A method and kit for detecting genetic elements

Abstract

The present application relates to multiplexed, asymmetric amplification of nucleic acid molecules containing genetic elements. In particular, the present application provides a method and a kit for detecting genetic elements by simultaneously and asymmetric amplification of nucleic acid molecules containing genetic elements.

Description

Technical Field

[0001] The present application relates to multiplexed, asymmetric amplification of nucleic acid molecules containing genetic elements. In particular, the present application provides a method and a kit for detecting genetic elements by simultaneously and asymmetric amplification of nucleic acid molecules containing genetic elements. Background Art

[0002] With the rapid increase in the global population, how to use limited land resources, water resources and other resources to increase food production and better solve the food problem has become a top priority for governments around the world. Genetically modified organisms refer to plants, animals, microorganisms and their products that are genetically modified and improved at the genetic level using genetic engineering for agricultural production or agricultural product processing. Genetically modified organisms have become a good choice for countries around the world to solve the food problem due to their high yield, disease resistance and other excellent traits. Since their introduction, they have been gradually promoted worldwide. While bringing huge social, economic and ecological benefits, the safety of genetically modified organisms has also attracted widespread attention from many countries and organizations in the world.

[0003] In order to safeguard people's right to know and right to choose, more than 40 countries have successively promulgated the labeling system of genetically modified products. The thresholds set by different countries are different. Some countries are mandatory, such as Australia, New Zealand, Brazil, Chile, India, etc., and some countries are voluntary, such as Argentina, Canada, the United States, etc. After 2001, China gradually improved the relevant laws and regulations on genetically modified organisms to ensure technical safety and industrial health. China takes a more cautious attitude towards genetically modified organisms. At present, it implements a mandatory labeling management system without a threshold, that is, as long as the product contains genetically modified ingredients, it needs to be marked with "genetically modified labeling" on the outer packaging. To implement the genetically modified biological safety management and labeling system, it is necessary to rely on the progress of science and technology to continuously develop new scientific and effective detection methods.

[0004] GMO detection is mainly aimed at the detection of transgenic elements such as promoters, screening marker genes, and terminators. At present, the detection technologies for genetically modified organisms are mainly divided into two categories: one is the detection technology at the protein level, such as protein blotting, enzyme-linked immunosorbent assay, and immunoassay strip method. Protein blotting is a method that visualizes the presence of the target protein through electrophoresis, antigen-antibody reaction, substrate color development, and radioautography. This technology has a good effect on the detection of insoluble proteins, but it is time-consuming and cumbersome to operate. Enzyme-linked immunosorbent assay is a method that uses antigen-antibody precipitation reaction combined with color development reaction. It is the most sensitive and quantitatively accurate method among all transgenic detection methods based on exogenous proteins. Immunoassay strip technology also uses antigen-antibody immune reaction. This technology has low requirements for operator expertise and detection equipment, fast detection, and small sample volume, but it still needs to be improved in terms of quantification and detection sensitivity. There are four main shortcomings of exogenous protein detection technology: First, it is only applicable to raw materials and primary processed products, and is difficult to apply to deep-processed products, because exogenous proteins are likely to be degraded, inactivated or even disappear due to various processing conditions, which makes the stability and repeatability of the detection face challenges, and also increases the false negative rate; second, it is impossible to achieve true high-throughput detection, which requires that genetically modified organisms or products contain specific genes and can only be used in a few authorized genetically modified products; third, some transferred exogenous genes do not express protein, so they cannot be detected; fourth, the operation is relatively cumbersome and not suitable for batch testing.

[0005] The other type is based on the detection method at the DNA level, the common ones are Southern hybridization, PCR, gene chip method and isothermal amplification method. Compared with protein, DNA has high stability and is widely present, so the GMO detection technology targeting DNA has a wider range in practical application, from seeds to end products, which is generally not affected by the degree of material processing. At the same time, the detection technology targeting DNA has high sensitivity and specificity. The polymerase chain reaction (PCR) effectively amplifies specific exogenous gene fragments in genetically modified organisms by designing primers, making them easier to identify and recognize. At present, the DNA-based PCR detection method is the most widely used technology in the process of genetically modified organism detection, and it is also the most accurate method, and will continue to play an irreplaceable role in the future.

[0006] International, national and industry standards have issued some methods for screening and detecting genetically modified organisms, but in long-term detection practice, it has been found that the detection throughput is low, the time consumption is long, and some primers have problems with poor specificity and sensitivity. Multiplex PCR amplification has shown the advantages of simple, rapid and multi-target amplification in the parallel amplification analysis of a small number of nucleic acid molecules. It has received extensive attention and in-depth research in recent years, but it is difficult to achieve high-throughput analysis of multiple different nucleic acid molecules in complex systems. The main reason is that multiple pairs of primers in the multiplex PCR method exist in the same system at the same time, which can easily cause non-specific amplification such as primer dimers. In addition, due to the difference in amplification efficiency of different primers, some of the target fragments will be preferentially amplified. With the increase of the multiplex PCR multiplicity (i.e., the number of primer pairs) and the increase of the number of amplification cycles, the limitations brought by this defect will become more obvious. Therefore, new rapid, accurate and high-throughput detection methods have become an important research direction for genetically modified organism detection methods. Summary of the invention

[0007] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0008] As used herein, the term "gene element" means any nucleic acid sequence related to gene expression, including genes (e.g., resistance genes, selection marker genes) and regulatory elements such as promoters, enhancers, operators, and terminators that regulate genes. Gene expression can be regulated (e.g., induced or repressed) at the stages of gene activation, transcription initiation, post-transcriptional processing, mRNA degradation, protein translation, post-translational processing modification, and protein degradation.

[0009] As used herein, the term "transgenic plant" refers to a plant in which a target gene or specific DNA fragment isolated from an animal, plant or microorganism is transferred to the genome of a plant by various methods using genetic engineering (DNA recombinant technology) techniques, together with appropriate regulatory elements, to obtain a plant in which the gene or DNA sequence can be stably expressed and inherited. In this article, the term "transgenic element" includes the exogenous gene to be integrated and the elements that regulate the gene.

[0010] As used herein, the term "complementary" means that two nucleic acid sequences can form hydrogen bonds between each other according to the base pairing principle (Waston-Crick principle), and thus form a duplex. In the present application, the term "complementary" includes "substantially complementary" and "completely complementary". As used herein, the term "completely complementary" means that each base in a nucleic acid sequence can be paired with a base in another nucleic acid chain without mismatching or gaps. As used herein, the term "substantially complementary" means that most of the bases in a nucleic acid sequence can be paired with a base in another nucleic acid chain, and it allows the presence of mismatches or gaps (for example, mismatches or gaps of one or several nucleotides). Generally, under conditions that allow nucleic acid hybridization, annealing or amplification, two nucleic acid sequences of "complementary" (for example, substantially complementary or completely complementary) will selectively / specifically hybridize or anneal, and form a duplex. Accordingly, the term "non-complementary" means that two nucleic acid sequences cannot hybridize or anneal under conditions that allow nucleic acid hybridization, annealing or amplification, and cannot form a duplex. As used herein, the term "not completely complementary" means that the bases in one nucleic acid sequence cannot be completely paired with the bases in another nucleic acid chain, and there is at least one mismatch or gap.

[0011] As used herein, the terms "hybridization" and "annealing" refer to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. In this application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Typically, two nucleic acid sequences that are fully complementary or substantially complementary can hybridize or anneal. The complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, particularly the temperature.

[0012] As used herein, the term "PCR reaction" has a meaning generally understood by those skilled in the art, and refers to a reaction (polymerase chain reaction) using a nucleic acid polymerase and a primer to amplify a target nucleic acid. As used herein, the term "multiplex amplification" refers to amplifying multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to amplification products obtained by amplifying a target nucleic acid in which the amounts of two complementary nucleic acid chains are different, and the amount of one nucleic acid chain is greater than the amount of the other nucleic acid chain.

[0013] As used herein, and as generally understood by those skilled in the art, the terms "forward" and "reverse" are merely for the convenience of describing and distinguishing two primers in a primer pair; they are relative and have no special meaning.

[0014] As used herein, the term "melting curve analysis" has a meaning generally understood by those skilled in the art, which refers to a method for analyzing the presence or identity of a double-stranded nucleic acid molecule by measuring the melting curve of the double-stranded nucleic acid molecule, which is generally used to assess the dissociation characteristics of the double-stranded nucleic acid molecule during heating. The method for performing melting curve analysis is well known to those skilled in the art (see, for example, The Journal of Molecular Diagnostics 2009, 11 (2): 93-101). In this application, the terms "melting curve analysis" and "melting analysis" have the same meaning and are used interchangeably.

[0015] In certain preferred embodiments of the present application, melting curve analysis can be performed by using a detection probe labeled with a reporter group and a quencher group. In short, at ambient temperature, the detection probe can form a duplex with its complementary sequence through base pairing. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the detection probe are separated from each other, and the quencher group cannot absorb the signal (e.g., fluorescent signal) emitted by the reporter group. At this time, the strongest signal (e.g., fluorescent signal) can be detected. As the temperature rises, the two chains of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the detection probe under dissociation is in a single-stranded free curling state. In this case, the reporter group (e.g., fluorescent group) and the quencher group on the dissociated detection probe are close to each other, so that the signal (e.g., fluorescent signal) emitted by the reporter group (e.g., fluorescent group) is absorbed by the quencher group. Therefore, as the temperature rises, the detected signal (e.g., fluorescent signal) gradually weakens. When the two chains of the duplex are completely dissociated, all detection probes are in a single-stranded free curling state. In this case, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) on all detection probes are absorbed by the quenching group. Therefore, the signals (e.g., fluorescent signals) emitted by the reporter groups (e.g., fluorescent groups) are basically undetectable. Therefore, by detecting the signals (e.g., fluorescent signals) emitted by the duplex containing the detection probe during the heating or cooling process, the hybridization and dissociation process of the detection probe and its complementary sequence can be observed, forming a curve in which the signal intensity changes with temperature. Further, by performing a derivative analysis on the obtained curve, a curve with the signal intensity change rate as the ordinate and the temperature as the abscissa can be obtained (i.e., the melting curve of the duplex). The peak in the melting curve is the melting peak, and the temperature corresponding to it is the melting point (T m ). Generally speaking, the higher the matching degree between the detection probe and the complementary sequence (for example, the fewer mismatched bases and the more matched bases), the higher the T of the duplex. m Therefore, by detecting the T m, the presence and identity of the sequence in the duplex that is complementary to the detection probe can be determined. As used herein, the terms "melting peak", "melting point" and "T m ” have the same meaning and can be used interchangeably.

[0016] The present invention realizes the amplification and detection of one or more gene elements by using a multiple asymmetric amplification system and a multicolor probe melting curve analysis technique. On this basis, the present application has developed a method for simply and quickly detecting gene elements, as well as a kit for implementing the method.

[0017] Therefore, in one aspect, the present application provides a method for detecting a genetic element, comprising:

[0018] (a) providing a sample containing one or more target nucleic acids, wherein the target nucleic acids include one or more genetic elements; and providing a first universal primer and a second universal primer, and providing at least one target-specific primer pair for each genetic element; wherein,

[0019] The first universal primer comprises a first universal sequence;

[0020] The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence;

[0021] The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the gene element, and the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence is not completely complementary to the complementary sequence of the forward primer; and

[0022] (b) amplifying the target nucleic acid in the sample by PCR reaction using the first universal primer and the second universal primer and the target-specific primer pair under conditions allowing nucleic acid amplification, thereby obtaining an amplification product corresponding to the sample;

[0023] (c) performing melting curve analysis on the amplified product obtained in step (b);

[0024] (d) judging whether a genetic element exists based on the melting curve analysis result of step (c); optionally, judging the species of the sample by judging whether a genetic element exists, or judging whether the sample is a transgenic sample by judging whether a genetic element exists.

[0025] In the method of the present application, the forward primer and the reverse primer respectively contain a forward nucleotide sequence and a reverse nucleotide sequence specific to the target nucleic acid, thereby, during the PCR reaction, the target-specific primer pair (forward primer and reverse primer) will anneal to the target nucleic acid, and initiate PCR amplification to produce an initial amplification product, which contains two nucleic acid chains (nucleic acid chain A and nucleic acid chain B) complementary to the forward primer and the reverse primer, respectively. Further, since the forward primer and the first universal primer both contain the first universal sequence, the nucleic acid chain A complementary to the forward primer can also be complementary to the first universal primer. Similarly, the nucleic acid chain B complementary to the reverse primer can also be complementary to the second universal primer.

[0026] Therefore, as the PCR reaction proceeds, the first universal primer and the second universal primer will anneal to the nucleic acid chain A and nucleic acid chain B of the initial amplification product, respectively, and further initiate PCR amplification. In this process, since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid chain A (nucleic acid chain complementary to the forward primer / first universal primer) and synthesize its complementary chain, but also anneal to nucleic acid chain B (nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain. That is, the first universal primer can simultaneously amplify nucleic acid chain A and nucleic acid chain B of the initial amplification product. At the same time, the second universal primer contains additional nucleotides at the 3' end of the first universal sequence. Therefore, although the second universal primer may also anneal to nucleic acid chain A (nucleic acid chain complementary to the forward primer / first universal primer, which has a sequence complementary to the forward primer), it is not matched with nucleic acid chain A at the 3' end (that is, it cannot be completely complementary at the 3' end). Thus, during the amplification process, the second universal primer will preferentially anneal to nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain, while basically being unable to extend and synthesize the complementary chain of nucleic acid chain A (the nucleic acid chain complementary to the first forward primer / first universal primer).

[0027] Therefore, as PCR amplification proceeds, the synthesis efficiency of the complementary chain of nucleic acid chain A (nucleic acid chain B) will be significantly lower than that of the complementary chain of nucleic acid chain B (nucleic acid chain A), resulting in the complementary chain of nucleic acid chain B (nucleic acid chain A) being synthesized and amplified in large quantities, while the synthesis and amplification of the complementary chain of nucleic acid chain A (nucleic acid chain B) are inhibited, thereby generating a large number of single-stranded products (nucleic acid chain A, which contains a sequence complementary to the forward primer / first universal primer and a sequence of the reverse primer / second universal primer), achieving asymmetric amplification of a target nucleic acid containing one or more genetic elements. Therefore, in steps (a) and (b) of the method of the present application, asymmetric amplification of one or more target nucleic acids in a sample is achieved.

[0028] In addition, since both the forward primer and the reverse primer contain the first universal sequence, during the PCR reaction, the primer dimer formed by the non-specific amplification of the forward primer and the reverse primer will generate a single-stranded nucleic acid whose 5' and 3' ends contain complementary reverse sequences after denaturation, and the single-stranded nucleic acid is easy to anneal itself in the annealing stage to form a stable panhandle structure, which prevents the first universal primer and the second universal primer from annealing and extending the single-stranded nucleic acid, thereby inhibiting further amplification of the primer dimer. Therefore, in the method of the present invention, the non-specific amplification of the primer dimer can be effectively inhibited.

[0029] In certain embodiments, the sample is obtained from a plant and / or an animal.

[0030] In certain embodiments, the plant from which the sample is obtained is selected from soybean, corn, cotton, potato, tomato, rice, papaya, or any combination thereof.

[0031] In certain embodiments, the genetic element is selected from a promoter, an enhancer, an operator, a resistance gene, a selection marker gene, a terminator, or any combination thereof.

[0032] In certain embodiments, the selectable marker gene is an exogenous gene (eg, a transgene) to the sample.

[0033] In certain embodiments, the target nucleic acid comprises a genetic element selected from the group consisting of tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi, 18S rDNA, and any combination of the aforementioned genetic elements (e.g., a combination of any 5, 10, 15, 20, 25, 30, 36 of the aforementioned genetic elements).

[0034] In certain embodiments, the target nucleic acid in the sample comprises the following genetic elements: tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi and 18SrDNA.

[0035] In certain embodiments, in step (a), for each gene element, a detection probe is further provided, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the gene element, and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence;

[0036] Furthermore, in step (c), the detection probe is used to perform a melting curve analysis on the amplification product corresponding to the sample obtained in step (b).

[0037] In certain embodiments, in step (b) of the method, the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and a PCR reaction is performed, and then, after the PCR reaction is completed, a detection probe is added to the product of step (b), and a melting curve analysis is performed; or, in step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase, and a PCR reaction is performed, and then, after the PCR reaction is completed, a melting curve analysis is performed.

[0038] In certain embodiments, the detection probe comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acids (PNA) or locked nucleic acids), or any combination thereof. In certain preferred embodiments, the detection probe comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the detection probe comprises non-natural nucleotides, such as deoxyxanthine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acids (LNA).

[0039] In certain embodiments, the detection probe is not limited by its length. In certain embodiments, the length of the detection probe is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.

[0040] In certain embodiments, the detection probe has a 3'-OH terminus; alternatively, the 3'-terminus of the detection probe is blocked; for example, by adding a chemical moiety (e.g., biotin or an alkyl group) to the 3'-OH of the last nucleotide of the detection probe, by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide, thereby blocking the 3'-terminus of the detection probe.

[0041] In certain embodiments, the detection probe is a self-quenching probe; for example, the detection probe is labeled with a reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at the 5' end or upstream. In certain embodiments, the reporter group and the quencher group are 10-80nt or longer apart.

[0042] In certain embodiments, the detection probe is a self-quenching probe; for example, the detection probe is labeled with a reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or a reporter group is labeled at its 3' end or downstream and a quencher group is labeled at the 5' end or upstream. In such embodiments, when the detection probe is not hybridized with other sequences, the quencher group is located at a position that can absorb or quench the signal of the reporter group (for example, the quencher group is located adjacent to the reporter group), thereby absorbing or quenching the signal emitted by the reporter group. In this case, the detection probe does not emit a signal. Further, when the detection probe is hybridized with its complementary sequence, the quencher group is located at a position that cannot absorb or quench the signal of the reporter group (for example, the quencher group is located away from the reporter group), thereby failing to absorb or quench the signal emitted by the reporter group. In this case, the detection probe emits a signal.

[0043] The design of such self-quenching detection probes is within the capabilities of those skilled in the art. For example, the 5' end of the detection probe may be labeled with a reporter group and the 3' end may be labeled with a quencher group, or the 3' end of the detection probe may be labeled with a reporter group and the 5' end may be labeled with a quencher group. Thus, when the detection probe exists alone, the reporter group and the quencher group are close to each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated from each other, so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal.

[0044] However, it should be understood that the reporter group and the quencher group do not have to be marked at the end of the detection probe. The reporter group and / or the quencher group can also be marked inside the detection probe, as long as the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence. For example, the reporter group can be marked upstream (or downstream) of the detection probe, and the quencher group can be marked downstream (or upstream) of the detection probe, and the two are separated by a sufficient distance (e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, or a longer distance). Thus, when the detection probe exists alone, due to the free curling of the probe molecule or the formation of the secondary structure (e.g., hairpin structure) of the probe, the reporter group and the quencher group approach each other and interact with each other, so that the signal emitted by the reporter group is absorbed by the quencher group, thereby causing the detection probe to not emit a signal; and, when the detection probe hybridizes with its complementary sequence, the reporter group and the quencher group are separated by a sufficient distance from each other, so that the signal emitted by the reporter group cannot be absorbed by the quencher group, thereby causing the detection probe to emit a signal. In certain preferred embodiments, the reporter group and the quencher group are 10-80nt or longer apart, for example, 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt. In certain preferred embodiments, the reporter group and the quencher group are no more than 80nt, no more than 70nt, no more than 60nt, no more than 50nt, no more than 40nt, no more than 30nt, or no more than 20nt apart. In certain preferred embodiments, the reporter group and the quencher group are at least 5 nt, at least 10 nt, at least 15 nt, or at least 20 nt apart.

[0045] Therefore, the reporter group and the quencher group can be labeled at any suitable position of the detection probe, as long as the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence. However, in certain preferred embodiments, at least one of the reporter group and the quencher group is located at the end (e.g., 5' or 3' end) of the detection probe. In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe or at a position 1-10nt away from the 5' end, and the reporter group and the quencher group are at a suitable distance so that before the detection probe hybridizes with its complementary sequence, the quencher group can absorb or quench the signal of the reporter group. In certain preferred embodiments, one of the reporter group and the quencher group is located at the 3' end of the detection probe or at a position 1-10nt away from the 3' end, and the reporter group and the quencher group are at a suitable distance so that before the detection probe hybridizes with its complementary sequence, the quencher group can absorb or quench the signal of the reporter group. In certain preferred embodiments, the reporter group and the quencher group may be at a distance as defined above (e.g., 10-80nt or longer). In certain preferred embodiments, one of the reporter group and the quencher group is located at the 5' end of the detection probe and the other is located at the 3' end.

[0046] In some embodiments, the reporter group in the detection probe is a fluorescent group (e.g., ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL FluorRed590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and the quencher group is a molecule or group that can absorb / quench the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and / or TAMRA).

[0047] In certain embodiments, the detection probe is resistant to nuclease activity (e.g., 5' nuclease activity, such as 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises a modification that resists nuclease activity, such as a thiophosphate bond, an alkyl phosphotriester bond, an aryl phosphotriester bond, an alkyl phosphoester bond, an aryl phosphoester bond, a hydrogenated phosphate bond, an alkyl phosphoramidate bond, an aryl phosphoramidate bond, a 2'-O-aminopropyl modification, a 2'-O-alkyl modification, a 2'-O-allyl modification, a 2'-O-butyl modification, and a 1-(4'-thio-PD-ribofuranosyl) modification.

[0048] In certain embodiments, the detection probe is linear, or has a hairpin structure.

[0049] In certain embodiments, the detection probes each independently have the same or different reporter groups. In certain embodiments, the detection probes have the same reporter group, and the product of step (b) is subjected to a melting curve analysis, and then the presence of the target nucleic acid is determined based on the melting peak in the melting curve; or, the detection probes have different reporter groups, and the product of step (b) is subjected to a melting curve analysis, and then the presence of the target nucleic acid is determined based on the signal type of the reporter group and the melting peak in the melting curve.

[0050] In certain embodiments, in step (c), the product of step (b) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time to obtain a curve showing how the signal intensity of each reporter group changes with temperature; then, the curve is derived to obtain the melting curve of the product of step (b).

[0051] In certain embodiments, the detection probe comprises a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 20, 30, 40, 50, 60, 70): SEQ ID NO: 1, 4, 7, 10 and, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 and 106.

[0052] In certain embodiments, the sample comprises 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids.

[0053] In certain embodiments, the target nucleic acid comprises 1-5, 5-10, 10-15, 15-20, 20-30 or more genetic elements.

[0054] In certain embodiments, the sample is DNA (eg, genomic DNA or cDNA), an RNA molecule (eg, mRNA), or any combination thereof.

[0055] In certain embodiments, the target nucleic acid is a DNA (eg, genomic DNA or cDNA), an RNA molecule (eg, mRNA), or any combination thereof.

[0056] In some embodiments, the sample has a concentration greater than 1 copy / reaction (e.g., 5×101 Copies / reaction, 5×10 2 Copies / reaction, 5×10 3 Copies / reaction, 5×10 4 Copies / reaction, 5×10 5 Copy / React).

[0057] In certain embodiments, in step (a) of the method, 1-5, 5-10, 10-15, 15-20, 20-30 or more target-specific primer pairs are provided.

[0058] In certain embodiments, the target nucleic acid to be amplified is single-stranded or double-stranded.

[0059] In certain embodiments, in step (b) of the method, the working concentrations of the first universal primer and the second universal primer are higher than the working concentrations of the forward primer and the reverse primer; for example, the working concentrations of the first universal primer and the second universal primer are 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentrations of the forward primer and the reverse primer.

[0060] In certain embodiments, in step (b) of the method, the working concentrations of the first universal primer and the second universal primer are the same; or, the working concentration of the first universal primer is lower than that of the second universal primer.

[0061] In certain embodiments, in step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different.

[0062] In certain embodiments, the sample or target nucleic acid comprises mRNA and the sample is subjected to a reverse transcription reaction prior to performing step (b) of the method.

[0063] In certain embodiments, in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to carry out a PCR reaction. In certain embodiments, the nucleic acid polymerase is a DNA polymerase, such as a thermostable DNA polymerase. In certain embodiments, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermusoshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga In some embodiments, the DNA polymerase is Taq polymerase.

[0064] In certain embodiments, the first universal primer consists of the first universal sequence, or comprises the first universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0065] In certain embodiments, the first universal sequence is located at or constitutes the 3' portion of the first universal primer.

[0066] In the embodiments of the present application, the first universal primer can be of any length as long as it can perform a PCR reaction. In certain embodiments, the length of the first universal primer is 5-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.

[0067] In certain embodiments, the first universal primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the first universal primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the first universal primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the first universal primer (or any of its components) comprises non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0068] In certain embodiments, the second universal primer consists of the second universal sequence, or comprises the second universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0069] In certain embodiments, the second universal sequence is located at or constitutes the 3' portion of the second universal primer.

[0070] In certain embodiments, the second universal sequence comprises the first universal sequence and additionally comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides 3' to the first universal sequence.

[0071] In the embodiments of the present application, the second universal primer can be of any length as long as it can perform a PCR reaction. In certain embodiments, the length of the second universal primer is 8-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50nt.

[0072] In certain embodiments, the second universal primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the second universal primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the second universal primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the second universal primer (or any of its components) comprises non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0073] In certain embodiments, in the forward primer, the forward nucleotide sequence is directly linked to the 3' end of the first universal sequence, or is linked to the 3' end of the first universal sequence through a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0074] In certain embodiments, the forward primer further comprises an additional sequence located at the 5' end of the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0075] In certain embodiments, the forward primer comprises or consists of a first universal sequence and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of a first universal sequence, a nucleotide linker, and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the first universal sequence, and a forward nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the first universal sequence, a nucleotide linker, and a forward nucleotide sequence from 5' to 3'.

[0076] In certain embodiments, the forward nucleotide sequence is located at or constitutes the 3' portion of the forward primer.

[0077] In certain embodiments, the length of the forward nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0078] In certain embodiments, the forward primer has a length of 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.

[0079] In certain embodiments, the forward primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the forward primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the forward primer (or any of its components) comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the forward primer (or any of its components) comprises non-natural nucleotides, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0080] In certain embodiments, in the reverse primer, the reverse nucleotide sequence is directly connected to the 3' end of the second universal sequence, or the reverse nucleotide sequence is connected to the 3' end of the second universal sequence via a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0081] In certain embodiments, the reverse primer further comprises an additional sequence located at the 5' end of the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

[0082] In certain embodiments, the reverse primer comprises or consists of a second universal sequence and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of a second universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, a second universal sequence, and a reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, a second universal sequence, a nucleotide linker, and a reverse nucleotide sequence from 5' to 3'.

[0083] In certain embodiments, the reverse nucleotide sequence is located at or constitutes the 3' portion of the reverse primer.

[0084] In certain embodiments, the length of the reverse nucleotide sequence is 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt.

[0085] In certain embodiments, the length of the reverse primer is 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-110nt, 110-120nt, 120-130nt, 130-140nt, 140-150nt.

[0086] In certain embodiments, the reverse primer or any of its components comprises or consists of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the reverse primer (or any of its components) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the reverse primer (or any of its components) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the reverse primer (or any of its components) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1-(2'-deoxy-β-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole or locked nucleic acid (LNA).

[0087] In certain embodiments, the second universal sequence is not completely complementary to the complementary sequence of the forward primer; for example, at least one nucleotide located at the 3' end of the second universal sequence, such as 1-5, 5-10, 10-15, 15-20 or more nucleotides, is not complementary to the complementary sequence of the forward primer.

[0088] In certain embodiments, the sequence of the first universal primer is shown as SEQ ID NO:109.

[0089] In certain embodiments, the sequence of the second universal primer is shown as SEQ ID NO:110.

[0090] In certain embodiments, the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the following or any combination thereof (e.g., a combination of any 20, 30, 40, 50, 60, 70 primer pairs): SEQ ID NO: 2 and 3; 5 and 6; 8 and 9; 11 and 12; 14 and 15; 17 and 18; 20 and 21; 23 and 24; 26 and 27; 29 and 30; 32 and 33; 35 and 36; 38 and 39; 41 and 42; 44 and 45; 47 and 48; 50 and 51; 53 and 54; 56 and 5 7; 59 and 60; 62 and 63; 65 and 66; 68 and 69; 71 and 72; 74 and 75; 77 and 78; 80 and 81; 83 and 84; 86 and 87; 89 and 90; 92 and 93; 95 and 96; 98 and 99; 101 and 102; 104 and 105; 107 and 108.

[0091] In certain embodiments, steps (a)-(b) of the method are performed by a protocol comprising the following steps (I)-(VI):

[0092] (I) providing a sample containing one or more target nucleic acids derived from the individual, wherein the target nucleic acids include one or more genetic elements; and providing a first universal primer and a second universal primer, and, for each genetic element, providing a target-specific primer pair; wherein the first universal primer, the second universal primer and the target-specific primer pair are as defined above;

[0093] (II) mixing the sample with the first universal primer and the second universal primer and a target-specific primer pair, and a nucleic acid polymerase;

[0094] (III) incubating the product of the previous step under conditions that allow nucleic acid denaturation;

[0095] (IV) incubating the product of the previous step under conditions that allow annealing or hybridization of the nucleic acids;

[0096] (V) incubating the product of the previous step under conditions that allow for nucleic acid extension; and

[0097] (VI) Optionally, repeat steps (III)-(V) one or more times.

[0098] In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105° C., thereby denaturing the nucleic acid.

[0099] In certain embodiments, in step (III), the product of step (II) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0100] In certain embodiments, in step (IV), the product of step (III) is incubated at a temperature of 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, or 65-70°C to allow nucleic acid annealing or hybridization.

[0101] In certain embodiments, in step (IV), the product of step (III) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min.

[0102] In certain embodiments, in step (V), the product of step (IV) is incubated at a temperature of 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, 70-75°C, 75-80°C, 80-85°C to allow nucleic acid extension.

[0103] In certain embodiments, in step (V), the product of step (IV) is incubated for 10-20 s, 20-40 s, 40-60 s, 1-2 min, 2-5 min, 5-10 min, 10-20 min or 20-30 min.

[0104] In certain embodiments, steps (IV) and (V) are performed at the same or different temperatures.

[0105] In certain embodiments, steps (III)-(V) are repeated at least once, e.g., at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times. In certain embodiments, when steps (III)-(V) are repeated one or more times, the conditions used in steps (III)-(V) of each cycle are independently the same or different.

[0106] In another aspect, the present application also provides a kit, comprising a primer set capable of asymmetrically amplifying a target nucleic acid containing a genetic element.

[0107] In certain embodiments, the primer set comprises: a first universal primer and a second universal primer, and, for each genetic element, at least one target-specific primer pair is provided, wherein:

[0108] The first universal primer comprises a first universal sequence;

[0109] The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence;

[0110] The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the gene element, and the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer.

[0111] In certain embodiments, the kit further comprises one or more detection probes capable of detecting the genetic element, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the genetic element, and is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence.

[0112] In certain embodiments, the kit further comprises one or more components selected from the group consisting of a nucleic acid polymerase, a reagent for performing nucleic acid amplification, a reagent for performing melting curve analysis, or any combination thereof.

[0113] In certain embodiments, the genetic element is selected from a promoter, an enhancer, an operator, a resistance gene, a selection marker gene, a terminator, or any combination thereof.

[0114] In certain embodiments, the selectable marker gene is an exogenous gene (eg, a transgene) to the sample.

[0115] In certain embodiments, the target nucleic acid comprises a genetic element selected from the group consisting of tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi, 18S rDNA, and any combination of the aforementioned genetic elements (e.g., a combination of any 5, 10, 15, 20, 25, 30, 36 of the aforementioned genetic elements).

[0116] In certain embodiments, the target nucleic acid comprises the following genetic elements: tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi and 18S rDNA.

[0117] In certain embodiments, the detection probe comprises a detection probe having a nucleotide sequence selected from the following or any combination thereof (e.g., any combination of 20, 30, 40, 50, 60, 70): SEQ ID NO: 1, 4, 7, 10 and, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103 and 106.

[0118] In certain embodiments, the sequence of the first universal primer is shown as SEQ ID NO:109.

[0119] In certain embodiments, the sequence of the second universal primer is shown as SEQ ID NO:110.

[0120] In certain embodiments, the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the following or any combination thereof (e.g., a combination of any 20, 30, 40, 50, 60, 70 primer pairs): SEQ ID NO: 2 and 3; 5 and 6; 8 and 9; 11 and 12; 14 and 15; 17 and 18; 20 and 21; 23 and 24; 26 and 27; 29 and 30; 32 and 33; 35 and 36; 38 and 39; 41 and 42; 44 and 45; 47 and 48; 50 and 51; 53 and 54; 56 and 5 7; 59 and 60; 62 and 63; 65 and 66; 68 and 69; 71 and 72; 74 and 75; 77 and 78; 80 and 81; 83 and 84; 86 and 87; 89 and 90; 92 and 93; 95 and 96; 98 and 99; 101 and 102; 104 and 105; 107 and 108.

[0121] It is easy to understand that the first universal primer, the second universal primer, the target-specific primer pair and the detection probe in the kit of the present application are used to implement the method as described above. Therefore, the detailed description of the first universal primer, the second universal primer, the target-specific primer pair and the detection probe above (including the description of various preferred features and exemplary features) is also applicable here.

[0122] In certain embodiments, the nucleic acid polymerase is a template-dependent nucleic acid polymerase, such as a DNA polymerase, in particular a thermostable DNA polymerase. In certain embodiments, the nucleic acid polymerase is as defined above.

[0123] In certain embodiments, the reagents for performing nucleic acid amplification include a working buffer for an enzyme (e.g., a nucleic acid polymerase), dNTPs (labeled or unlabeled), water, and an ion (e.g., Mg 2+ ), a single-stranded DNA binding protein, or any combination thereof.

[0124] In certain embodiments, the kit is used to detect a genetic element.

[0125] In certain preferred embodiments, the kit is used to detect transgenic elements.

[0126] In yet another aspect, the present application provides a use of the primer set as described above for preparing a kit for asymmetric amplification of a target nucleic acid containing a genetic element.

[0127] In certain embodiments, the kit further comprises a detection probe as described above.

[0128] In certain embodiments, the kit is used to perform the method as described above.

[0129] Advantageous Effects of the Invention

[0130] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0131] (1) Compared with conventional PCR detection technology for transgenic products, the technical solution of the present invention can detect multiple exogenous genes and endogenous genes of transgenic products in a single PCR reaction tube, which is simple to operate, efficient in detection, low in cost, and has broad application prospects.

[0132] (2) Compared with conventional transgenic multiplex PCR detection methods, the technical solution of the present invention can effectively inhibit the nonspecific amplification of primer dimers and significantly improve the specificity of amplification and the sensitivity of detection.

[0133] Therefore, the present invention has developed a new method for simultaneously and asymmetrically amplifying multiple transgenic target nucleic acids, which can simultaneously realize effective multiplex PCR amplification and asymmetric PCR amplification, meeting the requirements for simultaneous amplification and detection of multiple target nucleic acids in transgenic product detection work.

[0134] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 An exemplary embodiment of the method of the present invention for detecting endogenous genes and exogenous genes in transgenic organisms is schematically described to illustrate the basic principle of the method of the present invention.

[0136] Figure 1 A schematically describes the primer set and self-quenching fluorescence detection probe involved in this embodiment, wherein the primer set includes: a first universal primer and a second universal primer, and a target-specific primer pair including a forward primer and a reverse primer; wherein,

[0137] The first universal primer comprises a first universal sequence (Tag1);

[0138] The second universal primer comprises a second universal sequence (Tag2) comprising the first universal sequence and additionally comprising at least one nucleotide (e.g., 1-5, 5-10, 10-15, 15-20 or more nucleotides) at the 3' end of the first universal sequence;

[0139] The forward primer comprises a first universal sequence and a forward nucleotide sequence specific to a target nucleic acid containing an exogenous gene or an endogenous gene, and the forward nucleotide sequence is located at the 3' end of the first universal sequence;

[0140] The reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid containing an exogenous gene or an endogenous gene, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and,

[0141] The forward primer and the reverse primer can specifically amplify the corresponding target nucleic acid containing the exogenous gene or the endogenous gene; and,

[0142] The second universal sequence may not be completely complementary to the complementary sequence of the forward primer.

[0143] Figure 1 B schematically depicts the use of Figure 1 The principle that when the primer set A is used for amplification, the nonspecific amplification of primer dimers is inhibited, wherein the primer dimers formed due to the nonspecific amplification of the forward primer and the reverse primer will produce a single-stranded nucleic acid whose 5' and 3' ends contain reverse sequences complementary to each other after denaturation, and the single-stranded nucleic acid itself will form a panhandle structure in the annealing stage, preventing the first universal primer and the second universal primer from annealing and extending the single-stranded nucleic acid, thereby inhibiting further amplification of the primer dimers.

[0144] Figure 1 C schematically depicts the use of Figure 1 The principle of the primer set and detection probe of A for simultaneous detection of endogenous genes and exogenous transgenic elements. In this embodiment, a pair of forward primers and reverse primers and a self-quenching fluorescent detection probe are designed for each target nucleic acid containing an endogenous gene or an exogenous gene. The specific detection process is as follows:

[0145] First, PCR amplification is initiated by a target-specific primer pair with a low concentration to produce an initial amplification product, which contains two nucleic acid chains (nucleic acid chain A and nucleic acid chain B) that are complementary to the forward primer / first universal primer and the reverse primer / second universal primer, respectively; then, the initial amplification product is subjected to subsequent PCR amplification by the first universal primer and the second universal primer with a high concentration.

[0146] Since the reverse primer / second universal primer contains the first universal sequence, the first universal primer can not only anneal to nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer) and synthesize its complementary chain, but also anneal to nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain. That is, the first universal primer can amplify nucleic acid chain A and nucleic acid chain B at the same time.

[0147] The second universal primer contains an extra nucleotide at the 3' end of the first universal sequence, and therefore, it does not match the nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer) at the 3' end (i.e., it cannot be completely complementary at the 3' end). Thus, during the amplification process, the second universal primer will preferentially anneal to the nucleic acid chain B (the nucleic acid chain complementary to the reverse primer / second universal primer) and synthesize its complementary chain, and substantially cannot extend and synthesize the complementary chain of the nucleic acid chain A (the nucleic acid chain complementary to the forward primer / first universal primer).

[0148] Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary chain of nucleic acid chain A (nucleic acid chain B) will be significantly lower than that of the complementary chain of nucleic acid chain B (nucleic acid chain A), resulting in the complementary chain of nucleic acid chain B (nucleic acid chain A) being synthesized and amplified in large quantities, while the synthesis and amplification of the complementary chain of nucleic acid chain A (nucleic acid chain B) are inhibited, thereby generating a large number of target single-stranded products (nucleic acid chain A, which contains a sequence complementary to the forward primer / first universal primer and a sequence of the reverse primer / second universal primer), achieving asymmetric amplification. In addition, in order to further enhance the asymmetry of amplification, the ratio of the first universal primer to the second universal primer can also be adjusted so that the concentration of the first universal primer is lower than that of the second universal primer, so as to better enrich the target single-stranded product. By using multiple pairs of forward primers and reverse primers simultaneously in the same reaction system, multiple target nucleic acids containing endogenous genes and exogenous genes can be asymmetrically amplified at the same time, generating a large number of target nucleic acid single strands containing endogenous genes and exogenous transgenic elements.

[0149] After PCR amplification, the multiple self-quenching fluorescent detection probes added in advance are respectively combined with the corresponding target nucleic acid single strands containing endogenous genes and exogenous transgenic elements to form double-stranded hybrids of the detection probes and the target nucleic acid single strands. Due to the different stabilities of the double-stranded hybrids formed, different melting peaks can be obtained after melting curve analysis, and then the presence or absence of melting peaks can be used to determine whether endogenous genes and exogenous transgenic elements exist in each target nucleic acid single strand.

[0150] Figure 2 The results of melting curve analysis after amplification of the soybean endogenous lectin gene using the system of the present invention in Example 1 are shown. The black and gray solid lines represent the detection results using non-transgenic soybean genomic DNA and negative control as templates, respectively.

[0151] Figure 3 The results of melting curve analysis after amplification of 36 exogenous genes and endogenous genes using the detection system of the present invention in Example 2 are shown.

[0152] Figure 4The results of melting curve analysis after amplification of plasmid DNA samples of different concentrations using the detection system of the present invention in Example 2 are shown. The black solid line, gray dashed line, black dashed line, gray solid line, and gray dotted line represent the concentration of plasmid DNA samples of 5×10 4 Copies / reaction, 5×10 3 Copies / reaction, 5×10 2 Copies / reaction, 5×10 1 Results of melting curve analysis after PCR amplification of samples of copies / reaction and no-template control.

[0153] Figure 5 The results of melting curve analysis after amplification of transgenic soybean genomic DNA samples using the system of the present invention in Example 3 are shown. The black solid line and the gray solid line represent the detection results using transgenic soybean genomic DNA and negative control as templates, respectively. DETAILED DESCRIPTION

[0154] The present invention is now described with reference to the following embodiments which are intended to illustrate the present invention (but not to limit the present invention). It should be understood that these embodiments are only used to illustrate the principles and technical effects of the present invention, and do not represent all possibilities of the present invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in these embodiments. Those skilled in the art can implement other technical solutions based on the principles of the present invention using other similar materials or reaction conditions. Such technical solutions do not depart from the basic principles and concepts described in the present invention, and are encompassed within the scope of the present invention.

[0155] The detailed information of the detection probe, upstream oligonucleotide (forward primer), downstream oligonucleotide (reverse primer) and universal primer used in the examples of this application, as well as their working concentrations (except for specific examples where working concentrations are given again) are summarized in Table 1.

[0156] Table 1: Sequences and concentrations of primers and probes used in the examples

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Example 1. Detection of single transgenic elements

[0163] In this embodiment, the detection probe and upstream and downstream primers of the Lectin element in Table 1 were used to detect the soybean endogenous gene Lectin. The specific preparation method is as follows: 25 μL reaction system contains 1×Taq PCR buffer (TaKaRa, Beijing), 3mM MgCl2, 0.24mM dNTPs, 1U Taq HS (TaKaRa, Beijing), 0.2μM detection probe, 0.04μM upstream and downstream primers, 0.2μM universal primer Tag1, 1.6μM universal primer Tag2, 5μL non-transgenic soybean genomic DNA (concentration is about 1ng / μL) or 5μL water (negative control).

[0164] The PCR amplification program is: 50℃ UNG enzyme action for 5min; 95℃ pre-denaturation for 5min; 95℃ denaturation for 15s, 65℃-56℃ (1℃ decrease per cycle) annealing for 15s, 76℃ extension for 20s, 10 cycles; 95℃ denaturation for 15s, 55℃ annealing for 15s, 76℃ extension for 20s, 50 cycles; then melting curve analysis is performed, the program is: 95℃ denaturation for 1min, 37℃ insulation for 3min, melting curve analysis from 40℃-85℃, heating rate of 0.4℃ / s, and detection of fluorescence signal of HEX channel. The instrument used in this embodiment is SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.). The test results are as follows. Figure 2 shown.

[0165] Figure 2 The results showed that the non-transgenic soybean genome sample had a melting peak at 73.9°C in the HEX channel (black solid line), while the blank control (NTC) without template added had no melting peak (gray solid line). This result shows that the detection system designed according to the principle of the present invention can be used to detect a single transgenic element.

[0166] Example 2. Simultaneous detection of 36 transgenic elements

[0167] By using the reagents provided in Table 1 (36 detection probes, 36 upstream primers, 36 downstream primers and 2 universal primers), the method of the present invention can simultaneously detect 36 transgenic elements tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi, and 18S rDNA in two reactions. The fluorescence detection channels used, the GenBank accession numbers of the various elements, and the melting points of the detected melting peaks are summarized in Table 2 .

[0168] Table 2. Detection scheme

[0169]

[0170]

[0171] In brief, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included: 1×Taq PCR buffer, 6 mM MgCl2, 0.32 mM dNTPs, 2 U Taq HS (TaKaRa, Beijing), the probes and primers described in Table 1 (used at specified working concentrations), and 5 μL DNA template (mixed plasmid containing 36 transgenic elements) or water (negative control). The PCR amplification procedure and melting curve analysis procedure were the same as in Example 1, and the fluorescence signals of the five channels of FAM, HEX, ROX, CY5 and Quasar 705 were detected. The instrument used in this example was a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.). The test results are shown in the figure. Figure 3 and Figure 4 shown.

[0172] Figure 3The results showed that the two reactions produced a total of 36 melting peaks, corresponding to 36 transgenic elements, and each transgenic element was correctly identified. In addition, the melting point difference between adjacent melting peaks in each channel was more than 4°C, and there was no melting point overlap. This result shows that for samples containing different transgenic elements, the designed detection system can obtain correct detection results. For any sample to be tested, the designed detection system can determine whether the sample to be tested contains the corresponding transgenic element based on the presence or absence of a melting peak in the test results.

[0173] Figure 4 The results of the detection of DNA templates (mixed plasmids) with different concentration gradients diluted 10 times, where the black solid line, gray dotted line, black dotted line, and gray solid line represent the concentration of plasmid DNA of 5×10 4 Copies / reaction, 5×10 3 Copies / reaction, 5×10 2 copies / reaction and 5×10 1 The results of the melting curve analysis of the copies / reactions. The gray dotted line represents the results of the melting curve analysis of the sample without template control after PCR amplification. The results show that the system of the present invention can detect the number of copies / reactions as low as 5×10 1 Each transgenic element of the copy / reaction mixed plasmid can be correctly detected, with high detection sensitivity, which can meet the detection needs of genetically modified products.

[0174] Example 3. Simultaneous detection of multiple elements in transgenic soybeans

[0175] Using the detection scheme, detection system and detection procedure in Example 2, we ground the genetically modified soybeans provided by Xiamen Customs with liquid nitrogen, extracted the soybean genomic DNA using the classic phenol-chloroform extraction method, and then conducted the detection. The detection results are as follows: Figure 5 shown.

[0176] Figure 5 The results showed that the two reactions produced a total of 7 melting peaks. The HEX channel of reaction 1 detected the melting peaks corresponding to CTP2-CP4-EPSPS (59.4℃) and lectin (73.9℃), the ROX channel detected the melting peaks corresponding to pSsuAra (51.4℃) and tE9 (59.8℃), the CY5 channel detected the melting peak corresponding to cry1A(c) (50.0℃), the CY5 channel of reaction 2 detected the melting peak corresponding to pFMV35S (67.6℃), and the Quasar 705 channel detected the melting peak corresponding to 18S rDNA (67.0℃). The detection results are consistent with the detection results of the detection method provided in the national standard of the People's Republic of China GB / T 19495.4-2018.

[0177] The above experimental results show that the system of the present invention can simultaneously detect and identify multiple gene elements of transgenic organisms.

[0178] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for detecting a genetic element, comprising: (a) providing a sample containing one or more target nucleic acids, wherein the target nucleic acids include one or more genetic elements; and providing a first universal primer and a second universal primer, and providing at least one target-specific primer pair for each genetic element; wherein, The first universal primer comprises a first universal sequence; The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence; The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the gene element, and the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence is not completely complementary to the complementary sequence of the forward primer; and (b) amplifying the target nucleic acid in the sample by PCR reaction using the first universal primer and the second universal primer and the target-specific primer pair under conditions allowing nucleic acid amplification, thereby obtaining an amplification product corresponding to the sample; (c) performing melting curve analysis on the amplified product obtained in step (b); (d) judging whether a genetic element exists according to the melting curve analysis result of step (c); judging the species of the sample by judging whether the genetic element exists, or judging whether the sample is a transgenic sample by judging whether the genetic element exists.

2. The method of claim 1, wherein the sample is obtained from plants and / or animals.

3. The method of claim 2, wherein The plant from which the sample is obtained is selected from soybean, corn, cotton, potato, tomato, rice, papaya, or any combination thereof.

4. The method of claim 1, wherein The genetic element is selected from a promoter, an enhancer, an operator, a resistance gene, a selection marker gene, a terminator, or any combination thereof.

5. The method of claim 4, wherein The screening marker gene is an exogenous gene of the sample.

6. The method of claim 5, wherein The exogenous gene is a transgenic gene.

7. The method of claim 1, wherein The target nucleic acid comprises a genetic element selected from the following: tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi, 18S rDNA, and any combination of the aforementioned genetic elements.

8. The method of claim 1, wherein The target nucleic acid in the sample includes the following genetic elements: tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi and 18SrDNA.

9. The method of any one of claims 1 to 8, wherein in step (a), for each gene element, a detection probe is further provided, wherein the detection probe comprises a nucleotide sequence specific to the target nucleic acid and is capable of annealing or hybridizing with a region of the target nucleic acid containing the gene element, and the detection probe is labeled with a reporter group and a quencher group, wherein: The reporter group is capable of emitting a signal, and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridized with its complementary sequence is different from the signal emitted when not hybridized with its complementary sequence; Furthermore, in step (c), the detection probe is used to perform a melting curve analysis on the amplification product corresponding to the sample obtained in step (b).

10. The method of claim 9, wherein the method has one or more technical features selected from the following: (1) In step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, and a nucleic acid polymerase, and a PCR reaction is carried out, and then, after the PCR reaction is completed, a detection probe is added to the product of step (b), and a melting curve analysis is performed; or, in step (b), the sample is mixed with the first universal primer, the second universal primer, the target-specific primer pair, the detection probe, and a nucleic acid polymerase, and a PCR reaction is carried out, and then, after the PCR reaction is completed, a melting curve analysis is performed; (2) The detection probe comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (3) The length of the detection probe is 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-200 nt, 200-300 nt, 300-400 nt, 400-500 nt, 500-600 nt, 600-700 nt, 700-800 nt, 800-900 nt, 900-1000 nt; (4) The detection probe has a 3'-OH end; or, the 3'-end of the detection probe is blocked; (5) The detection probe is a self-quenching probe; (6) The reporter group in the detection probe is a fluorescent group; and the quencher group is a molecule or group that can absorb / quench the fluorescence; (7) The detection probe has resistance to nuclease activity; (8) The detection probe is linear or has a hairpin structure; (9) The detection probes each independently have the same or different reporter groups; (10) In step (c), the product of step (b) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a curve showing the change of the signal intensity of each reporter group with the change of temperature; then, the curve is derived to obtain a melting curve of the product of step (b); (11) The detection probe includes a detection probe having a nucleotide sequence selected from the following: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 and, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 103 and SEQ ID NO:

106.

11. The method of claim 10, wherein the method has one or more technical features selected from the following: (1) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (2) The non-natural nucleotide is a peptide nucleic acid or a locked nucleic acid; (3) blocking the 3'-end of the detection probe by adding a chemical moiety to the 3'-OH of the last nucleotide of the detection probe, by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a dideoxynucleotide; (4) The detection probe is labeled with a reporter group at its 5' end or upstream and a quencher group at its 3' end or downstream, or a reporter group at its 3' end or downstream and a quencher group at its 5' end or upstream; (5) The reporter group and the quencher group are separated by a distance of 10-80 nt or longer; (6) The fluorescent gene is selected from ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXASRED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705; (7) The quenching group is selected from DABCYL, BHQ, ECLIPSE, and / or TAMRA; (8) The detection probe has 5' nuclease activity; (9) The main chain of the detection probe contains a modification that resists nuclease activity; (10) The backbone of the detection probe comprises a phosphorothioate bond, an alkyl phosphotriester bond, an aryl phosphotriester bond, an alkyl phosphonate bond, an aryl phosphonate bond, a hydrogenated phosphate bond, an alkyl phosphoramidate bond, an aryl phosphoramidate bond, a 2'-O-aminopropyl modification, a 2'-O-alkyl modification, a 2'-O-allyl modification, a 2'-O-butyl modification, and a 1-(4'-thio-PD-ribofuranosyl) modification; (11) The detection probes have the same reporter group, and a melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined based on the melting peak in the melting curve; or, the detection probes have different reporter groups, and a melting curve analysis is performed on the product of step (b), and then the presence of the target nucleic acid is determined based on the signal type of the reporter group and the melting peak in the melting curve.

12. The method of claim 1, wherein The method has one or more technical features selected from the following: (1) The sample contains 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids; (2) The target nucleic acid comprises 1-5, 5-10, 10-15, 15-20, 20-30 or more genetic elements; (3) The sample is a DNA, RNA molecule or any combination thereof; (4) The target nucleic acid is a DNA, RNA molecule or any combination thereof; (5) The concentration of the sample is greater than 1 copy / reaction; (6) in step (a) of the method, providing 1-5, 5-10, 10-15, 15-20, 20-30 or more target-specific primer pairs; (7) The target nucleic acid to be amplified is single-stranded or double-stranded; (8) In step (b) of the method, the working concentrations of the first universal primer and the second universal primer are higher than the working concentrations of the forward primer and the reverse primer; (9) In step (b) of the method, the working concentrations of the first universal primer and the second universal primer are the same; or, the working concentration of the first universal primer is lower than that of the second universal primer; (10) In step (b) of the method, the working concentrations of the forward primer and the reverse primer are the same or different; (11) the sample or target nucleic acid comprises mRNA, and the sample is subjected to a reverse transcription reaction before performing step (b) of the method; and (12) In step (b) of the method, a nucleic acid polymerase is used to perform a PCR reaction.

13. The method of claim 12, wherein the method has one or more technical features selected from the following: (1) The DNA is genomic DNA or cDNA; (2) the working concentrations of the first universal primer and the second universal primer are 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentrations of the forward primer and the reverse primer; (3) The nucleic acid polymerase is a template-dependent nucleic acid polymerase; (4) The nucleic acid polymerase is a DNA polymerase.

14. The method of claim 13, wherein the DNA polymerase is a thermostable DNA polymerase.

15. The method of claim 14, the thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcusliteralis, Thermus antranildanii, Thermus caldophllus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermusruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermusthermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Thermococcus litoralis, Thermococcus barossi, Thermococcusgorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcus horikoshii, Pyrococcusabyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifex aeolieus.

16. The method of claim 13, wherein the DNA polymerase is Taq polymerase.

17. The method of claim 1, wherein The method has one or more technical features selected from the following: (1) The first universal primer consists of a first universal sequence, or comprises the first universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the first universal sequence; (2) the first universal sequence is located in or constitutes the 3' portion of the first universal primer; (3) The length of the first universal primer is 5-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt; (4) The first universal primer or any component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (5) the second universal primer consists of a second universal sequence, or comprises the second universal sequence and an additional sequence, wherein the additional sequence is located at the 5' end of the second universal sequence; (6) the second universal sequence is located in or constitutes the 3' portion of the second universal primer; (7) the second universal sequence comprises the first universal sequence and additionally comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides at the 3' end of the first universal sequence; (8) the length of the second universal primer is 8-15 nt, 15-20 nt, 20-30 nt, 30-40 nt, or 40-50 nt; and (9) The second universal primer or any component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

18. The method of claim 17, wherein the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

19. The method of claim 1, wherein The method has one or more technical features selected from the following: (1) In the forward primer, the forward nucleotide sequence is directly linked to the 3' end of the first universal sequence, or is linked to the 3' end of the first universal sequence through a nucleotide linker; (2) the forward primer further comprises an additional sequence located at the 5' end of the first universal sequence; (3) The forward primer comprises or consists of the first universal sequence and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the first universal sequence, a nucleotide linker and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the additional sequence, the first universal sequence and the forward nucleotide sequence from 5' to 3'; or, comprises or consists of the additional sequence, the first universal sequence, a nucleotide linker and the forward nucleotide sequence from 5' to 3'; (4) the forward nucleotide sequence is located in or constitutes the 3' portion of the forward primer; (5) The length of the forward nucleotide sequence is 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt; (6) The length of the forward primer is 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140 nt, 140-150 nt; (7) The forward primer or any component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; (8) In the reverse primer, the reverse nucleotide sequence is directly linked to the 3' end of the second universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the second universal sequence via a nucleotide linker; (9) the reverse primer further comprises an additional sequence located at the 5' end of the second universal sequence; (10) The reverse primer comprises or consists of the second universal sequence and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of the second universal sequence, a nucleotide linker and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the second universal sequence and the reverse nucleotide sequence from 5' to 3'; or, comprises or consists of an additional sequence, the second universal sequence, a nucleotide linker and the reverse nucleotide sequence from 5' to 3'; (11) the reverse nucleotide sequence is located in or constitutes the 3' portion of the reverse primer; (12) The length of the reverse nucleotide sequence is 10-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt; (13) The length of the reverse primer is 15-20 nt, 20-30 nt, 30-40 nt, 40-50 nt, 50-60 nt, 60-70 nt, 70-80 nt, 80-90 nt, 90-100 nt, 100-110 nt, 110-120 nt, 120-130 nt, 130-140nt, 140-150 nt; (14) the reverse primer or any component thereof comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and (15) The second universal sequence is not completely complementary to the complementary sequence of the forward primer.

20. The method of claim 19, wherein The method has one or more technical features selected from the following: (1) The nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides; (2) the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides; (3) The naturally occurring nucleotide is a deoxyribonucleotide or a ribonucleotide; (4) At least one nucleotide at the 3' end of the second universal sequence is not complementary to the complementary sequence of the forward primer.

21. The method of claim 1, wherein the sequence of the first universal primer is shown in SEQ ID NO:

109.

22. The method of claim 1, wherein the sequence of the second universal primer is shown in SEQ ID NO:

110.

23. The method of claim 1, wherein the target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 8 and SEQ ID NO: 9; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 20 and SEQ ID NO: 21; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 27; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 32 and SEQ ID NO: 33; SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 38 and SEQ ID NO: 39; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 49 and SEQ ID NO:

50. SEQ ID NO: 44 and SEQ ID NO: 45; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 50 and SEQ ID NO: 51; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 56 and SEQ ID NO: 57; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 62 and SEQ ID NO: 63; SEQ ID NO: 65 and SEQ ID NO: 66; SEQ ID NO: 68 and SEQ ID NO: 69; SEQ ID NO: 71 and SEQ ID NO: 72; SEQ ID NO: 74 and SEQ ID NO: 75; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 80 and SEQ ID NO: 81; SEQ ID NO: 83 and SEQ ID NO: 84; SEQ ID NO: 86 and SEQ ID NO: 87; SEQ ID NO: SEQ ID NO: 89 and SEQ ID NO: 90; SEQ ID NO: 92 and SEQ ID NO: 93; SEQ ID NO: 95 and SEQ ID NO: 96; SEQ ID NO: 98 and SEQ ID NO: 99; SEQ ID NO: 101 and SEQ ID NO: 102;SEQ ID NO: 104 and SEQ ID NO: 105; SEQ ID NO: 107 and SEQ ID NO: 108.; 24. The method of claim 1, wherein Steps (a)-(b) of the method are carried out by a scheme comprising the following steps (I)-(VI): (I) providing a sample containing one or more target nucleic acids, wherein the target nucleic acids include one or more genetic elements; and providing a first universal primer and a second universal primer, and providing a target-specific primer pair for each genetic element; wherein the first universal primer, the second universal primer and the target-specific primer pair are as defined in claim 1; (II) mixing the sample with the first universal primer and the second universal primer and a target-specific primer pair, and a nucleic acid polymerase; (III) incubating the product of the previous step under conditions that permit nucleic acid denaturation; (IV) incubating the product of the previous step under conditions that permit annealing or hybridization of the nucleic acids; (V) incubating the product of the previous step under conditions permissive for nucleic acid extension; and (VI) Repeating steps (III) to (V) one or more times.

25. The method of claim 24, wherein the method has one or more technical features selected from the following: (1) in step (III), incubating the product of step (II) at a temperature of 80-105° C. to denature the nucleic acid; (2) in step (III), incubating the product of step (II) for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min; (3) in step (IV), incubating the product of step (III) at a temperature of 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, or 65-70°C to allow nucleic acid annealing or hybridization; (4) in step (IV), incubating the product of step (III) for 10-20 s, 20-40 s, 40-60 s, 1-2 min, or 2-5 min; (5) in step (V), incubating the product of step (4) at a temperature of 35-40°C, 40-45°C, 45-50°C, 50-55°C, 55-60°C, 60-65°C, 65-70°C, 70-75°C, 75-80°C, 80-85°C to allow nucleic acid extension; (6) in step (V), incubating the product of step (IV) for 10-20 s, 20-40 s, 40-60 s, 1-2 min, 2-5 min, 5-10 min, 10-20 min or 20-30 min; (7) performing steps (IV) and (V) at the same or different temperatures; and (8) Repeat steps (III) to (V) at least once.

26. The method of claim 24, wherein when steps (III)-(V) are repeated one or more times, the conditions used in steps (III)-(V) of each cycle are independently the same or different.

27. The method of claim 25, wherein steps (III) to (V) are repeated 2 times, 5 times, 10 times, 20 times, 30 times, 40 times or 50 times.

28. A kit comprising a primer set capable of asymmetrically amplifying a target nucleic acid containing a genetic element; in, The primer set comprises: a first universal primer and a second universal primer, and, for each gene element, at least one target-specific primer pair is provided, wherein: The first universal primer comprises a first universal sequence; The second universal primer comprises a second universal sequence, the second universal sequence comprises the first universal sequence and additionally comprises at least one nucleotide at the 3' end of the first universal sequence; The target-specific primer pair can amplify the target nucleic acid as a template to produce a nucleic acid product containing the gene element, and the target-specific primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises a first universal sequence and a forward nucleotide sequence specific to the target nucleic acid, and the forward nucleotide sequence is located at the 3' end of the first universal sequence; the reverse primer comprises a second universal sequence and a reverse nucleotide sequence specific to the target nucleic acid, and the reverse nucleotide sequence is located at the 3' end of the second universal sequence; and the second universal sequence cannot be completely complementary to the complementary sequence of the forward primer.

29. The kit of claim 28, further comprising one or more detection probes capable of detecting the gene element, the detection probes comprising a nucleotide sequence specific to the target nucleic acid and capable of annealing or hybridizing to a region of the target nucleic acid containing the gene element, and being labeled with a reporter group and a quencher group, wherein: The reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the signal emitted by the detection probe when hybridizing with its complementary sequence is different from the signal emitted when not hybridizing with its complementary sequence.

30. The kit of claim 28, further comprising one or more components selected from the group consisting of a nucleic acid polymerase, a reagent for performing nucleic acid amplification, a reagent for performing melting curve analysis, or any combination thereof.

31. The kit of claim 28, wherein the genetic element is selected from a promoter, an enhancer, an operator, a selection marker gene, a terminator, or any combination thereof. The kit of claim 31 , wherein the screening marker gene is an exogenous gene of the sample.

33. The kit of claim 28, wherein the target nucleic acid comprises a genetic element selected from the group consisting of tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi, 18S rDNA, and any combination of the foregoing genetic elements.

34. The kit of claim 28, wherein the target nucleic acid comprises the following genetic elements: tOCS, pRice-Eactin, pNOS, bar, NPTII, cry3A, CTP2-CP4-EPSPS, waxy, Lectin, pSsuAra, tE9, pCaMV35S, pTA29, CryIA(c), CHY, UGPase, SadI, PMI, tCaMV35S, PAT, tg7, CP4-EPSPS, SAH7, Alfalfa-Acc, tNOS, CryIA(b), LAT52, GAG56, GLuA3, SPS, HMG, GOX, zSSIIb, pFMV35S, pUbi and 18S rDNA.

35. The kit of claim 29, wherein the detection probe comprises a detection probe having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 and, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 103 and SEQ ID NO:

106.

36. The kit of claim 28, wherein The kit has one or more features selected from the following: (1) The sequence of the first universal primer is shown in SEQ ID NO: 109; (2) The sequence of the second universal primer is shown in SEQ ID NO: 110; (3) The target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 8 and SEQ ID NO: 9; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 20 and SEQ ID NO: 21; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 27; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 32 and SEQ ID NO: 33; SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 38 and SEQ ID NO: 39; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: SEQ ID NO: 81 ; SEQ ID NO: 83 and SEQ ID NO: 84; SEQ ID NO: 86 and SEQ ID NO: 87; SEQ ID NO: 89 and SEQ ID NO: 90; SEQ ID NO: 91 and SEQ ID NO: 92; SEQ ID NO: 93 and SEQ ID NO: 94; SEQ ID NO: 95 and SEQ ID NO: 96; SEQ ID NO: 97 and SEQ ID NO: 98; SEQ ID NO: 100 and SEQ ID NO: 101; SEQ ID NO: 102 and SEQ ID NO: 103; SEQ ID NO: 104 and SEQ ID NO: 105; SEQ ID NO: 106 and SEQ ID NO: 107; SEQ ID NO: 108 and SEQ ID NO: 109; NO: 90; SEQ ID NO: 92 and SEQ ID NO: 93; SEQ ID NO: 95 and SEQ ID NO: 96; SEQ ID NO: 98 and SEQ ID NO: 99; SEQ ID NO: 101 and SEQ ID NO: 102;SEQ ID NO: 104 and SEQ ID NO: 105; SEQ ID NO: 107 and SEQ ID NO: 108.; 37. The kit of claim 30, wherein The kit has one or more features selected from the following: (1) The nucleic acid polymerase is a template-dependent nucleic acid polymerase; (2) The reagents for nucleic acid amplification include enzyme working buffer, dNTPs, water, a solution containing ions, a single-stranded DNA binding protein, or any combination thereof; (3) The kit is used to detect genetic elements; (4) The kit is used to detect transgenic elements.

38. Use of the primer set defined in any one of claims 28 to 37 for preparing a kit for asymmetric amplification of a target nucleic acid containing a genetic element.

39. The use according to claim 38, wherein the kit further comprises a detection probe as defined in any one of claims 9 to 11.

40. The use according to claim 38, wherein the kit is used to implement the method described in any one of claims 1 to 27.

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