CN114686574A

CN114686574A - Method and kit for detecting deletion of large nucleotide fragment

Info

Publication number: CN114686574A
Application number: CN202011563858.0A
Authority: CN
Inventors: 黄秋英; 李庆阁; 刘雪铖
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2022-07-01

Abstract

The present application provides a method for detecting nucleotide fragment deletions (particularly large fragment deletions) that is capable of simultaneously detecting the presence of one or more (e.g., 1, 2, 5, 10, 15, 19, 20, 25, 30, 35, 40 or more) nucleotide fragment deletions in a target nucleic acid by simultaneously asymmetrically amplifying multiple target nucleic acids present in a sample. In addition, the present application provides a kit capable of simultaneously detecting the presence of one or more (e.g., 1, 2, 5, 10, 15, 19, 20, 25, 30, 35, 40 or more) nucleotide fragment deletions in a nucleic acid molecule in a single round of reaction.

Description

Method and kit for detecting deletion of large nucleotide fragment

Technical Field

The present application relates to multiplex asymmetric detection of nucleic acid molecules. In particular, the present application provides a method for detecting nucleotide fragment deletions (particularly large fragment deletions) that is capable of simultaneously detecting the presence of one or more (e.g., 1, 2, 5, 10, 15, 19, 20, 25, 30, 35, 40, or more) nucleotide fragment deletions in a target nucleic acid by simultaneously asymmetrically amplifying multiple target nucleic acids present in a sample. In addition, the present application provides a kit capable of simultaneously detecting the presence of one or more (e.g., 1, 2, 5, 10, 15, 19, 20, 25, 30, 35, 40 or more) nucleotide fragment deletions in a nucleic acid molecule in a single round of reaction.

Background

Large fragment deletions and duplications of genes are also known as Copy Number Variations (CNV). With the development and application of CNV microarray chip technology and second-generation sequencing technology, Copy Number Variation (CNV) has been found to be widely present in the human chromosomal genome. CNVs are mainly caused by genomic recombination, generally meaning an increase or decrease in copy number of large genomic fragments of several kb to several Mb in length, and mainly manifest as repeats, deletions and insertions at a submicroscopic level. The current detection methods of CNV mainly comprise: the method comprises the following steps of a multiple ligation dependent probe amplification (MLPA) technology, microarray comparative gene hybridization (array-CGH), a Single Nucleotide Polymorphism (SNP) typing chip, a high-throughput sequencing technology, a Fluorescence In Situ Hybridization (FISH) technology, a real-time fluorescent quantitative PCR technology (RT-qPCR) and the like. The array-CGH, the SNP typing chip, the high-throughput sequencing technology and the like are mainly used for detecting unknown CNV in the whole genome range. These techniques are advanced, accurate and efficient in detecting CNVs in the genome-wide range, but the detection cost is high and expensive instruments are required, so they are not the best choice in detecting CNVs of certain specific genes or specific regions. The fluorescence in situ hybridization technology is widely applied to the detection of chromosome aneuploidy and chromosome aberration at present, but is very small in application to the detection of copy number variation of a single specific gene due to the complex operation process and high cost. The RT-qPCR combined with Gap-PCR method is classical, but the efficiency is too low to detect a plurality of fragments in one reaction system at the same time. MLPA was first reported by Schouten et al in 2002 and is the most widely used gene copy number detection technology at present. The method can detect the copy number of a specific gene or a specific region, and can detect the copy number of up to 50 different gene segments in the same reaction tube, however, the method needs a plurality of operation steps such as hybridization, connection, PCR amplification, capillary electrophoresis analysis of PCR products and the like, has troublesome operation, long time consumption and higher requirements on the quality and the concentration of genome DNA. Patent CN107058541A discloses a CNVplex high-throughput ligation probe amplification method (HLPA) -based kit for detecting copy number variation of phenylketonuria-related genes, the detection principle and detection process of which are similar to MLPA technology, and can detect copy number variation of multiple gene segments simultaneously. However, this method has the same drawbacks as the MLPA technique.

In view of the above, various methods for detecting copy number variation of genes have been reported, but each of them has some limitations. Therefore, there is still a need to develop a method for detecting gene copy number variation (especially detecting gene fragment deletion) more rapidly, simply, sensitively, specifically, stably, reliably, economically and efficiently.

Disclosure of Invention

In the present application, the inventors are able to simultaneously detect the presence of one or more (e.g., 1, 2, 5, 10, 15, 19, 20, 25, 30, 35, 40 or more) nucleotide fragment deletions in a target nucleic acid by simultaneously asymmetrically amplifying multiple target nucleic acids present in a sample. On the basis, the inventor of the application develops a rapid, simple, sensitive, specific, stable and reliable high-throughput nucleic acid detection method which can simultaneously detect the deletion of a plurality of nucleotide fragments in a single tube.

In a first aspect of the present application, there is provided a method of detecting the presence of n nucleotide fragment deletions in a target nucleic acid in a sample, wherein n is an integer ≧ 1 (e.g., n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more), and the method comprises the steps of:

(a) providing a universal primer, and a first target-specific primer pair and a second target-specific primer pair for each nucleotide fragment deletion to be detected; wherein the content of the first and second substances,

the universal primer comprises a first universal sequence;

a first target-specific primer pair capable of amplifying a wild-type target nucleic acid and comprising a forward primer 1 and a reverse primer 1, wherein the forward primer 1 comprises a second universal sequence and a wild-type forward nucleotide sequence specific for the wild-type target nucleic acid, and the wild-type forward nucleotide sequence is located 3' of the second universal sequence; the reverse primer 1 comprises a first universal sequence and a wild-type reverse nucleotide sequence specific for the wild-type target nucleic acid, and the wild-type reverse nucleotide sequence is located 3' of the first universal sequence;

a second target-specific primer pair capable of amplifying a deleted target nucleic acid and comprising a forward primer 2 and a reverse primer 2, wherein the forward primer 2 comprises a second universal sequence and a deleted forward nucleotide sequence specific for the deleted target nucleic acid, and the deleted forward nucleotide sequence is located 3' of the second universal sequence; the reverse primer 2 comprises a first universal sequence and a deleted reverse nucleotide sequence specific to the deleted target nucleic acid, and the deleted reverse nucleotide sequence is located 3' of the first universal sequence;

and, the first universal sequence is capable of hybridizing or annealing to a complement of the second universal sequence under conditions that allow for hybridization or annealing of nucleic acids, and the second universal sequence differs from the first universal sequence by the inclusion of one or more nucleotides at the 3' end of the first universal sequence that are each independently deleted or substituted; and, the first universal sequence is not fully complementary to the complement of the forward primer 1 or forward primer 2;

(b) amplifying the target nucleic acid in the sample by a PCR reaction using the universal primer and the first and second target-specific primer pairs under conditions that allow for nucleic acid amplification;

(c) performing a melting curve analysis on the product of step (b); and determining whether each of the deletion type target nucleic acids and/or the wild type target nucleic acid is present in the sample and whether each of the nucleotide fragment deletions is present in the target nucleic acid in the sample based on the results of the melting curve analysis.

In the method of the present invention, the forward primer and the reverse primer comprise a forward nucleotide sequence and a reverse nucleotide sequence, respectively, specific to said target nucleic acid, whereby, during the PCR reaction, a target-specific primer pair (forward primer and reverse primer) will anneal to the target nucleic acid and initiate PCR amplification, resulting in an initial amplification product comprising two nucleic acid strands (nucleic acid strand a and nucleic acid strand B) complementary to the forward primer and reverse primer, respectively.

Further, since both the reverse primer and the universal primer comprise the first universal sequence, the nucleic acid strand B complementary to the reverse primer is also complementary to the universal primer. Thus, during a PCR reaction, the universal primer is able to anneal to the nucleic acid strand B and normally initiate PCR amplification (i.e., normally synthesize the complementary strand of nucleic acid strand B). At the same time, since the first universal sequence is capable of hybridizing or annealing to the complement of the second universal sequence under conditions that allow nucleic acid hybridization or annealing, the universal primer (which comprises the first universal sequence) is also capable of annealing to the nucleic acid strand a that is complementary to the forward primer (which comprises the second universal sequence) during the PCR reaction. However, since the second universal sequence differs from the first universal sequence in that one or more nucleotides located at the 3 'end of the first universal sequence are each independently deleted or replaced, the universal primer (particularly the 3' end thereof) is not capable of being fully complementary to the nucleic acid strand a, which results in the inhibition of PCR amplification of the nucleic acid strand a by the universal primer (i.e., the inhibition of synthesis of the complementary strand of the nucleic acid strand a).

Thus, as the PCR reaction proceeds, the universal primers will anneal to the nucleic acid strand a and nucleic acid strand B of the initial amplification product, respectively, and further initiate PCR amplification, wherein synthesis of the complementary strand of nucleic acid strand B will proceed normally, while synthesis of the complementary strand of nucleic acid strand a will be inhibited. Therefore, as PCR amplification proceeds, the synthesis efficiency of the complementary strand of the nucleic acid strand a (nucleic acid strand B) will be significantly lower than that of the nucleic acid strand B (nucleic acid strand a), resulting in the synthesis and amplification of the complementary strand of the nucleic acid strand B (nucleic acid strand a) in a large amount, while the synthesis and amplification of the complementary strand of the nucleic acid strand a (nucleic acid strand B) is suppressed, thereby producing a large amount of single-stranded product (nucleic acid strand a, which contains a sequence complementary to the forward primer/second universal sequence and a sequence of the reverse primer/universal primer), achieving asymmetric amplification of the target nucleic acid.

In addition, since the first universal sequence in the reverse primer is capable of hybridizing or annealing to the complementary sequence of the second universal sequence in the forward primer under conditions that allow nucleic acid hybridization or annealing, primer dimers formed by non-specific amplification of the forward and reverse primers during the PCR reaction will, after denaturation, produce single-stranded nucleic acids whose 5 'and 3' ends contain sequences that are capable of complementary annealing to each other, which readily self-anneal during the annealing stage, forming a stable panhandle structure that prevents annealing and extension of the single-stranded nucleic acids by the universal primers, thereby inhibiting further amplification of the primer dimers. Therefore, in the method of the present invention, nonspecific amplification of primer dimer can be effectively suppressed. Thus, the method of the present invention is particularly suitable for performing multiplex amplification of a target nucleic acid containing a deletion of a nucleotide fragment. For example, in the methods of the invention, universal primers can be used in combination with a plurality of target-specific primer pairs to achieve multiplex amplification of one or more target nucleic acids containing nucleotide fragment deletions.

Thus, in certain preferred embodiments, the methods of the invention are capable of simultaneously amplifying 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids, e.g., at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target nucleic acids. In certain preferred embodiments, the methods of the invention are capable of amplifying 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids simultaneously and asymmetrically. In such embodiments, accordingly, a first target-specific primer pair and a second target-specific primer pair are provided for each nucleotide fragment deletion in step (a). Thus, in such embodiments, 1-5, 5-10, 10-15, 15-20, 20-50 or more first and/or second target-specific primer pairs are provided in step (1), e.g., at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more first and/or second target-specific primer pairs.

It will be readily appreciated that for different nucleotide fragment deletions, different forward and reverse primers may be used. However, when there is sequence identity or similarity between different nucleotide fragment deletions or between wild-type and nucleotide-deleted fragments, different target-specific primer pairs may have the same forward or reverse primer.

In order to facilitate multiplex asymmetric amplification and to effectively inhibit non-specific amplification of primer dimers, in certain preferred embodiments, the working concentration of the universal primer is higher than the working concentration of the forward primer 1, forward primer 2, reverse primer 1 and reverse primer 2. In certain preferred embodiments, the working concentration of the universal primer is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 18-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold or more higher than the working concentration of the forward primer 1, the forward primer 2, the reverse primer 1, and the reverse primer 2. In certain preferred embodiments, the working concentration of the universal primer is 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentration of the forward primer 1, the forward primer 2, the reverse primer 1 and the reverse primer 2.

In the method of the present invention, the working concentrations of the forward primer 1, the forward primer 2, the reverse primer 1 and the reverse primer 2 may be the same or different. In certain preferred embodiments, the working concentration of forward primer 1, forward primer 2, reverse primer 1 and reverse primer 2 is the same. In certain preferred embodiments, the working concentrations of forward primer 1, forward primer 2, reverse primer 1 and reverse primer 2 are different. In certain preferred embodiments, the working concentration of the forward primer 1 and forward primer 2 is lower than the working concentration of the reverse primer 1 and reverse primer 2.

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.

In certain embodiments, in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to perform the PCR reaction; in certain embodiments, the nucleic acid polymerase is a DNA polymerase, e.g., a thermostable DNA polymerase; in certain embodiments, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus tiramilis, Thermus antandranii, Thermus caldophlus, Thermus chloridophilus, Thermus flavus, Thermus igniterae, Thermus lacteus, Thermus osimami, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus fulvoranserina, Thermus thermophilus, Thermotoga maritima, Thermotoga neoparatana, Thermomyces affiulus, Thermococcus pacificus, Thermococcus maculatus, Thermomyces purpurea, Thermomyces nigra, Thermomyces flaveria afolicus, Thermococcus flavus, Thermococcus purpurea, Thermoctoria, Thermocapiaria, Thermocosissima pacifia, Thermococcus, Thermoctoria, Thermocapium, Thermocapiaria pacifia purpurea, Thermococcus, Thermoctoria, Thermocapium, Theragrichia purpurea, Thermococcus, Theragrichia purpurea, Theragria purpurea, Thermococcus, Theragria, Theragrichia, Theragria purpuria, Theragria, Theragrichia, Theragria purpurea, Theragria purpurea, Theragria purpuria, Theragria purpuria, Theragria purpura, Theragria, Thermorpeumorquium, Thermorquium, Thermorpeumorpeutical. In certain embodiments, the DNA polymerase is Taq polymerase.

In certain embodiments, the nucleotide fragment deletion refers to a deletion of at least 200 consecutive bases.

In certain embodiments, the nucleotide fragment deletion refers to a deletion of at least 500 consecutive bases.

In certain embodiments, the nucleotide fragment deletion is a large fragment deletion of the phenylalanine hydroxylase (PAH) gene. In certain embodiments, the PAH gene large fragment deletion is selected from ex1del5.3kb, ex3del6.6kb, Ex4del, Ex5del, Ex6del, ex1del3.7kb, Ex3del4.7kb, Ex5_6del, Ex4_7del, Ex4_5del, or any combination thereof.

In certain embodiments, in step (a), the reverse primer 1 is no more than 500bp away from the forward primer 1 when hybridized to the wild type target nucleic acid, e.g., 200-500 bp; and/or, when hybridized to a wild-type target nucleic acid, the reverse primer 2 is at least 500bp, e.g., at least 600bp, at least 700bp, at least 800bp, at least 900bp, at least 1000bp or more from the forward primer 1 and/or forward primer 2.

In certain embodiments, in step (a), the reverse primer 1 hybridizes to a nucleotide fragment suspected of being deleted and the forward primer 1 hybridizes upstream of the nucleotide fragment suspected of being deleted when hybridized to a wild-type target nucleic acid; the reverse primer 2 is different from the reverse primer 1, and the forward primer 1 and the forward primer 2 may be the same or different.

In certain embodiments, in step (a), the forward primer 1 hybridizes to a nucleotide fragment suspected of being deleted and the reverse primer 1 hybridizes downstream of the nucleotide fragment suspected of being deleted when hybridized to a wild-type target nucleic acid; the forward primer 1 is different from the forward primer 2, and the reverse primer 1 and the reverse primer 2 may be the same or different.

In certain embodiments, in step (a), the forward primer 1 and reverse primer 1 both hybridize to a nucleotide fragment suspected of being deleted when hybridized to a wild-type target nucleic acid; the reverse primer 2 is different from the reverse primer 1, and the forward primer 1 is different from the forward primer 2; and, when hybridizing to a deletion target nucleic acid sequence, the deletion target sequence can comprise a sequence complementary to the deletion target nucleic acid that comprises a breakpoint or is located upstream or downstream of the breakpoint.

In certain embodiments, in step (a), for each nucleotide fragment deletion, a wild-type detection probe and a deleted-type detection probe are also provided, respectively, wherein the wild-type detection probe comprises a wild-type targeting sequence specific for the wild-type target nucleic acid and the wild-type targeting sequence is capable of annealing or hybridizing to all or part of the region of the nucleotide fragment of the wild-type target nucleic acid suspected of being deleted; the deletion detection probe comprises a deletion targeting sequence specific to the deletion target nucleic acid, and the deletion targeting sequence comprises a sequence complementary to the deletion target nucleic acid sequence;

in certain embodiments, the deletion detection probe is capable of annealing to or hybridizing to the products of the forward primer 1 and reverse primer 1 amplification of the wild-type target nucleic acid, and the wild-type detection probe is capable of annealing to or hybridizing to the products of the forward primer 2 and reverse primer 2 amplification of the deletion target nucleic acid;

in certain preferred embodiments, the wild-type detection probe is the same as the deletion-type detection probe;

in certain preferred embodiments, the targeting sequence of the detection probe is fully hybridized to the wild-type target nucleic acid, and the targeting sequence portion of the detection probe is partially hybridized to the deleted target nucleic acid;

in certain preferred embodiments, the detection probe hybridizes to the wild-type target nucleic acid simultaneously to a portion of the wild-type target nucleic acid where the nucleotide fragment is suspected to be deleted and a portion of the nucleotide fragment where the deletion does not occur, and the detection probe hybridizes to the deletion target nucleic acid upstream or downstream of the breakpoint of the deletion target nucleic acid;

in such embodiments, the detection probe forms a melting point (T) of a duplex with the wild-type target nucleic acid and the deleted target nucleic acid_m) Different.

In certain embodiments, the deletion detection probe is capable of hybridizing to a wild-type target nucleic acid and does not hybridize at a position between the hybridization position of the forward primer 1 and the hybridization position of the reverse primer 1;

and, the deletion detection probe and the wild-type detection probe are each independently labeled with a reporter and a quencher, wherein the reporter is capable of emitting a signal and the quencher is capable of absorbing or quenching the signal emitted by the reporter; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement;

in step (c), the amplification products obtained in step (b) are subjected to melting curve analysis using the detection probes.

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

In certain embodiments, the detection probe may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., Peptide Nucleic Acids (PNAs) or locked nucleic acids), or any combination thereof. In certain preferred embodiments, the detection probe comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the detection probe comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the detection probe comprises a non-natural nucleotide, such as deoxyhypoxanthine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA).

In some embodiments, the length of the wild-type detection probe and the deletion-type detection probe is 15-1000nt, such as 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, respectively.

In some embodiments, the length of the targeting sequence in the wild-type detection probe and the deletion-type detection probe is 10-500nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500nt, respectively.

In certain embodiments, the deletion detection probe and the wild-type detection probe each independently have a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or alkyl) 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.

In certain embodiments, the deletion-type detection probe and the wild-type detection probe are each independently a self-quenching probe; in such embodiments, the quencher is positioned to absorb or quench the signal from the reporter (e.g., the quencher is positioned adjacent to the reporter) when the detection probe is not hybridized to the other sequence, thereby absorbing or quenching the signal from the reporter. In this case, the detection probe does not emit a signal. Further, when the detection probe hybridizes to its complement, the quencher is located at a position that is unable to absorb or quench the signal from the reporter (e.g., the quencher is located at a position remote from the reporter), and thus unable to absorb or quench the signal from the reporter. In this case, the detection probe emits a signal.

The design of such self-quenching detection probes is within the ability of those skilled in the art. For example, the detection probe may be labeled with a reporter group at the 5 'end and a quencher group at the 3' end, or the detection probe may be labeled with a reporter group at the 3 'end and a quencher group at the 5' end. Whereby, when the detection probe is present alone, the reporter and the quencher are in proximity to each other and interact such that a signal emitted by the reporter is absorbed by the quencher, thereby causing no signal to be emitted by the detection probe; and when the detection probe hybridizes to its complementary sequence, the reporter and the quencher are separated from each other such that a signal from the reporter is not absorbed by the quencher, thereby causing the detection probe to emit a signal.

However, it will be appreciated that the reporter and quencher need not be labeled at the terminus of the detection probe. The reporter and/or quencher may also be labeled within the detection probe, so long as the detection probe emits a signal upon hybridization to its complementary sequence that is different from the signal emitted without hybridization to its complementary sequence. For example, the reporter can be labeled upstream (or downstream) of the detection probe and the quencher can be labeled downstream (or upstream) of the detection probe at a sufficient distance (e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, or longer). Whereby, when the detection probe is present alone, the reporter and the quencher are in proximity to each other and interact due to free coil of the probe molecule or formation of a secondary structure (e.g., hairpin structure) of the probe such that the signal emitted by the reporter is absorbed by the quencher, thereby rendering the detection probe non-emitting a signal; and, when the detection probe hybridizes to its complement, the reporter and the quencher are separated from each other by a sufficient distance such that the signal from the reporter is not absorbed by the quencher, thereby causing the detection probe to emit a signal. In certain preferred embodiments, the reporter and quencher are separated by a distance of 10-80nt or more, e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80 nt. In certain preferred embodiments, the reporter and quencher are separated by 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 20 nt. In certain preferred embodiments, the reporter and quencher are separated by at least 5nt, at least 10nt, at least 15nt, or at least 20 nt.

Thus, the reporter and quencher can be labeled at any suitable position on the detection probe, so long as the detection probe emits a signal upon hybridization to its complementary sequence that is different from the signal emitted without hybridization to its complementary sequence. However, in certain preferred embodiments, at least one of the reporter and quencher is at the terminus (e.g., the 5 'or 3' terminus) of the detection probe. In certain preferred embodiments, one of the reporter and the quencher is located at the 5 'end of the detection probe or 1-10nt from the 5' end, and the reporter and the quencher are separated by a suitable distance such that the quencher is capable of absorbing or quenching the signal of the reporter prior to hybridization of the detection probe to its complementary sequence. In certain preferred embodiments, one of the reporter and the quencher is located at the 3 'end of the detection probe or 1-10nt from the 3' end, and the reporter and the quencher are suitably spaced apart such that the quencher is capable of absorbing or quenching the signal of the reporter prior to hybridization of the detection probe to its complementary sequence. In certain preferred embodiments, the reporter and quencher can be separated by a distance as defined above (e.g., a distance of 10-80nt or more). In certain preferred embodiments, one of the reporter and quencher is at the 5 'end of the detection probe and the other is at the 3' end.

In certain embodiments, the deletion detection probe and the wild-type detection probe each independently have the same or different reporter groups. In certain embodiments, the deletion detection probe and the wild-type detection probe have the same reporter group, and the product of step (b) is subjected to a melting curve analysis, and then the melting point (T) and the position of the melting peak in the melting curve are determined_m) The magnitude of the value to determine the presence of the target nucleic acid; or, the deletion-type detection probe and the wild-type detection probe have different reporter groups, and the product of the step (b) is subjected to melting curve analysis, and then melting peak positions and melting points (T) in the melting curve are determined according to the kinds of signals of the reporter groups_m) The magnitude of the value determines the presence of the target nucleic acid.

In certain embodiments, the reporter in the wild-type and the deletion-type detection probes are each independently a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL)

Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS Red, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and, the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (e.g., DABCYL, BHQ)BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).

In certain embodiments, the deletion detection probe and the wild-type detection probe are not resistant to nuclease activity, or each independently are resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications.

In certain embodiments, the deletion detection probe and the wild-type detection probe are each independently linear or have a hairpin structure. In certain preferred embodiments, the detection probe is linear. In certain preferred embodiments, the detection probe has a hairpin structure. Hairpin structures may be natural or artificially introduced. In addition, detection probes having hairpin structures can be constructed using methods conventional in the art. For example, the detection probe can form a hairpin structure by adding complementary 2 oligonucleotide sequences at the 2 termini (5 'and 3' ends) of the detection probe. In such embodiments, the complementary 2 oligonucleotide sequences constitute the arms (stems) of the hairpin structure. The arms of the hairpin structure may have any desired length, for example the length of the arms may be 2-15nt, for example 3-7nt, 4-9nt, 5-10nt, 6-12 nt.

In certain embodiments, in step (c), the product of step (b) is subjected to a gradual increase or decrease in temperature and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining a profile of the signal intensity of each reporter group as a function of temperature; then, the curve is derived to obtain the melting curve of the product of step (b).

In certain embodiments, the presence of a deletion target nucleic acid and/or a wild-type target nucleic acid corresponding to a melting peak (melting point) in a melting curve is determined based on the melting peak (melting point).

In certain embodiments, in step (a), at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of said deletion detection probes and/or wild-type detection probes are provided.

In certain embodiments, the deletion detection probes comprise detection probes having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or any combination thereof: 6, 11, 16, 21, 26, 31, 36, 41, 47 and 52 in SEQ ID NO.

In certain embodiments, the wild-type detection probe comprises a detection probe having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or any combination thereof: 5, 10, 15, 20, 25, 30, 35, 40, 46 and 51 SEQ ID NOs.

In certain embodiments, the sample comprises or is a mixture of DNA, or RNA, or nucleic acids.

In certain embodiments, the deleted target nucleic acid sequence and/or the wild-type target nucleic acid sequence is DNA or RNA.

In certain embodiments, the deleted target nucleic acid sequence and/or the wild-type target nucleic acid sequence is single-stranded or double-stranded.

In certain preferred embodiments, the universal primer consists of a first universal sequence. In certain preferred embodiments, the universal primer further comprises an additional sequence located 5' to the first universal sequence. In certain preferred embodiments, the additional sequence comprises one or more nucleotides, such as 1-5, 5-10, 10-15, 15-20 or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In the present application, the universal primers are used for performing PCR amplification, and thus preferably the first universal sequence is located in or constitutes the 3' portion of the universal primers.

In the embodiments of the present application, the universal primer may be any length as long as it can perform a PCR reaction. For example, the universal primer may be 5-50nt in length, such as 5-15nt, 15-20nt, 20-30nt, 30-40nt, or 40-50 nt.

In certain embodiments of the present application, the universal primer (or any component thereof) may comprise or consist of naturally occurring nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain preferred embodiments, the universal primer (or any component thereof) comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the universal primer (or any component thereof) comprises a modified nucleotide, such as a modified deoxyribonucleotide or ribonucleotide, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the universal primer (or any component thereof) comprises a non-natural nucleotide, such as deoxyinosine, inosine, 1- (2' -deoxy-. beta. -D-ribofuranosyl) -3-nitropyrrole, 5-nitroindole, or Locked Nucleic Acid (LNA). ' in certain embodiments of the application, at least 1 target-specific primer pair is provided in step (1), e.g., at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, at least 12, at least 15, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more target-specific primer pairs. In certain preferred embodiments, 1-5, 5-10, 10-15, 15-20, 20-50 or more target-specific primer pairs are provided in step (1). In certain preferred embodiments, the method is capable of simultaneously amplifying 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids. In certain preferred embodiments, the methods are capable of amplifying 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids simultaneously and asymmetrically.

In certain embodiments, in the forward primer 1, the wild type forward nucleotide sequence is directly linked to the 3 'end of the second universal sequence, or is linked 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.

In certain embodiments, in the forward primer 2, the deletion-type forward nucleotide sequence is directly linked to the 3 'end of the second universal sequence, or is linked to the 3' end of the second universal sequence through a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the forward primer 1 and the forward primer 2 further each independently comprise an additional sequence located 5' to the second universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the forward primer 1 and the forward primer 2 each independently comprise or consist of from 5 'to 3' a second universal sequence and a forward nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', a second universal sequence, a nucleotide linker and a forward nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a second universal sequence, and a forward nucleotide sequence; alternatively, each independently comprises or consists of from 5 'to 3', an additional sequence, a second universal sequence, a nucleotide linker and a forward nucleotide sequence.

In certain embodiments, the wild-type forward nucleotide sequence is located in or constitutes the 3' portion of the forward primer 1.

In certain embodiments, the deleted forward nucleotide sequence is located on or constitutes the 3' portion of the forward primer 2.

In certain embodiments, the wild-type forward nucleotide sequence and the deletion-type forward nucleotide sequence are each independently 10-100nt in length, e.g., 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100 nt.

In certain embodiments, the length of the forward primer 1 and the forward primer 2 is 15-150nt, such as 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 + 150 nt.

In certain embodiments, the forward primer 1 and forward primer 2 each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof.

In certain embodiments, in the reverse primer 1, the wild-type reverse nucleotide sequence is directly linked to the 3 'end of the first universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the first universal sequence via a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, in the reverse primer 2, the wild type reverse nucleotide sequence is directly linked to the 3 'end of the first universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the first universal sequence via a nucleotide linker. In certain embodiments, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the reverse primer 1 and the reverse primer 2 further each independently comprise an additional sequence located 5' to the first universal sequence. In certain embodiments, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides.

In certain embodiments, the reverse primer 1 and reverse primer 2 each independently comprise or consist of from 5 'to 3' a first universal sequence and a reverse nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', a first universal sequence, a nucleotide linker, and an inverted nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a first universal sequence, and an inverted nucleotide sequence; alternatively, each independently comprises or consists of from 5 'to 3', an additional sequence, a first universal sequence, a nucleotide linker and an inverted nucleotide sequence.

In certain embodiments, the wild-type reverse nucleotide sequence is located at or constitutes the 3' portion of the reverse primer 1.

In certain embodiments, the deleted reverse nucleotide sequence is located at or constitutes the 3' portion of the reverse primer 2.

In certain embodiments, the wild-type inverted nucleotide sequence and the deletion-type inverted nucleotide sequence are each independently 10 to 100nt in length, e.g., 10 to 20nt, 20 to 30nt, 30 to 40nt, 40 to 50nt, 50 to 60nt, 60 to 70nt, 70 to 80nt, 80 to 90nt, 90 to 100 nt.

In certain embodiments, the length of the reverse primer 1 and the length of the reverse primer 2 are each independently 15-150nt, such as 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-.

In certain embodiments, the reverse primer 1 and reverse primer 2 each independently or any component thereof comprises or consists of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof.

In certain embodiments, the forward primers 2 each independently hybridize to the distal upstream end of a deletion detection probe, or to the proximal upstream vicinity of a deletion detection probe, or have a partially overlapping sequence with the targeting sequence of a deletion detection probe after hybridization to a deletion target nucleic acid; and/or, the forward primers 1 each independently hybridize to the upstream distal end of the wild-type detection probe, or to the upstream proximal portion of the wild-type detection probe, or to a sequence that has partial overlap with the targeting sequence of the wild-type detection probe after hybridization to the wild-type target nucleic acid sequence.

In certain embodiments, the first universal sequence is not fully complementary to the complement of forward primer 1 and forward primer 2; for example, 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 is not complementary to the complementary sequences of the forward primer 1 and forward primer 2.

In certain embodiments, the difference in the second universal sequence from the first universal sequence comprises or consists of 1-5, 5-10, 10-15, 15-20 or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, each independently deleted or substituted at the 3' end of the first universal sequence.

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

(I) providing a sample containing one or more target nucleic acids containing one or more nucleotide fragment deletions and, for each nucleotide fragment deletion to be detected, providing a universal primer, a first target-specific primer pair, a second target-specific primer pair, and, optionally, a wild-type detection probe and a deletion-type detection probe; wherein the universal primer and target-specific primer pair are as defined above and the wild-type detection probe and the deletion-type detection probe are as defined above;

(II) mixing the sample with the universal primer, a first target-specific primer pair, a second target-specific primer pair, and a nucleic acid polymerase; optionally, adding a wild-type detection probe and a deletion-type detection probe;

(III) incubating the product of the previous step under conditions that allow denaturation of the nucleic acids;

(IV) incubating the product of the previous step under conditions that allow annealing or hybridization of the nucleic acid;

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

(VI) optionally, repeating steps (III) - (V) one or more times.

In certain embodiments, in step (III), the product of step (II) is incubated at a temperature of 80-105 ℃ to denature the nucleic acid.

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

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

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

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

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

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

In certain embodiments, steps (III) - (V) are repeated at least once, such as 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 for each cycle of steps (III) - (V) are each independently the same or different.

In another aspect, the present application provides a primer set comprising: universal primers, and, at least 2 target-specific primer pairs (e.g., 2 pairs, 3 pairs, 4 pairs, 5 pairs, 8 pairs, 10 pairs, or more); wherein the content of the first and second substances,

the universal primer comprises a first universal sequence;

and, the first universal sequence is capable of hybridizing or annealing to a complement of the second universal sequence under conditions that allow nucleic acid hybridization or annealing, and the second universal sequence differs from the first universal sequence by one or more nucleotides at the 3' end of the first universal sequence each independently being deleted or substituted; and, the first universal sequence is not fully complementary to the complement of the forward primer 1 or forward primer 2.

It will be readily appreciated that such primer sets may be used to carry out the methods of the invention as described in detail above. Thus, the various technical features detailed above for the universal primer, the target-specific primer pair, the target nucleic acid, the sample are equally applicable to the technical solutions in the present application involving the primer set. Thus, in certain preferred embodiments, the primer set comprises a universal primer and/or a target-specific primer pair as defined above.

In certain embodiments, the sequence of the universal primer is shown in SEQ ID NO. 1.

In certain embodiments, the first target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 2, 3, 4, 5, 8, 10 primer pairs), or any combination thereof: 2 and 4; 7 and 9; 12 and 14; 17 and 18; 22 and 23; 27 and 28; 32 and 33; 37 and 38; 42 and 43; 38 and 49.

In certain embodiments, the second target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 2, 3, 4, 5, 8, 10 primer pairs), or any combination thereof: 3 and 4 are SEQ ID NOs; 8 and 9; 13 and 14; 17 and 19; 22 and 24; 27 and 29; 32 and 34; 37 and 39; 44 and 45; 48 and 50.

In certain embodiments, the primer set further comprises a third target-specific primer pair, the third target-specific primer pair being capable of amplifying a deletion-type target nucleic acid.

In certain embodiments, the primer set further comprises a third target-specific primer pair and a fourth target-specific primer pair. The third target-specific primer pair is capable of amplifying a deleted-type target nucleic acid and the fourth target-specific primer pair is capable of amplifying a deleted-type target nucleic acid, or the third target-specific primer pair is capable of amplifying a wild-type target nucleic acid and the fourth target-specific primer pair is capable of amplifying a deleted-type target nucleic acid.

In addition, for convenience, the primer set of the present invention may be combined with one or more reagents required for carrying out the method (detection method) of the present invention to prepare a kit. It will be readily appreciated that such kits may be used to carry out the methods of the invention as described in detail above. Thus, the various technical features described in detail above for the various components are equally applicable to the various components of the kit. Also, such kits may further comprise other reagents necessary to carry out the methods of the invention.

In another aspect, the present application provides a kit comprising the primer set as described above, and 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 nucleic acid hybridization, a reagent for performing nucleic acid extension, a reagent for performing melt curve analysis, or any combination thereof.

In certain embodiments, the nucleic acid polymerase is a template-dependent nucleic acid polymerase, e.g., a DNA polymerase, particularly a thermostable DNA polymerase. In certain embodiments, the nucleic acid polymerase is as defined above.

In certain embodiments, the reagents for performing nucleic acid amplification compriseWorking buffer for enzymes (e.g.nucleic acid polymerases), dNTPs (labeled or unlabeled), water, ions (e.g.Mg)²⁺) A single-stranded DNA binding protein, or any combination thereof.

In certain embodiments, the reagents for performing a melt curve analysis comprise detection probes. In certain embodiments, the detection probe comprises a wild-type detection probe and a deletion-type detection probe. In certain embodiments, the detection probe is as defined above.

In certain embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 wild-type detection probes and/or deletion-type detection probes.

In certain embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 primer sets.

In another aspect, the present application provides the use of a kit as described above for asymmetric amplification of a wild type target nucleic acid and/or a deleted target nucleic acid.

In certain embodiments, the kit is for detecting the presence or level of a nucleotide fragment deletion in a target nucleic acid in a sample, or for diagnosing whether a subject has the nucleotide fragment deletion and/or has a disease resulting from the nucleotide fragment deletion.

In certain embodiments, the nucleotide fragment deletion refers to a deletion of at least 200 consecutive bases; in certain embodiments, the nucleotide fragment deletion refers to a deletion of at least 500 consecutive bases; in certain embodiments, the nucleotide fragment deletion is a large fragment deletion of the phenylalanine hydroxylase (PAH) gene; in certain embodiments, the PAH gene large fragment deletion is selected from ex1del5.3kb, ex3del6.6kb, Ex4del, Ex5del, Ex6del, ex1del3.7kb, Ex3del4.7kb, Ex5_6del, Ex4_7del, Ex4_5del, or any combination thereof. In certain embodiments, the disease caused by the deletion of the nucleotide fragment is phenylalanine hydroxylase deficiency. In certain embodiments, the sample comprises DNA, or RNA, or a mixture of nucleic acids. In certain embodiments, the nucleic acid molecule is DNA or RNA; and/or, the nucleic acid molecule is single-stranded or double-stranded. In certain embodiments, the sample is a sample obtained from a subject, e.g., blood, saliva, tissue, or hair. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.

Definition of terms

As used herein, the term "nucleotide fragment deletion" has the meaning generally understood by a person skilled in the art, which refers to the deletion of a plurality of consecutive bases (typically at least 100bp, such as at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp, at least 1000bp, at least 2000bp, at least 3000bp, at least 4000bp, at least 5000bp, at least 6000bp, at least 7000bp, at least 8000bp, at least 9000bp, at least 104bp, at least 105bp, at least 106bp, or even more bases) in a nucleic acid molecule of interest, such as a genomic nucleic acid molecule. In certain preferred embodiments of the present application, a nucleotide fragment deletion refers to a deletion of at least 200 consecutive bases. In certain preferred embodiments, a nucleotide fragment deletion refers to a deletion of at least 500 consecutive bases. In certain preferred embodiments, a nucleotide fragment deletion refers to a deletion of at least 1000 consecutive bases. It is known in the art that nucleotide fragment deletions result in the fragmentation and religation of nucleic acid molecules, which will create a new base sequence at the site of this religation (i.e., break point). Thus, as used herein, the term "breakpoint" when used to describe a deletion of a nucleotide fragment refers to the location in a deleted nucleic acid molecule where the deletion of the nucleotide fragment occurs as compared to a wild-type nucleic acid molecule. This means that on one side of the breakpoint the deleted nucleic acid molecule has the same sequence as the wild type nucleic acid molecule and on the other side of the breakpoint the deleted nucleic acid molecule has a nucleotide fragment that is deleted compared to the sequence of the wild type nucleic acid molecule.

As used herein, the terms "target nucleic acid sequence", "target nucleic acid", and "target sequence" refer to the target nucleic acid sequence to be detected. In particular, in the present application, the expression "deletion-type target nucleic acid" refers to a target nucleic acid containing a deletion of a nucleotide fragment to be detected; correspondingly, the expression "wild-type target nucleic acid sequence" refers to a target nucleic acid sequence which does not contain the deletion of the nucleotide fragment to be detected. In the present application, the terms "target nucleic acid sequence", "target nucleic acid" and "target sequence" have the same meaning and are used interchangeably.

As used herein, the terms "targeting sequence" and "target-specific sequence" refer to a sequence capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that allow for hybridization, annealing, or amplification of the nucleic acid, which comprises a sequence complementary to the target nucleic acid sequence. In the present application, the terms "targeting sequence" and "target-specific sequence" have the same meaning and are used interchangeably. It is readily understood that the targeting or target-specific sequence is specific for the target nucleic acid sequence. In other words, under conditions that allow nucleic acid hybridization, annealing, or amplification, the targeting or target-specific sequence hybridizes or anneals only to a particular target nucleic acid sequence, and not to other nucleic acid sequences.

As used herein, the term "forward nucleotide sequence" refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence that is capable of hybridizing (or annealing) to the target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification and, when hybridized to the target nucleic acid sequence, is located upstream of a detection probe. Accordingly, the term "reverse nucleotide sequence" refers to an oligonucleotide sequence comprising a sequence complementary to a target nucleic acid sequence that is capable of hybridizing (or annealing) to the target nucleic acid sequence under conditions that allow nucleic acid hybridization (or annealing) or amplification and, when hybridized to the target nucleic acid sequence, is located downstream of a detection probe.

As used herein, the term "upstream" is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning commonly understood by those skilled in the art. For example, the expression "one nucleic acid sequence is located upstream of another nucleic acid sequence" means that, when arranged in the 5' to 3' direction, the former is located at a more advanced position (i.e., a position closer to the 5' end) than the latter. As used herein, the term "downstream" has the opposite meaning as "upstream".

The term "complementary" as used herein means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the base pairing principle (Watton-Crick principle) and thereby forming a duplex. In the present application, the term "complementary" includes "substantially complementary" and "fully complementary". As used herein, the term "fully complementary" means that each base in one nucleic acid sequence is capable of pairing with a base in another nucleic acid strand without mismatches or gaps. As used herein, the term "substantially complementary" means that a majority of the bases in one nucleic acid sequence are capable of pairing with bases in another nucleic acid strand, which allows for the presence of mismatches or gaps (e.g., mismatches or gaps of one or several nucleotides). Typically, two nucleic acid sequences that are "complementary" (e.g., substantially complementary or fully complementary) will selectively/specifically hybridize or anneal and form a duplex under conditions that allow the nucleic acids to hybridize, anneal, or amplify. Accordingly, the term "non-complementary" means that two nucleic acid sequences do not hybridize or anneal under conditions that allow for hybridization, annealing, or amplification of the nucleic acids, and do not form a duplex. As used herein, the term "not being fully complementary" means that the bases in one nucleic acid sequence are not capable of fully pairing with the bases in another nucleic acid strand, at least one mismatch or gap being present.

As used herein, the term "substitution" means that a certain nucleotide is replaced by another nucleotide in a DNA molecule. In general, substitutions can be divided into two classes, transitions and transversions, wherein a transition means the substitution of one purine nucleotide for another purine nucleotide or a pyrimidine nucleotide for another pyrimidine nucleotide (e.g., A for G, G for A, C for T, and T for C); transversion (transversion) refers to the substitution of a purine nucleotide for a pyrimidine nucleotide or a pyrimidine nucleotide for a purine nucleotide (e.g., a is substituted for T or C, G is substituted for T or C, T is substituted for a or G, and C is substituted for a or G).

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

As used herein, "conditions that allow nucleic acid hybridization" have the meaning commonly understood by those skilled in the art and can be determined by conventional methods. For example, two nucleic acid molecules having complementary sequences can hybridize under suitable hybridization conditions. Such hybridization conditions may involve the following factors: temperature, pH, composition, ionic strength of the hybridization buffer, etc., and can be determined based on the length and GC content of the two complementary nucleic acid molecules. For example, when the length of two complementary nucleic acid molecules is relatively short and/or the GC content is relatively low, low stringency hybridization conditions can be used. High stringency hybridization conditions can be used when the length of two nucleic acid molecules that are complementary is relatively long and/or the GC content is relatively high. Such hybridization conditions are well known to those skilled in the art and can be found, for example, in Joseph Sambrook, et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and m.l.m.anderson, Nucleic Acid Hybridization, Springer-Verlag New York inc.n.y. (1999). In the present application, "hybridization" and "annealing" have the same meaning and are used interchangeably. Accordingly, the expressions "conditions allowing hybridization of nucleic acids" and "conditions allowing annealing of nucleic acids" also have the same meaning and are used interchangeably.

As used herein, the expression "conditions that allow nucleic acid amplification" has the meaning generally understood by those skilled in the art, which refers to conditions that allow a nucleic acid polymerase (e.g., a DNA polymerase) to synthesize one nucleic acid strand using the other nucleic acid strand as a template and form a duplex. Such conditions are well known to those skilled in the art and may involve the following factors: temperature, pH, composition, concentration, ionic strength, etc. of the hybridization buffer. Suitable nucleic acid amplification conditions can be determined by conventional methods (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). In the method of the present invention, the "conditions which allow nucleic acid amplification" are preferably working conditions of a nucleic acid polymerase (e.g., a DNA polymerase).

As used herein, the term "nucleic acid denaturation" has the meaning commonly understood by those skilled in the art, which refers to the process of dissociation of a double-stranded nucleic acid molecule into single strands. The expression "conditions which allow denaturation of nucleic acids" means conditions which allow dissociation of double-stranded nucleic acid molecules into single strands. Such conditions can be routinely determined by those skilled in the art (see, e.g., Joseph Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, n.y. (2001)). For example, the nucleic acid can be denatured by conventional techniques such as heating, alkali treatment, urea treatment, enzymatic methods (e.g., methods using helicase), and the like. In the present application, preferably, the nucleic acid is denatured under heating. For example, nucleic acids can be denatured by heating to 80-105 ℃.

As used herein, the term "PCR reaction" has the meaning commonly understood by those skilled in the art, which refers to a reaction that uses a nucleic acid polymerase and primers to amplify a target nucleic acid (polymerase chain reaction). As used herein, the term "multiplex amplification" refers to the amplification of multiple target nucleic acids in the same reaction system. As used herein, the term "asymmetric amplification" refers to amplification of a target nucleic acid resulting in an amplification product that is not identical in the amount of two complementary nucleic acid strands, one nucleic acid strand being in a larger amount than the other nucleic acid strand.

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

In certain preferred embodiments of the present application, the melting curve analysis may be performed by using a detection probe labeled with a reporter group and a quencher group. Briefly, at ambient temperature, the detection probe is capable of forming a duplex with its complementary sequence by base pairing. In this case, the reporter (e.g., fluorophore) and the quencher on the detection probe are separated from each other, and the quencher cannot absorb a signal (e.g., a fluorescent signal) emitted from the reporter, and at this time, the strongest signal (e.g., a fluorescent signal) can be detected. As the temperature is increased, both strands of the duplex begin to dissociate (i.e., the detection probe gradually dissociates from its complementary sequence), and the dissociated detection probe is in a single-stranded free coiled-coil state. In this case, the reporter (e.g., fluorophore) and the quencher on the detection probe under dissociation are brought into close proximity to each other, whereby a signal (e.g., a fluorescent signal) emitted from the reporter (e.g., fluorophore) is absorbed by the quencher. Thus, as the temperature increases, the detected signal (e.g., the fluorescence signal) becomes progressively weaker. When both strands of the duplex are completely dissociated, all detection probes are in a single-stranded free coiled-coil state. In this case, the signal (e.g., fluorescent signal) from the reporter (e.g., fluorophore) on all of the detection probes is absorbed by the quencher. Thus, a signal (e.g., a fluorescent signal) emitted by a reporter (e.g., a fluorophore) is substantially undetectable. Thus, by detecting the signal (e.g., fluorescent signal) emitted by the duplex containing the detection probe during the temperature increase or decrease, the hybridization and dissociation processes of the detection probe and its complementary sequence can be observed, forming a curve whose signal intensity varies with temperature. Further, the derivative analysis is carried out on the obtained curve, and the signal intensity change rate can be obtained as the ordinateThe temperature is plotted on the abscissa (i.e., the melting curve of the duplex). The peak in the melting curve is the melting peak and the corresponding temperature is the melting point (T) of the duplex_mValue). In general, the higher the degree of match of the detection probe to the complementary sequence (e.g., the fewer mismatched bases, the more bases paired), the T of the duplex_mThe higher the value. Thus, by detecting T of the duplex_mValue, 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_mThe value "has the same meaning and may be used interchangeably.

Advantageous effects of the invention

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

compared with the prior multiple Gap-PCR, the invention adopts a multiple asymmetric primer dimer-free system, thereby effectively reducing non-specific amplification, particularly amplification of primer dimers; meanwhile, the universal primers are adopted for subsequent amplification, so that the amplification difference caused by different amplification efficiencies of different specific primers is effectively reduced, the amplification among different targets can be balanced, and the detection sensitivity of the system is effectively improved.

Compared with MLPA technology, array-CGH, SNP typing chips, high-throughput sequencing technology and the like, the system does not need to be operated after PCR, and is simpler, faster, lower in cost and high in throughput.

Therefore, the invention provides a simple, high-efficiency and low-cost method for detecting the deletion of a plurality of nucleotide large fragments, which can simultaneously detect the deletion of a plurality of nucleotide fragments and wild types thereof. The maximum number of nucleotide fragment deletions and their wild-type that can be detected by the method of the invention is not limited by the type of fluorescent label used (number of instrument fluorescent detection channels). That is, the method of the present invention can realize simultaneous detection (multiplex detection) of a larger number of nucleotide fragment deletions and wild-types thereof on the basis of the number of fluorescence detection channels of a limited fluorescence labeling species instrument, which is particularly advantageous.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 schematically depicts an embodiment of the method of the invention for detecting deletion of large fragments of nucleotides to illustrate the basic principle of the method of the invention.

FIG. 1A schematically depicts a primer set and detection probe used in this embodiment, the primer set comprising: a universal primer, and a target-specific primer pair comprising a forward primer and a reverse primer; the detection probe is a double-labeled self-quenching fluorescent probe, the 5 'end is respectively labeled with a fluorescent group, the 3' end is labeled with a quenching group, the detection probe can be hybridized with different target nucleic acids to form double-stranded hybrids, and each double-stranded hybrid has a fixed melting point.

FIG. 1B schematically depicts the principle that non-specific amplification of primer dimers is inhibited when amplification is performed using the primer set of FIG. 1A, wherein primer dimers formed by non-specific amplification of the forward and reverse primers, after denaturation, will produce single-stranded nucleic acids whose 5 'and 3' ends can complementarily anneal to each other, which will form a stable panhandle structure during the annealing stage, preventing annealing and extension of the single-stranded nucleic acids by the universal primers, thereby inhibiting further amplification of primer dimers.

FIG. 1C schematically depicts the principle of multiplexing, asymmetric amplification and detection using the primer set of FIG. 1A (universal primer, first target-specific primer pair comprising forward primer 1 and reverse primer 1, second target-specific primer pair comprising forward primer 2 and reverse primer 2) and detection probe.

Wherein the universal primer comprises a first universal sequence (Tag 1);

the first target-specific primer pair is capable of amplifying a wild-type target nucleic acid, wherein the forward primer 1 comprises a second universal sequence (Tag2) and a wild-type forward nucleotide sequence specific for the wild-type target nucleic acid, and the wild-type forward nucleotide sequence is located 3' of the universal sequence; the reverse primer 1 comprises a first universal sequence and a wild-type reverse nucleotide sequence specific for a wild-type target nucleic acid, and the wild-type reverse nucleotide sequence is located 3' of the first universal sequence;

a second target-specific primer pair capable of amplifying the deleted target nucleic acid, wherein forward primer 2 comprises a second universal sequence (Tag2) and a deleted forward nucleotide sequence specific for the deleted target nucleic acid, and the deleted forward nucleotide sequence is located 3' of the universal sequence; the reverse primer 2 comprises a first universal sequence and a deleted reverse nucleotide sequence specific to the deleted target nucleic acid, and the deleted reverse nucleotide sequence is located at the 3' end of the first universal sequence;

and, the first universal sequence is capable of hybridizing or annealing to a complement of the second universal sequence under conditions that allow for hybridization or annealing of nucleic acids, and the second universal sequence differs from the first universal sequence by the inclusion of one or more nucleotides at the 3' end of the first universal sequence that are each independently deleted or substituted; and, the first universal sequence is not fully complementary to the complementary sequence of the forward primer.

In this embodiment, first, PCR amplification is initiated by a first target-specific primer pair and a second target-specific primer pair having a low concentration to produce an initial amplification product comprising two nucleic acid strands (nucleic acid strand a and nucleic acid strand B) complementary to a forward primer 1 and a reverse primer 1/a universal primer, respectively, and two nucleic acid strands (nucleic acid strand C and nucleic acid strand D) complementary to a forward primer 2 and a reverse primer 2/a universal primer; subsequently, the initial amplification product is subjected to a subsequent PCR amplification by the high concentration of universal primers.

Since the reverse primer 1, the reverse primer 2 and the universal primer each contain the first universal sequence, the nucleic acid strand B complementary to the reverse primer 1 and the nucleic acid strand D complementary to the reverse primer 2 can also be complementary to the universal primer. Thus, during a PCR reaction, the universal primer is able to anneal to nucleic acid strands B and D and normally initiate PCR amplification (i.e., normally synthesize the complementary strands of nucleic acid strands B and D).

At the same time, since the first universal sequence is capable of hybridizing or annealing to the complement of the second universal sequence under conditions that allow nucleic acid hybridization or annealing, the universal primer (which comprises the first universal sequence) is also capable of annealing to nucleic acid strand a that is complementary to forward primer 1 (which comprises the second universal sequence) and to nucleic acid strand C that is complementary to forward primer 2 (which comprises the second universal sequence) during the PCR reaction. However, since the second universal sequence is different from the first universal sequence (in which one or more nucleotides at the 3 'end of the first universal sequence are each independently deleted or replaced), the universal primer (particularly at the 3' end thereof) is not capable of being fully complementary to the nucleic acid strands a and C, which results in the universal primer being inhibited from PCR amplification of the nucleic acid strands a and C (i.e., synthesis of complementary strands of the nucleic acid strands a and C is inhibited).

Therefore, as the PCR amplification proceeds, the synthesis efficiency of the complementary strand of the nucleic acid strand a (nucleic acid strand B) will be significantly lower than that of the nucleic acid strand B (nucleic acid strand a), resulting in the synthesis and amplification of the complementary strand of the nucleic acid strand B (nucleic acid strand a) in a large amount, while the synthesis and amplification of the complementary strand of the nucleic acid strand a (nucleic acid strand B) is suppressed, thereby producing a large amount of the target single-stranded product (nucleic acid strand a, which contains a sequence complementary to the forward primer 1/the second universal sequence and a sequence of the reverse primer 1/the universal primer), achieving asymmetric amplification. The asymmetric amplification of the nucleic acid strands C and D is the same.

And performing melting curve analysis after the PCR amplification is finished, and hybridizing the detection probe and the corresponding PCR single-stranded product to form different double-stranded hybrids. The T of the double-stranded hybrid formed by the detection probe is caused according to the difference of the length and the GC content of the detection probe_mDifferent values, by melting curve analysis, can determine to have a specific T_mThe presence of a double-stranded hybrid is evaluated, and the presence of a target nucleic acid molecule corresponding to the duplex can be determined. Designing different wild type detection probe and deletion type detection probe respectively aiming at wild type and deletion type of different target nucleic acid, and enabling the wild type detection probe and the deletion type detection probe to form a plurality of T after hybridizing with corresponding targets_mValue, detection of multiple objects within a single channel may therefore be achieved.

FIG. 2 shows the results of detection of the Ex4del and Ex3del4.7kb deletions for the PAH genes. Wherein, the black solid line is the detection result of the wild type sample; the black dotted line indicates the detection result of the Ex3del4.7kb deletion type sample; the gray dotted line is the detection result of the Ex4del heterozygous deletion type sample; the solid grey line is the detection result of the No Template Control (NTC).

FIG. 3 shows the results of simultaneous detection of deletion of ten PAH genes using the reagents described in Table 1 and the detection protocol described in Table 2.

FIG. 4 shows the results of sensitive assays for different concentrations of wild type genome using the reagents described in Table 1 and the assay protocol described in Table 2.

Information on the sequences to which this application refers (SEQ ID NOS: 1-52) is provided in Table 1.

Table 1: EXAMPLES sequence information of reagents involved in the reaction System (SEQ ID NOS: 1-52)

Note: the underlined bases are replaced with the corresponding LNAs.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. It is to be understood that these embodiments are merely illustrative of the principles and technical effects of the present invention, and do not represent all possibilities for the invention. The present invention is not limited to the materials, reaction conditions or parameters mentioned in these examples. Other embodiments may be practiced by those skilled in the art using other similar materials or reaction conditions in accordance with the principles of the invention. Such solutions do not depart from the basic principles and concepts described herein, and are intended to be within the scope of the invention.

Detection probes, upstream oligonucleotide (forward primer), downstream oligonucleotide (reverse primer) used in the examples of the present applicationAnd details of the universal primers, as well as their working concentrations (except for the working concentrations given in the specific examples) are summarized in table 1. The detection objects are PAH gene large fragment deletion types common to 10 Chinese people, and the HGVS names of the detection objects are c.4163-406 del3758 (abbreviated as ' Ex1del3.7kb '), c.1932-60 +3337delins56 (abbreviated as ' Ex1del5.3kb '), c.353-8714_441+2813del11612 (abbreviated as ' Ex4del '), c.442-5521_509+2161delins3 (abbreviated as ' Ex5 del), c.353-1069_510+4247del16271 (abbreviated as ' Ex4_5 del), c.510-6708_706+888delins2 (abbreviated as ' Ex6 del), c.169-4948_352+ 8 (abbreviated as ' Ex 6kb '), c.169-473k48 _352+ 4742 (abbreviated as ' Ex4 dell 3.7kb '), respectively^]C.456_706 _ 138del11653 (abbreviated as "Ex 5_6 del"), c. [ 353-; 441+8099_842+518del](abbreviated as "Ex 4_7 del").

Example 1 detection of Large fragment deletion of two PAH genes

The scheme of this example is shown in figure 1. In this example, the Ex4del deletion and the Ex3del4.7kb deletion were detected and genotyped in the same channel using 3 primers (forward primer 8F and reverse primers 8R and 8R ') and 2 probes (probes 8P and 8P') designed for the Ex4del deletion in table 1 and 3 primers (forward primer 10F and reverse primers 10R and 10R ') and 2 probes (probes 10P and 10P') designed for the Ex3del4.7kb deletion. The preparation method comprises the following steps: mu.L of the reaction system contained 1 XTaq PCR buffer (TaKaRa, Beijing), 5mM MgCl₂0.2mM dNTPs, 1U Taq HS (TaKaRa, Beijing), the amounts of each of the forward and reverse primers and the detection probe are shown in Table 1, and 2. mu.M of the universal primer, 5. mu.L of human genomic DNA (concentration about 10 ng/. mu.L) or 5. mu.L of water (negative control).

The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 65-56 ℃ (1 ℃ drop per cycle) for 15s, extension at 76 ℃ for 20s, 10 cycles; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, and extension at 76 ℃ for 20s for 50 cycles; melting curve analysis was then performed, with the program: denaturation at 95 ℃ for 1min, heat preservation at 37 ℃ for 3min, melting curve analysis from 45 ℃ to 85 ℃, heating rate of 0.4 ℃/s, and detection of fluorescence signals of CY5 channels. The instrument used in this example was a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi medical science and technology Co., Ltd.). The results of the detection are shown in FIG. 2.

The results in FIG. 2 show that the wild type sample has only the corresponding wild type melting peak (solid black line in FIG. 2); ex4del heterozygous deletion samples correspond to deletion melting peaks in addition to wild type melting peaks (fig. 2 grey dashed line); the ex3del4.7kb heterozygous deletion sample corresponds to a deletion fusion (black dashed line in fig. 2) peak in addition to the wild type fusion peak; whereas the blank control (NTC) without added template did not have any melting peaks (solid grey line in fig. 2). The result shows that the designed detection system can be used for detecting the large fragment deletion of the PAH gene.

Example 2.10 Simultaneous detection and genotyping of Large fragment deletions of the PAH Gene

By using the primers and probes provided in table 1 (14 forward primers, 17 reverse primers and 1 universal primer, 10 deletion detection probes, 10 wild type detection probes), the method of the invention enables simultaneous detection of 10 nucleotide fragment deletions and 10 corresponding wild types in a single PCR reaction (twenty-fold detection). The fluorescence detection channels used and melting points of the melting peaks detected are summarized in table 2.

Table 2: detection scheme

Briefly, in this example, 25 μ L of a PCR reaction system was used for PCR amplification and melting curve analysis, including: 1 × Taq PCR buffer (TaKaRa, Beijing), 13mM MgCl₂1.2mM dNTPs, 4U Taq HS (TaKaRa, Beijing), various reagents described in Table 1 (used at the indicated working concentrations), 5. mu.L human genomic DNA (samples include: wild-type sample, Ex1del5.3kb heterozygous deletion-type sample, Ex3del6.6kb heterozygous deletion-type sample, Ex4del heterozygous deletion-type sample)Ex5del heterozygous deletion type samples, Ex6del heterozygous deletion type samples, Ex1del3.7kb heterozygous deletion type samples, Ex3del4.7kb heterozygous deletion type samples, Ex5 — 6del heterozygous deletion type samples, Ex4 — 7del heterozygous deletion type samples, Ex4 — 5del heterozygous deletion type samples) PCR amplification programs were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 65-56 ℃ (1 ℃ drop per cycle) for 40s, extension at 76 ℃ for 20s, 10 cycles; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 40s, and extension at 76 ℃ for 20s for 50 cycles; extending the medium at 35 ℃ for 30 min; melting curve analysis was then performed, with the program: denaturation at 95 ℃ for 1min, heat preservation at 37 ℃ for 3min, melting curve analysis from 40 ℃ to 95 ℃, heating rate of 0.4 ℃/s, and detection of fluorescence signals of FAM, HEX, ROX and CY 54 channels. The instrument used in this example was a SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi medical science and technology, Inc.). The results of the detection are shown in FIGS. 3 and 4.

The results in FIG. 3 show that the wild-type sample (black solid line in FIG. 3) yielded only 10 wild-type melting peaks; 10 deletion types of PAH gene heterozygous deletion type samples (black dashed line in FIG. 3), which can generate 10 wild type melting peaks and deletion type corresponding melting peaks; whereas the blank control (NTC) without added template did not have any melting peaks (solid grey line in fig. 3). This result indicates that the designed detection system can obtain correct results and has strong specificity to the corresponding genotype for various samples containing different PAH gene deletions. In summary, for any sample to be detected, the designed detection system can determine whether the sample to be detected contains PAH gene deletion and deletion type according to the number of melting peaks and the melting point in the detection result.

FIG. 4 shows the results of the detection of 10-fold dilution gradient (50 ng/reaction-50 pg/reaction) wild-type genome samples, which shows that 10 wild-type melting peaks can be detected from the wild-type template of the concentration gradient, and the detection results are the corresponding wild-type results. Therefore, the method and the kit can detect the human genomic DNA with the concentration of 50 pg/reaction genomic DNA, namely 15 copies of the human genomic DNA, and have high detection sensitivity.

The experimental results show that the designed detection system and the designed reagent (particularly the designed primer and the designed probe) can realize simultaneous detection and genotyping of ten PAH gene deletions and wild types thereof in a single determination.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

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Claims

1. A method of detecting the presence of n nucleotide fragment deletions in a target nucleic acid in a sample, wherein n is an integer ≧ 1 (e.g., n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more), and comprising the steps of:

the universal primer comprises a first universal sequence;

(c) performing a melting curve analysis on the product of step (b); and determining whether each of the deletion-type target nucleic acids and/or the wild-type target nucleic acid is present in the sample and whether each of the nucleotide fragment deletions is present in the target nucleic acids in the sample based on the results of the melting curve analysis.

2. The method of claim 1, wherein the method has one or more technical features selected from the group consisting of:

(1) the method is used for detecting 1-5, 5-10, 10-15, 15-20, 20-50 or more target nucleic acids;

(2) in step (a) of the method, 1-5, 5-10, 10-15, 15-20, 20-50 or more first target-specific primer pairs and/or second target-specific primer pairs are provided;

(3) in step (b) of the method, the working concentration of the universal primer is higher than the working concentrations of the forward primer 1, the forward primer 2, the reverse primer 1 and the reverse primer 2; for example, the working concentration of the universal primer is 1-5 times, 5-10 times, 10-15 times, 15-20 times, 20-50 times or more higher than the working concentration of the forward primer 1, the forward primer 2, the reverse primer 1 and the reverse primer 2;

(4) in step (b) of the method, the working concentrations of the forward primer 1, the forward primer 2, the reverse primer 1 and the reverse primer 2 are the same or different from each other;

(5) 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;

(6) in step (b) of the method, a nucleic acid polymerase (particularly a template-dependent nucleic acid polymerase) is used to perform the PCR reaction; preferably, the nucleic acid polymerase is a DNA polymerase, such as a thermostable DNA polymerase; preferably, the thermostable DNA polymerase is obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antalidanii, Thermus caldophlus, Thermus chloriphilus, Thermus flavus, Thermus agniterae, Thermus lacteus, Thermus osidamia, Thermus ruber, Thermus scodifuctus, Thermus silvanicus, Thermus thermophilus, Thermotogamarimaritima, Thermotoga neocolina, Thermosiperus africans, Thermococcus leucotrichuria, Thermococcus leucotrichum, Thermococcus thermophilus, Thermococcus maritima, Thermococcus purpurea, Thermococcus africans, Thermococcus flavus, Thermococcus purpurea, Thermococcus purpurea, Pyrococcus, Thermococcus purpurea, Pyrococcus, Thermococcus purpurea, Thermococcus pacifia, Thermocascus, Pyrococcus, Thermocascus purpurea, Thermocascus purpuria, Thermocascus, Thermoascus purpuria, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus, Thermoascus, Pyrococcus; preferably, the DNA polymerase is Taq polymerase;

(7) the nucleotide fragment deletion refers to deletion of at least 200 consecutive bases;

preferably, the nucleotide fragment deletion refers to a deletion of at least 500 consecutive bases;

preferably, the nucleotide fragment deletion is a large fragment deletion of phenylalanine hydroxylase (PAH) gene; preferably, the PAH gene large fragment deletion is selected from ex1del5.3kb, ex3del6.6kb, Ex4del, Ex5del, Ex6del, ex1del3.7kb, ex3del4.7kb, Ex5_6del, Ex4_7del, Ex4_5del, or any combination thereof;

(8) in step (a), the reverse primer 1 is not more than 500bp away from the forward primer 1 when hybridizing to the wild-type target nucleic acid, e.g., 200-500 bp; and/or, when hybridizing to a wild type target nucleic acid, the reverse primer 2 is at least 500bp, e.g., at least 600bp, at least 700bp, at least 800bp, at least 900bp, at least 1000bp or more from the forward primer 1 and/or forward primer 2.

3. The method of claim 1 or 2, wherein the method has any one of the technical features selected from the group consisting of:

(1) in step (a), when hybridizing to a wild-type target nucleic acid, the reverse primer 1 hybridizes to a nucleotide fragment suspected of being deleted and the forward primer 1 hybridizes upstream of the nucleotide fragment suspected of being deleted; the reverse primer 2 is different from the reverse primer 1, and the forward primer 1 and the forward primer 2 can be the same or different;

(2) in step (a), when hybridizing to a wild-type target nucleic acid, the forward primer 1 hybridizes to a nucleotide fragment suspected of being deleted and the reverse primer 1 hybridizes downstream of the nucleotide fragment suspected of being deleted; the forward primer 1 is different from the forward primer 2, and the reverse primer 1 and the reverse primer 2 may be the same or different;

(3) in step (a), the forward primer 1 and the reverse primer 1 both hybridize to a nucleotide fragment suspected of being deleted when hybridized to a wild-type target nucleic acid; the reverse primer 2 is different from the reverse primer 1, and the forward primer 1 is different from the forward primer 2; and, when hybridizing to a deletion target nucleic acid sequence, the deletion target sequence can comprise a sequence complementary to the deletion target nucleic acid that comprises a breakpoint or is located upstream or downstream of the breakpoint.

4. The method according to any one of claims 1 to 3, wherein in step (a) one wild-type detection probe and one deletion-type detection probe are further provided for each nucleotide fragment deletion, respectively, wherein the wild-type detection probe comprises a wild-type targeting sequence specific for the wild-type target nucleic acid and the wild-type targeting sequence is capable of annealing or hybridizing to all or part of the region of the nucleotide fragment of the wild-type target nucleic acid suspected to be deleted; the deletion detection probe comprises a deletion targeting sequence specific to the deletion target nucleic acid, and the deletion targeting sequence comprises a sequence complementary to the deletion target nucleic acid sequence;

preferably, the method has any one of the following features:

(I) the deletion detection probe is capable of annealing or hybridizing to the products of the forward primer 1 and reverse primer 1 amplification of the wild-type target nucleic acid, and the wild-type detection probe is capable of annealing or hybridizing to the products of the forward primer 2 and reverse primer 2 amplification of the deletion target nucleic acid;

preferably, the wild-type detection probe is the same as the deletion-type detection probe;

more preferably, the targeting sequence of the detection probe is fully hybridized to the wild-type target nucleic acid and the targeting sequence of the detection probe is partially hybridized to the deleted target nucleic acid;

further preferably, the detection probe hybridizes to the wild-type target nucleic acid simultaneously with a portion of the nucleotide fragment suspected of being deleted and a portion of the nucleotide fragment not suspected of being deleted in the wild-type target nucleic acid, and the detection probe hybridizes to the deletion-type target nucleic acid upstream or downstream of the breakpoint in the deletion-type target nucleic acid;

(II) the deletion detection probe is capable of hybridizing to a wild-type target nucleic acid and its hybridization position is not between the hybridization position of the forward primer 1 and the hybridization position of the reverse primer 1;

and, in step (c), performing a melting curve analysis on each of the amplification products obtained in step (b) using the detection probe;

preferably, the method has one or more technical features selected from the group consisting of:

(1) mixing the sample with the universal primer, the first target-specific primer pair, the second target-specific primer pair and the nucleic acid polymerase in the step (b), carrying out PCR reaction, then adding the deletion-type detection probe and the wild-type detection probe into the product of the step (b) after the PCR reaction is finished, and carrying out melting curve analysis; or, in the step (b), mixing the sample with the universal primer, the first target-specific primer pair, the second target-specific primer pair, the deletion-type detection probe, the wild-type detection probe, and the nucleic acid polymerase, performing a PCR reaction, and then, after the PCR reaction is completed, performing a melting curve analysis;

(2) the deletion detection probe and the wild-type detection probe each independently comprise or consist of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide (e.g., a Peptide Nucleic Acid (PNA) or a locked nucleic acid), or any combination thereof;

(3) the length of the wild-type detection probe and the length of the deletion-type detection probe are respectively 15-1000nt, such as 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 and 900-1000 nt;

(4) the length of the targeting sequence in the wild-type detection probe and the deletion-type detection probe is 10-500nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt, 90-100nt, 100-150nt, 150-200nt, 200-250nt, 250-300nt, 300-350nt, 350-400nt, 400-450nt, 450-500 nt;

(5) the deletion detection probe and the wild-type detection probe each independently have a 3' -OH terminus; alternatively, the 3' -end of the detection probe is blocked; for example, the 3' -end of the detection probe can be blocked by adding a chemical moiety (e.g., biotin or alkyl) 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;

(6) the deletion detection probe and the wild-type detection probe are each independently 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 is labeled with a reporter group at its 3 'end or downstream and a quencher group at its 5' end or upstream; preferably, the reporter and quencher are separated by a distance of 10-80nt or more;

(7) the reporter groups in the wild-type and the deletion-type detection probes are each independently a fluorophore (e.g., ALEX-350, FAM, VIC, TET, CAL)

Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS Red, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and, the quenching group is a molecule or group capable of absorbing/quenching the fluorescence (e.g., DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA);

(8) the deletion detection probe and the wild-type detection probe are either not resistant to nuclease activity or are each independently resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity); for example, the backbone of the detection probe comprises modifications that are resistant to nuclease activity, such as phosphorothioate linkages, alkylphosphotriester linkages, arylphosphotriester linkages, alkylphosphonate linkages, arylphosphonate linkages, hydrogenphosphate linkages, alkylaminophosphate linkages, arylaminophosphate linkages, 2' -O-aminopropyl modifications, 2' -O-alkyl modifications, 2' -O-allyl modifications, 2' -O-butyl modifications, and 1- (4' -thio-PD-ribofuranosyl) modifications;

(9) the deletion detection probe and the wild-type detection probe are each independently linear or have a hairpin structure;

(10) the deletion detection probe and the wild-type detection probe each independently have the same or different reporter groups; preferably, the deletion-type detection probe and the wild-type detection probe 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 from a melting peak in the melting curve; or, the deletion-type detection probe and the wild-type detection probe have different reporter groups, and the product of step (b) is subjected to 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;

(11) in the step (c), gradually heating or cooling the product of the step (b) and monitoring the signal emitted by the reporter group on each detection probe in real time, thereby obtaining a curve of the signal intensity of each reporter group changing along with the change of the temperature; then, deriving the curve to obtain a melting curve of the product of step (b);

(12) determining the presence of the deletion-type target nucleic acid and/or the wild-type target nucleic acid corresponding to a melting peak (melting point) based on the melting peak (melting point) in the melting curve;

(13) in step (a), at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 of said deletion detection probes and/or wild-type detection probes are provided.

(14) The deletion detection probes include detection probes having a nucleotide sequence selected from the group consisting of: 6, 11, 16, 21, 26, 31, 36, 41, 47 and 52;

(15) the wild-type detection probes include detection probes having a nucleotide sequence selected from the group consisting of seq id no, or any combination thereof (e.g., any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10): 5, 10, 15, 20, 25, 30, 35, 40, 46 and 51 of SEQ ID NO.

5. The method of any one of claims 1-4, wherein the method has one or more characteristics selected from the group consisting of:

(1) the sample comprises or is a mixture of DNA, or RNA, or nucleic acids;

(2) the deleted target nucleic acid sequence and/or the wild-type target nucleic acid sequence is DNA or RNA;

(3) the deleted target nucleic acid sequence and/or the wild-type target nucleic acid sequence is single-stranded or double-stranded.

6. The method of any one of claims 1-5, wherein the method has one or more technical features selected from the group consisting of:

(1) the universal primer consists of a first universal sequence or alternatively, comprises a first universal sequence and an additional sequence, the additional sequence being located 5' of the first universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(2) the first universal sequence is located on or constitutes the 3' portion of the universal primer;

(3) the length of the universal primer is 5-15nt, 15-20nt, 20-30nt, 30-40nt or 40-50 nt;

(4) the universal primer, or any component thereof, comprises or consists of a naturally occurring nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide), a modified nucleotide, a non-natural nucleotide, or any combination thereof.

7. The method of any one of claims 1-6, wherein the method has one or more characteristics selected from the group consisting of:

(1) in the forward primer 1, the wild-type forward nucleotide sequence is directly connected to the 3 'end of the second universal sequence, or is connected to the 3' end of the second universal sequence through a nucleotide linker; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(2) in the forward primer 2, the deletion type forward nucleotide sequence is directly connected to the 3 'end of the second universal sequence, or is connected to the 3' end of the second universal sequence through a nucleotide linker; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(3) the forward primer 1 and forward primer 2 further each independently comprise an additional sequence located 5' to the second universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(4) the forward primer 1 and the forward primer 2 each independently comprise or consist of from 5 'to 3' a second universal sequence and a forward nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', a second universal sequence, a nucleotide linker and a forward nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a second universal sequence, and a forward nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a second universal sequence, a nucleotide linker, and a forward nucleotide sequence;

(5) the wild type forward nucleotide sequence is located in or constitutes the 3' portion of the forward primer 1;

(6) the deletion type forward nucleotide sequence is located in or constitutes the 3' part of the forward primer 2;

(7) the length of the wild type forward nucleotide sequence and the length of the deletion type forward nucleotide sequence are 10-100nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt and 90-100nt respectively;

(8) the lengths of the forward primer 1 and the forward primer 2 are 15-150nt, such as 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, 140-150 nt;

(9) the forward primer 1 and the forward primer 2 each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(10) in the reverse primer 1, the wild-type reverse nucleotide sequence is directly linked to the 3 'end of the first universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the first universal sequence through a nucleotide linker; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(11) in the reverse primer 2, the wild-type reverse nucleotide sequence is directly linked to the 3 'end of the first universal sequence, or the reverse nucleotide sequence is linked to the 3' end of the first universal sequence through a nucleotide linker; preferably, the nucleotide linker comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(12) the reverse primer 1 and the reverse primer 2 further each independently comprise an additional sequence located 5' to the first universal sequence; preferably, the additional sequence comprises 1-5, 5-10, 10-15, 15-20 or more nucleotides;

(13) the reverse primer 1 and the reverse primer 2 each independently comprise or consist of from 5 'to 3' a first universal sequence and a reverse nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', a first universal sequence, a nucleotide linker, and an inverted nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a first universal sequence, and an inverted nucleotide sequence; or, each independently comprises or consists of from 5 'to 3', an additional sequence, a first universal sequence, a nucleotide linker, and an inverted nucleotide sequence;

(14) the wild type reverse nucleotide sequence is located in or constitutes the 3' portion of the reverse primer 1;

(15) the deleted reverse nucleotide sequence is located in or constitutes the 3' portion of the reverse primer 2;

(16) the length of the wild-type reverse nucleotide sequence and the length of the deletion-type reverse nucleotide sequence are respectively 10-100nt, such as 10-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt, 80-90nt and 90-100 nt;

(17) the lengths of the reverse primer 1 and the reverse primer 2 are 15-150nt, such as 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, 140-150 nt;

(18) the reverse primer 1 and the reverse primer 2 each independently or any component thereof comprises or consists of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(19) the forward primers 2, after hybridizing to the deletion target nucleic acid, each independently hybridize to the upstream distal end of the deletion detection probe, or to the upstream vicinity of the deletion detection probe, or have a partially overlapping sequence with the targeting sequence of the deletion detection probe; and/or, the forward primer 1, upon hybridization to the wild-type target nucleic acid sequence, each independently hybridizes upstream distally of the wild-type detection probe, or upstream proximal to the wild-type detection probe, or has a partially overlapping sequence with the targeting sequence of the wild-type detection probe;

(20) the first universal sequence is not completely complementary to the complementary sequences of the forward primer 1 and the forward primer 2; for example, 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 is not complementary to the complement of the forward primer 1 and forward primer 2; and

(21) the difference between the second universal sequence and the first universal sequence comprises or consists in that 1 to 5, 5 to 10, 10 to 15, 15 to 20 or more nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, located at the 3' end of the first universal sequence are each independently deleted or substituted.

8. The method of any one of claims 1-7, wherein steps (a) - (c) of the method are performed by a protocol comprising the following steps (I) - (VI):

(I) providing a sample containing one or more target nucleic acids containing one or more nucleotide fragment deletions and, for each nucleotide fragment deletion to be detected, providing a universal primer, a first target-specific primer pair, a second target-specific primer pair, and, optionally, a wild-type detection probe and a deletion-type detection probe; wherein the universal primer and target-specific primer pair are as defined in claim 1 and the wild-type detection probe and the deletion-type detection probe are as defined in claim 3;

(VI) optionally, repeating steps (III) - (V) one or more times;

(1) in step (III), incubating the product of step (II) at a temperature of 80-105 ℃ to denature the nucleic acid;

(2) incubating the product of step (b) in step (III) for 10-20s, 20-40s, 40-60s, 1-2min, or 2-5 min;

(3) in step (IV), incubating the product of step (III) at a temperature of 35-40 ℃, 40-45 ℃, 45-50 ℃, 50-55 ℃, 55-60 ℃, 60-65 ℃, or 65-70 ℃ to allow annealing or hybridization of the nucleic acid;

(4) incubating the product of step (III) in step (IV) for 10-20s, 20-40s, 40-60s, 1-2min, or 2-5 min;

(5) in step (V), incubating the product of step (IV) at a temperature of 35-40 ℃, 40-45 ℃, 45-50 ℃, 50-55 ℃, 55-60 ℃, 60-65 ℃, 65-70 ℃, 70-75 ℃, 75-80 ℃, 80-85 ℃ to allow nucleic acid extension;

(6) in step (V), incubating the product of step (IV) for 10-20s, 20-40s, 40-60s, 1-2min, 2-5min, 5-10min, 10-20min or 20-30 min;

(7) performing steps (IV) and (V) at the same or different temperatures; and

(8) repeating steps (III) - (V) at least once, such as 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; preferably, when repeating steps (III) - (V) one or more times, the conditions used for steps (III) - (V) of each cycle are each independently the same or different.

9. A primer set, comprising: universal primers, and, at least 2 target-specific primer pairs (e.g., 2 pairs, 3 pairs, 4 pairs, 5 pairs, 8 pairs, 10 pairs, or more); wherein the content of the first and second substances,

the universal primer comprises a first universal sequence;

preferably, the primer set has one or more technical features selected from the group consisting of:

(1) the primer set comprises 1-5, 5-10, 10-15, 15-20, 20-50 or more target-specific primer pairs;

(2) the universal primers are as defined in claim 6;

(3) the target-specific primer pair is as defined in claim 7; and

(4) the target nucleic acid is as defined in claim 5;

preferably, the sequence of the universal primer is shown as SEQ ID NO. 1;

preferably, the first target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 2, 3, 4, 5, 8, 10 primer pairs), or any combination thereof: 2 and 4; 7 and 9; 12 and 14; 17 and 18; 22 and 23; 27 and 28; 32 and 33; 37 and 38; 42 and 43; 38 and 49;

preferably, the second target-specific primer pair comprises a primer pair having a nucleotide sequence selected from the group consisting of seq id no (e.g., any combination of 2, 3, 4, 5, 8, 10 primer pairs), or any combination thereof: 3 and 4 of SEQ ID NO; 8 and 9; 13 and 14; 17 and 19; 22 and 24; 27 and 29; 32 and 34; 37 and 39; 44 and 45; 48 and 50.

10. A kit comprising the primer set of claim 9, and 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 a melting curve analysis, or any combination thereof;

preferably, the nucleic acid polymerase is a template-dependent nucleic acid polymerase, such as a DNA polymerase, in particular a thermostable DNA polymerase; preferably, the nucleic acid polymerase is as defined in claim 2;

preferably, the reagents for performing nucleic acid amplification include, working buffers for enzymes (e.g., nucleic acid polymerases), dNTPs (labeled or unlabeled), water, ions (e.g., Mg) containing²⁺) A single-stranded DNA binding protein, or any combination thereof;

preferably, the reagent for performing a melting curve analysis comprises a detection probe; preferably, the detection probes comprise a wild-type detection probe and a deletion-type detection probe; preferably, the detection probe is as defined in claim 3;

preferably, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 wild-type and/or deletion-type detection probes;

preferably, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 primer sets.

11. Use of a kit according to claim 10 for asymmetric amplification of a wild type target nucleic acid and/or a deleted target nucleic acid;

preferably, the kit is for detecting the presence or level of a nucleotide fragment deletion in a target nucleic acid in a sample, or for diagnosing whether a subject has the nucleotide fragment deletion and/or suffers from a disease caused by the nucleotide fragment deletion;

preferably, the nucleotide fragment deletion refers to a deletion of at least 200 consecutive bases; preferably, the nucleotide fragment deletion refers to a deletion of at least 500 consecutive bases; preferably, the nucleotide fragment deletion is a large fragment deletion of phenylalanine hydroxylase (PAH) gene; preferably, the PAH gene large fragment deletion is selected from ex1del5.3kb, ex3del6.6kb, Ex4del, Ex5del, Ex6del, ex1del3.7kb, ex3del4.7kb, Ex5_6del, Ex4_7del, Ex4_5del, or any combination thereof;

preferably, the disease caused by deletion of the nucleotide fragment is phenylalanine hydroxylase deficiency;

preferably, the sample comprises DNA, or RNA, or a mixture of nucleic acids;

preferably, the nucleic acid molecule is DNA or RNA; and/or, the nucleic acid molecule is single-stranded or double-stranded;

preferably, the sample is a sample obtained from a subject, e.g., blood, saliva, tissue, or hair; preferably, the subject is a mammal, e.g., a primate, e.g., a human.