mM.

In some embodiments of the present application, the initial reaction system corresponding to constant temperature amplification contains 0.1 μM-0.5 μM of the chimeric primer, 0.1U/μL-10U/μL of the reverse transcriptase, 0.02U/μL-2U /μL of the ribonuclease, 30ng/μL-300ng/μL of the strand-displacing DNA polymerase, and 100ng/μL-1000ng/μL of the single-stranded DNA-binding protein.

In some embodiments of the present application, the conditions for isothermal amplification include: the temperature is 25°C-45°C, and the time is 30min-120min.

In some embodiments of the present application, the nucleic acid fragment to be detected is an RNA fragment.

In some embodiments of the present application, the nucleic acid fragment to be detected is a DNA fragment, and the detection method further includes placing the DNA fragment at 55°C-95°C for 2min-5min, and then combining it with the The chimeric primer pair, reverse transcriptase, ribonuclease, strand displacement DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer are mixed for constant temperature amplification and detection.

In the second aspect of the present application, the present invention provides a nucleic acid detection kit mediated by chimeric primers, said kit including the pair of chimeric primers defined in the first aspect, optionally, also including the first aspect Reverse transcription, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA-binding protein, and amplification reaction buffer as defined in .

Compared with the traditional technology, the present application has the following beneficial effects:

The detection method provided by this application, the detection mechanism refers to Figure 1, mainly introduces the chimeric primer into the constant temperature amplification system, and highlights the RNA part of the hybrid DNA double strand formed by ribonuclease degradation and amplification. The partial binding site of RNA, and the protruding gap generated by the subsequent degradation of RNA by ribonuclease, can realize the exponential amplification of the target gene under the action of the strong strand displacement activity of strand displacement DNA polymerase.

Traditional PCR technology requires high-temperature denaturation, medium-temperature annealing and extension, which can only be realized by using expensive PCR amplification equipment. However, the present application can realize nucleic acid amplification at a single temperature without high-temperature denaturation of nucleic acid, and does not need an expensive PCR amplification instrument, and the operation is simple and the cost is low.

Compared with the helicase-dependent constant temperature amplification technology that can only perform DNA amplification, the nucleic acid detection method provided by the present application can realize both RNA amplification and DNA amplification. At the same time, unlike the traditional recombinase helicase constant temperature amplification technology that requires recombinase or helicase to open the double strands of DNA in the amplification process, this application does not require recombinase or helicase Participation, the constant temperature amplification of nucleic acid can be realized without relying on ATP to provide energy, and there is no need to purify the product in the process of product detection. The amplification product of this application can be directly detected by gel electrophoresis.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, and to understand the present application and its beneficial effects more completely, the following briefly introduces the drawings that need to be used in the description of the embodiments. Apparently, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative efforts.

Figure 1 is a schematic diagram of the principle of chimeric primer-mediated nucleic acid constant temperature amplification technology;

Fig. 2 is the result of RNA amplification of the new coronavirus in embodiment 1;

Fig. 3 is the result of DNA amplification of the N gene plasmid DNA of the new coronavirus in Example 2.

Detailed ways

The present application will be further described in detail below in conjunction with the accompanying drawings, implementation modes and examples. It should be understood that these implementations and examples are only used to illustrate the present application and are not intended to limit the scope of the application. The purpose of providing these implementations and examples is to make the disclosure of the application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, and is not limited to the implementation modes and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present application to obtain The equivalent forms also fall within the protection scope of the present application. Furthermore, in the following description, numerous specific details are given in order to provide a fuller understanding of the application, and it is understood that the application may be practiced without one or more of these details.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used herein in the specification of the application are for the purpose of describing the embodiments and examples only, and are not intended to limit the application.

the term

Unless otherwise stated or contradictory, terms and phrases used herein have the following meanings:

As used herein, the terms "and/or", "or/and", "and/or" include any one of two or more of the associated listed items, and any of the associated listed items. and all combinations including any combination of any two of the relevant listed items, any more of the relevant listed items, or all of the relevant listed items. It should be noted that when at least three items are connected with at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that in this application, the technical solution Undoubtedly include the technical solutions that are all connected by "logic and", and also undoubtedly include the technical solutions that are all connected by "logic or". For example, "A and/or B" includes three parallel schemes of A, B and A+B. For another 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, all are connected by "logic or") technical solution), also includes any and all combinations of A, B, C, and D, that is, includes any combination of any two or any three of A, B, C, and D, and also includes A, B, and C , four combinations of D (that is, all use the technical scheme of "logic and" connection).

The present application refers to "multiple", "multiple", "multiple", "multiple", etc., unless otherwise specified, means that the number is greater than 2 or equal to 2. For example, "one or more" means one or more than two.

As used herein, "combinations thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

In this article, "suitable" mentioned in "suitable combination", "suitable way", "any suitable way" and so on can implement the technical solutions of the application, solve the technical problems of the application, and realize the expectations of the application. The technical effect shall prevail.

Herein, "preferred", "better", "better" and "advisable" are only descriptions of better implementations or examples, and it should be understood that they do not limit the protection scope of the present application.

In the present application, "further", "further", "particularly" and the like are used for description purposes, indicating differences in content, but should not be construed as limiting the protection scope of the present application.

In the present application, "optionally", "optionally" and "optionally" refer to dispensable, that is to say, any one selected from the two parallel schemes of "with" or "without". If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction relationship, each "optional" is independent.

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

In this application, the technical features described in open form include closed technical solutions consisting of the listed features, and open technical solutions including the listed features.

In this application, when it comes to a numerical range (that is, a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical range, and includes the two numerical endpoints of the numerical range (i.e. the minimum value and the maximum value). value), and every numeric value between those two numeric endpoints. Unless otherwise specified, when the numerical range only refers to the integers within the numerical range, including the integers at the two endpoints of the numerical range, and every integer between the two endpoints, in this article, it is equivalent to directly enumerating each An integer, such as t being an integer selected from 1 to 10, means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Furthermore, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameters in this application, unless otherwise specified, are allowed to be treated at a constant temperature, and are also allowed to vary within a certain temperature range. It should be understood that the isothermal treatment described allows the temperature to fluctuate within the precision of the instrument control. It is allowed to fluctuate within the range of ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

In the present application, both % (w/w) and wt % mean percentage by weight, % (v/v) means percentage by volume, and % (w/v) means percentage by volume by mass.

All documents mentioned in this application are incorporated by reference in this application as if each individual document were individually indicated to be incorporated by reference. Unless it conflicts with the application purpose and/or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When referring to cited documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When citing documents are involved in this application, the examples and preferred modes of the cited related technical features can also be incorporated into this application as a reference, but only if the application can be implemented. It should be understood that when the referenced content conflicts with the description in the present application, the present application shall prevail or be amended adaptively according to the description in the present application.

The first aspect of the application

The embodiment of the present application provides a chimeric primer-mediated nucleic acid detection method, the detection method comprising the following steps:

Provide nucleic acid fragments to be tested;

A chimeric primer pair is designed for the target fragment in the nucleic acid fragment; in the chimeric primer pair, one chimeric primer specifically binds to the target fragment, and the other chimeric primer binds to the complementary DNA of the target fragment Fragment-specific binding, each chimeric primer contains an RNA fragment at the 5' end and a DNA fragment at the 3' end, and the length of the chimeric primer is 30nt-40nt;

Mix the nucleic acid fragment, the pair of chimeric primers, reverse transcriptase, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer, amplify at a constant temperature, and detect.

The detection mechanism of the present application can be seen in Figure 1: under the action of reverse transcriptase, the chimeric primer 1 anneals to the template RNA and synthesizes the first cDNA, and the obtained cDNA complements the RNA to form an RNA/DNA hybrid (step A and step B), Under the action of RNase, the RNA strand in the RNA/DNA hybrid is degraded to obtain a hybrid cDNA with an RNA sequence at the 5' end, and the single-stranded DNA binding protein binds to the hybrid cDNA to maintain its stability (step C) . Chimeric primer 2 anneals to the hybrid cDNA and is extended under the action of strand-displacing DNA polymerase to form a hybrid double-stranded DNA with RNA at the 5' ends of both strands (step D); while the DNA/RNA hybrid The RNA part in the strand is continuously degraded by RNase, presenting the binding site of the RNA part in the mixed primer (step E), so that the RNA part in the unbound chimeric primer can continuously obtain the binding site, and then bind to On the template DNA, the DNA polymerase with strand displacement activity continues to play its role, and carries out the DNA strand displacement reaction (step F), displacing the DNA single strand synthesized in the previous reaction and synthesizing a new hybrid DNA at the same time, replacing Two single-strands are obtained that can complement each other to form a double-stranded DNA, and the newly synthesized double-stranded DNA undergoes a cycle of RNA degradation, new primer annealing, and strand replacement to complete a large number of template amplifications.

In the present application, reverse transcriptase is the DNA polymerase that depends on RNA, has the DNA polymerase activity of 5'-3' terminal RNA guidance and the polymerase activity of DNA guidance, has no RNase H activity; Strand displacement DNA polymerase is to have strong Strand displacement activity and 5'→3' DNA polymerase activity, but no 5'→3' exonuclease activity and 3'→5' exonuclease activity.

In the present application, the chimeric primer is a combination of RNA and DNA, including an RNA segment at the 5' end and a DNA segment at the 3' end. RNA fragments are followed by DNA fragments. The length of the chimeric primer is 30nt-40nt (such as 30nt, 31nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt), wherein the optimal length of the RNA fragment is 10nt-20nt (such as 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt), the optimal length of the DNA fragment is 15nt-20nt (for example, 15nt, 16nt, 16nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt , 24nt, 25nt).

In one example, the reverse transcriptase is one or more of AMV reverse transcriptase and M-MLV reverse transcriptase.

In one example, the ribonuclease is one or more of the nucleases ribonuclease A, ribonuclease T1 and ribonuclease H.

DNA聚合酶和Bsu DNA聚合酶I中的一种或者多种。In one example, the strand-displacing DNA polymerase is Bst DNA polymerase I,

One or more of DNA polymerase and Bsu DNA polymerase I.

In one example, the amplification reaction buffer comprises KCl 10mM-100mM, MgCl ₂ 2mM-20mM, dNTPs 0.1mM-1mM, dithiothreitol 1mM-10mM and BSA 50ng/μL-200ng/μL, and Tris-HCl buffer solution with a pH value of 8.0-8.5, 10mM-200mM. In the amplification reaction buffer of the present application, the concentration of KCl is, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100mM, the concentration of MgCl ₂ is, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20mM, the concentration of dNTPs is, for example, 0.1mM, 0.2, 0.4, 0.6, 0.8, 1mM, dithiothreo The concentration of sugar alcohol is, for example, 1, 3, 5, 7, 9, 10 mM, and the concentration of BSA is, for example, 50, 70, 90, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 ng/μL , the pH value of the Tris-HCl buffer is, for example, 8, 8.1, 8.2, 8.3, 8.4, 8.5, and the concentration is, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mM.

In one example, the initial reaction solution for constant temperature amplification contains 0.1 μM-0.5 μM of the chimeric primer, 0.1U/μL-10U/μL of the reverse transcriptase, 0.02U/μL-2U/μL of the ribose Nuclease, 30ng/μL-300ng/μL of the strand-displacing DNA polymerase and 100ng/μL-1000ng/μL of the single-stranded DNA binding protein. In the initial reaction solution of the amplification reaction, the concentration of the chimeric primer is, for example, 0.1, 0.2, 0.3, 0.4, 0.5 μM, and the concentration of the reverse transcriptase is, for example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10U/μL, the concentration of ribonuclease is, for example, 0.02, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1 , 1.2, 1.4, 1.6, 1.8, 2U/μL, the concentration of strand displacement DNA polymerase is, for example, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300ng/μL, the concentration of single-stranded DNA binding protein is, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950, 1000ng/μL.

In one example, the conditions for isothermal amplification include: the temperature is 25°C-45°C, and the time is 30min-120min. The temperature of constant temperature amplification is, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45°C, The time is, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 min.

In one example, the nucleic acid fragment to be detected is an RNA fragment. For example, the nucleic acid fragment to be detected is the novel coronavirus nucleic acid, or the pseudoviral RNA of the novel coronavirus. Detection based on the nucleic acid of the novel coronavirus is mainly based on non-diagnostic purposes.

In one example, the nucleic acid fragment to be detected is a DNA fragment, and the detection method further includes placing the DNA fragment at 55°C-95°C for 2min-5min, and then combining it with the chimeric Primer pairs, reverse transcriptase, ribonuclease, strand displacement DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer are mixed for constant temperature amplification and detection. For example, first place the DNA fragments at 55, 60, 65, 70, 75, 80, 85, 90, 95°C for 2, 2.5, 3, 3.5, 4, 4.5, 5 minutes. For example, the DNA fragment is the N gene plasmid DNA of the novel coronavirus.

In one example, the chimeric primer pair is, for example, set forth in SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 shown.

In the second aspect of the application

The present application provides a kit for nucleic acid constant temperature amplification mediated by chimeric primers. The kit includes the primer pair defined in the first aspect, reverse transcription, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding Protein and amplification reaction reagents.

specific embodiment

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. For the experimental methods that do not indicate specific conditions in the following examples, please refer to the guidelines given in this application, or according to the experimental manual or routine conditions in this field, or according to the conditions suggested by the manufacturer, or refer to the existing conditions in this field. known experimental methods.

In the following specific examples, the measurement parameters related to raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Involves temperature and time parameters, allowing for acceptable deviations due to instrumental test accuracy or operational accuracy.

Example 1

The present embodiment provides a kind of detection method of novel coronavirus pseudovirus RNA nucleic acid, involves as follows:

 Viral DNA/RNA Kit购自北京全式金；逆转录酶、RNaseH、Bsu链置换DNA聚合酶I、单链DNA结合蛋白等购自菲鹏生物。(1) Reagent: Virus quality control material containing the genome of the novel coronavirus (SARS-CoV-2) (constructed by Jingliang Gene Technology (Shenzhen) Co., Ltd.);

Viral DNA/RNA Kit was purchased from Beijing Quanshijin; reverse transcriptase, RNaseH, Bsu strand-displacing DNA polymerase I, single-stranded DNA binding protein, etc. were purchased from Feipeng Biotech.

(2) Chimeric primer pair: According to the published SARS-CoV-2 novel coronavirus gene sequence, design a specific chimeric primer pair based on the N gene, and the chimeric primer pair is provided by Sangon (Shanghai) Bioengineering Co. The company synthesized and purified the sequence of the chimeric primer pair as follows:

Table 1

(3) RNA extraction: For the detailed extraction process, please refer to the kit instructions.

A brief introduction is as follows: Take 200 μL virus quality control product, add BB5 buffer containing proteinase K, mix well for 15 seconds, and then lyse at 56°C for 15 minutes. Add 250 μL of absolute ethanol, mix thoroughly for 15 seconds and then place at room temperature for 5 minutes. Transfer the resulting mixed liquid into a spin column at 12,000g for centrifugation for 1 minute, cut off the effluent, and repeat the above operation until all the liquid has passed through the spin column. Add 500 μL of WB5 to wash once, centrifuge at 12,000 g for 1 minute; repeat this step once; after centrifuging at 12,000 g for 2 minutes, completely remove residual ethanol. Add 30 μL of RNase-free Water, and centrifuge at 12000g for 1 minute to elute.

(4) Amplification reaction solution (50 μL): 20 mM Tris-HCl buffer solution with a pH value of 8.2; KCl 80 mM; MgCl ₂ 8 mM; dNTPs 0.2 mM; dithiothreitol (DTT) 2 mM; The final concentration of binding primer is 0.2 μM; 7.5U reverse transcriptase; 1U RNase H; the final concentration of strand displacement DNA polymerase is 240ng/μL; the final concentration of single-stranded DNA binding protein is 300ng/μL, 1×SYBR Green I Fluorescent dyes.

(5) Add 5 μL of novel coronavirus quality control nucleic acid RNA to the reaction system, mix well and react at 42°C for 60 minutes.

(6) Result analysis: The amplification results are shown in Figure 2. Except that the 10 bp at the 5' end is RNA sequence and the 10 bp at the 3' end is DNA, the chimeric primer pair 1 has no amplification signal, and the other three pairs of chimeric primers have different degrees of amplification. Especially when the chimeric primer pair 4 consisting of 20bp RNA fragments at the 5' end and 20bp DNA fragments at the 3' end, the fluorescent signal is strong and the amplification efficiency is high.

The amplified target sequence of the present embodiment 1 is shown in SEQ ID No.9 as follows:

5'-AGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAAT-3'.

Example 2

This embodiment provides a detection method for plasmid DNA containing the N gene of the novel coronavirus, and the contents involved are as follows:

(1) Reagents: The novel coronavirus N gene plasmid was provided by the Protein Engineering Department of Yahuilong Biotechnology Co., Ltd.; reverse transcriptase, RNase H, Bsu strand-displacing DNA polymerase I, single-stranded DNA binding protein, etc. were purchased from Feipeng biology.

(2) Primers: The primers used in this example are consistent with Example 1.

(3) Amplification reaction solution (50 μL): 20 mM Tris-HCl buffer with a pH value of 8.2; KCl 80 mM; MgCl ₂ 8 mM; dNTPs 0.2 mM; dithiothreitol (DTT) 2 mM; The final concentration of primers was 0.2 μM; 7.5 U of reverse transcriptase; 1 U of RNase H; the final concentration of strand-displacing DNA polymerase was 240 ng/μL, and the final concentration of single-stranded DNA-binding protein was 300 ng/μL.

(4) 100ng/μL of the novel coronavirus N gene plasmid was heat-treated at 95°C for 5 minutes in advance, and then taken out and immediately placed on ice for later use.

(5) Add 1 μL of the treated novel coronavirus N gene plasmid to the constant temperature reaction solution, mix well and react at 37°C for 60 minutes.

(6) Result analysis: the results are shown in Figure 3, except that chimeric primer 1, which has 10 bp at the 5' end as an RNA fragment and 10 bp at the 3' end as a DNA fragment, has no amplification signal, the other 3 pairs of chimeric primers have different degrees of amplification. In particular, the chimeric primer pair 4 consisting of 20 bp at the 5' end of an RNA fragment and 20 bp at the 3' end of a DNA fragment has a strong fluorescent signal and high amplification efficiency.

The amplified target sequence in the present embodiment 2 is shown in SEQ ID No.9 as follows:

5'-AGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAAT-3'.

In combination with the above Example 1 and Example 2, the present application uses chimeric primers with a length of 30nt-40nt comprising a 5'-end RNA fragment and a 3'-end DNA fragment to replace DNA in reverse transcriptase, RNase-H, and strands Under the catalysis of the polymerase, the amplification of DNA nucleic acid and RNA nucleic acid is realized at 37°C and 42°C respectively.

The various technical features of the above-mentioned embodiments and examples can be combined in any suitable manner. For the sake of brevity, all possible combinations of the various technical features in the above-mentioned embodiments and examples are not described. However, as long as these There is no contradiction in the combination of technical features, and all should be considered within the scope of the description.

The above-mentioned embodiments only express several implementation modes of the present application, which is convenient for a specific and detailed understanding of the technical solution of the present application, but should not be construed as limiting the protection scope of the patent application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teaching content of the present application, those skilled in the art may make various changes or modifications to the present application, and the obtained equivalent forms also fall within the protection scope of the present application. It should also be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in this application are within the protection scope of the appended claims of this application. Therefore, the scope of protection of the patent application should be based on the content of the appended claims, and the description and drawings can be used to interpret the content of the claims.

Claims (10)

1. a detection method of nucleic acid mediated by chimeric primers, characterized in that, the detection method comprises the steps: Provide nucleic acid fragments to be tested; A chimeric primer pair is designed for the target fragment in the nucleic acid fragment; in the chimeric primer pair, one chimeric primer specifically binds to the target fragment, and the other chimeric primer binds to the complementary DNA of the target fragment Fragment-specific binding, each chimeric primer contains an RNA fragment at the 5' end and a DNA fragment at the 3' end, and the length of the chimeric primer is 30nt-40nt; Mix the nucleic acid fragment, the pair of chimeric primers, reverse transcriptase, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer, amplify at a constant temperature, and detect. 2. the detection method of the nucleic acid mediated by chimeric primer according to claim 1, is characterized in that, in described chimeric primer, the length of the RNA fragment of 5 ' end is 10nt-20nt, the length of the DNA fragment of 3 ' end 15nt-20nt. 3. The nucleic acid detection method mediated by chimeric primers according to claim 1, wherein the reverse transcriptase is one or more of AMV reverse transcriptase and M-MLV reverse transcriptase. 4. the detection method of the nucleic acid mediated by chimeric primers according to claim 1, is characterized in that, described ribonuclease is one or more in ribonuclease A, ribonuclease T1 and ribonuclease H kind. 5. the detection method of the nucleic acid mediated by chimeric primers according to claim 1, is characterized in that, described strand displacement DNA polymerase is Bst DNA polymerase I,

One or more of DNA polymerase and Bsu DNA polymerase I. 6. the nucleic acid detection method of chimeric primer mediation according to claim 1, is characterized in that, described amplification reaction buffer comprises KCl 10mM-100mM, MgCl ₂ 2mM-20mM, dNTPs 0.1mM-1mM, two Thiothreitol 1mM-10mM and BSA 50ng/μL-200ng/μL, and Tris-HCl buffer with a pH value of 8-8.5 and 10mM-200mM. 7. The detection method of nucleic acid mediated by chimeric primers according to claim 1, wherein the conditions for constant temperature amplification meet one or more of the following conditions: (1) The initial reaction solution for constant temperature amplification contains 0.1 μM-0.5 μM of the chimeric primer, 0.1U/μL-10U/μL of the reverse transcriptase, 0.02U/μL-2U/μL of the ribonuclease, 30ng/μL-300ng/μL of the strand-displacing DNA polymerase and 100ng/μL-1000ng/μL of the single-stranded DNA binding protein; and, (2) Conditions for constant temperature amplification include: the temperature is 25°C-45°C, and the time is 30min-120min. 8. The nucleic acid detection method mediated by chimeric primers according to any one of claims 1 to 7, wherein the nucleic acid fragment to be detected is an RNA fragment. 9. according to the detection method of the nucleic acid mediated by chimeric primer according to any one of claims 1 to 7, it is characterized in that, the nucleic acid fragment to be detected is a DNA fragment, and the detection method also comprises firstly converting the DNA Place the fragment at 55°C-95°C for 2min-5min, and then combine it with the chimeric primer pair, reverse transcriptase, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer The solution is mixed for constant temperature amplification and detection. 10. A nucleic acid detection kit mediated by chimeric primers, characterized in that the kit comprises the pair of chimeric primers defined in any one of claims 1 to 9, optionally, also includes the chimeric primer pair defined in any one of claims 1 to 9. Reverse transcriptase, ribonuclease, strand-displacing DNA polymerase, single-stranded DNA binding protein and amplification reaction buffer as defined in any one of 9.

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