Multiplex nucleic acid detection methods, combinations, and kits

Abstract

The present application relates to a method, combination and kit for multiplex nucleic acid detection. The method comprises: a method for multiplex nucleic acid detection, comprising the following steps: mixing a combination containing a first primer set and a first probe with a sample from a subject; amplifying a target sequence possibly existing in the sample; performing a melting curve analysis on the product after amplification in a corresponding detection channel; and determining whether one or more of the targets exist according to the result of the melting curve analysis.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a solution for detecting melting curves of multiple targets. Background Technology

[0002] Polymerase chain reaction (PCR) is a molecular biology technique that replicates DNA enzymatically without the use of a living organism. PCR is commonly used in medical and biological research laboratories for a variety of tasks, such as the diagnosis of infectious diseases, gene cloning, phenotyping of laboratory animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, and paternity testing. Due to its unparalleled replication and precision capabilities, PCR is considered by molecular biologists to be the preferred method for nucleic acid detection. In the late 1990s, the introduction of Real-Time Quantitative PCR (qPCR) technology and related products by ABI in the United States further developed PCR into a highly sensitive, highly specific, and precisely quantitative nucleic acid sequence analysis technique.

[0003] Currently, the most commonly used primer and probe design method on qPCR platforms is the TaqMan hydrolysis probe method. Its working principle mainly utilizes an oligonucleotide probe that specifically binds to the template and is labeled with a fluorescent group (donor) and a quencher group (acceptor) at both ends. Simultaneously, a specific PCR primer is designed upstream and downstream of the probe. Before the PCR reaction begins, due to the fluorescence resonance energy transfer (FRET) principle, the fluorescent signal emitted by the fluorescent group at one end of the TaqMan probe is absorbed by the quencher group at the other end, thus preventing the fluorescence signal from being detected by the instrument. After PCR amplification begins, the TaqMan probe specifically binds to the template. When the DNA polymerase (Taq polymerase) extends to the site where the probe binds to the template, its 5-3' exonuclease activity cleaves the TaqMan probe, causing the fluorescent group on the probe to move away from the quencher group and no longer form a FRET structure. Therefore, the signal emitted by the fluorescent group can be detected by the instrument.

[0004] With the increasing application of PCR technology and the growing demand for high-throughput and rapid detection, it is often desirable to achieve PCR detection of multiple targets in the same reaction system. Typically, multiple detection is achieved by setting multiple TaqMan hydrolysis probes labeled with fluorescent groups of different wavelengths. However, depending on the number of fluorescence channels in the detection instrument, this method can only detect a maximum of about four different targets.

[0005] To achieve ultra-multiplex PCR detection, existing technologies typically combine PCR with other methods. Some reports describe combining PCR with hybridization chips for multiplex target detection using different amplification product lengths. For example, Chinese patent CN107090519A discloses a method for a multiplex RT-PCR combined with gene chip detection kit for common respiratory pathogens. This method utilizes the principle of nucleic acid hybridization, arranging and immobilizing single-stranded probes targeting various targets in a specific order on a gene chip to form a probe array. The multiplex PCR product to be tested is then hybridized with the probes on the chip, causing the target amplification product to hybridize and emit a fluorescent signal. Although this method can simultaneously detect 20 respiratory pathogens, the gene chip preparation process is complex and expensive, hindering clinical application. Furthermore, the process is cumbersome, requiring multiple opening and cleaning steps, which can easily lead to contamination and false positives.

[0006] There are also reports of combining PCR detection technology with capillary electrophoresis for multiplex target detection using different amplification product lengths. For example, Chinese patent CN103074450A discloses a kit and method for simultaneously detecting thirty diarrheal pathogens. This method utilizes a combination of PCR amplification and capillary electrophoresis, extracting the PCR amplification products and performing capillary electrophoresis analysis. The obtained spectra are compared with standard spectra to determine the type of diarrheal pathogen. However, this method requires capillary electrophoresis equipment in addition to PCR amplification detection equipment, significantly increasing the detection cost and hindering its widespread clinical application.

[0007] Therefore, there is an urgent clinical need for a method that can perform multiplex molecular detection efficiently and at low cost, so as to quickly and effectively detect a variety of common clinical targets such as bacteria, viruses, and gene mutations. Summary of the Invention

[0008] To address the aforementioned problems, the inventors conducted research on primer and probe design for qPCR detection technology and proposed a new solution for multiple target detection, thereby realizing this invention.

[0009] Accordingly, in a first aspect, the present invention provides a method for multiple target detection, comprising the following steps:

[0010] The combination containing the first primer set and the first probe is mixed with a sample from the subject;

[0011] Amplify any target sequences that may be present in the sample;

[0012] Melting curve analysis of the amplified product was performed in the corresponding detection channel; and

[0013] Based on the results of the melting curve analysis, determine whether one or more of the targets exist.

[0014] The first primer set includes upstream and downstream primers for a first target, and upstream and downstream primers for a second target.

[0015] The upstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region; the downstream primer of the first target includes a target sequence binding region located at the 3' end.

[0016] The upstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region; the downstream primer of the second target includes a target sequence binding region located at the 3' end.

[0017] The first and second signal detection regions are designed not to be complementary to the target sequence.

[0018] Furthermore, all or part of the sequence of the first probe is the same as the first signal detection region, and all or part of the sequence of the first probe is the same as the second signal detection region, thereby enabling the amplified product to be amplified (annealed and extended) after amplification using the first primer set; the first probe has a first detection group and a second detection group, and after integration into the double-stranded structure, the first detection group and the second detection group can thus generate a signal change.

[0019] It is understandable that during PCR amplification, primer pairs targeting different targets specifically bind to their respective target sequences and extend to generate pre-amplified products. Subsequently, probes can complementaryly pair with these pre-amplified products and continue PCR amplification, integrating the first and second detection groups into the resulting double-stranded amplified products. This causes a signal change that can be detected by the instrument. After PCR amplification is complete, melting curve analysis is performed. During temperature changes, the double strands of the PCR product carrying the first and second detection groups will unwind at a certain temperature, causing a signal change that can be detected by the instrument. By combining the type of detection group carried by the probe with the different melting temperatures of the PCR products of each target, multiple target detection can be achieved.

[0020] In a variation of the first aspect of the invention, the upstream primer of the first target includes a target sequence binding region located at the 3' end; the downstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region.

[0021] Furthermore, the upstream primer of the second target includes a target sequence binding region located at the 3' end; the downstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region.

[0022] In another variation of the first aspect of the invention, the upstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region; the downstream primer of the first target includes a target sequence binding region located at the 3' end.

[0023] Furthermore, the upstream primer of the second target includes a target sequence binding region located at the 3' end; the downstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region.

[0024] In some embodiments, the method of the present invention further includes a step of quantifying the detected target. For example, the target can be quantified by detecting a real-time fluorescence signal.

[0025] In a preferred embodiment, the combination of the present invention further comprises a second primer set and a second probe.

[0026] The second primer set includes upstream and downstream primers for the third target, and upstream and downstream primers for the fourth target.

[0027] Wherein, the upstream or downstream primer of the third target includes a third signal detection region located upstream of the target sequence binding region; the upstream or downstream primer of the fourth target includes the fourth signal detection region located upstream of the target sequence binding region;

[0028] The third and fourth signal detection regions are designed not to be complementary to the target sequence.

[0029] Furthermore, all or part of the sequence of the second probe is the same as the third signal detection region, and all or part of the sequence of the second probe is the same as the fourth signal detection region, thereby enabling the amplified product to be amplified (annealed and extended) after amplification using the second primer set; the second probe carries a third detection group and a fourth detection group, which, after integration into the double-stranded structure, can thus generate a signal change, and the signal generated by the second probe is different in type (e.g., wavelength) from the signal generated by the first probe.

[0030] It is understandable that by using the above method, the signal corresponding to the first probe can be detected and melting curve analysis can be performed in one detection channel to determine whether the target corresponding to the first primer set exists; at the same time, the signal corresponding to the second probe can be detected and melting curve analysis can be performed in another detection channel to determine whether the target corresponding to the second primer set exists.

[0031] Similarly, the combination of the present invention may further include a third probe, or even a fourth probe, so as to detect the target targeted by the corresponding third primer set and fourth primer set in their respective different detection channels.

[0032] In some embodiments, each primer set in the first aspect may contain upstream and downstream primer pairs for three, four or more targets, each upstream or downstream primer having its own signal detection region; in each primer set, the corresponding probe is capable of amplifying (annealing and extending) the amplification product of that primer set, but not the amplification product of other primer sets.

[0033] It is understandable that during the melting curve analysis process, different targets corresponding to each primer set are distinguished in each detection channel based on different melting temperatures.

[0034] Adjusting the melting temperature of the amplification product is within the capabilities of those skilled in the art. For example, the melting temperature of amplification products for different targets can be differentiated by adjusting the length of the spacer bases between the signal detection region and the target sequence binding region (e.g., by adding a certain number of G / C bases); specifically, when the number of spacer bases increases, the melting temperature will increase accordingly; while when the number of spacer bases decreases, the melting temperature will decrease accordingly.

[0035] In the first aspect of the method, melting curve analysis can be performed within the range of 60-95℃. In each detection channel, the melting temperature of the amplification product corresponding to each target differs by at least 2℃.

[0036] Furthermore, the first aspect of the present invention also provides a combination for multiple target detection, comprising:

[0037] The first primer set includes an upstream and a downstream primer targeting a first target, and an upstream and a downstream primer targeting a second target; and

[0038] A first probe, whose sequence is wholly or partially identical to a first signal detection region and whose sequence is wholly or partially identical to a second signal detection region, is capable of amplifying (annealing and extending) the amplified product after the target sequence is amplified using the first primer set. The first probe carries a first detection group and a second detection group, which are capable of generating a signal change after integration into the double-stranded structure.

[0039] The first signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the first target; the second signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the second target; the first and second signal detection regions are designed not to be complementary to the target sequence.

[0040] In this invention, the length of the probe can be 15-30 nt.

[0041] In this invention, the length of the upstream or downstream primer, including the signal detection region, can be 30-80 nt.

[0042] In this invention, the length of the upstream or downstream primer that does not include the signal detection region can be 15-30 nt.

[0043] In some implementations, in the combination of the first aspect, the signal detection regions within each primer set are identical to each other. For example, in the first primer set, the first signal detection region is the same as the second signal detection region.

[0044] In some implementations, within each primer set of the first aspect combination, the target sequence binding region and the signal detection region located upstream therefrom may be spaced 0-40 nt apart.

[0045] In a specific embodiment, the first and second detection groups on the probe of the first aspect of the present invention are not located at the 3' end.

[0046] In a specific embodiment, the first detection group and the second detection group on the probe of the first aspect of the present invention are spaced 5-25 nt apart.

[0047] In a second aspect, the present invention provides a method for multiple target detection, comprising the following steps:

[0048] The combination containing the first primer set and the first probe is mixed with a sample from the subject;

[0049] Amplify any target sequences that may be present in the sample;

[0050] Melting curve analysis of the amplified product was performed in the corresponding detection channel; and

[0051] Based on the results of the melting curve analysis, determine whether one or more of the targets exist.

[0052] The first primer set includes upstream and downstream primers for a first target, and upstream and downstream primers for a second target.

[0053] The upstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region; the downstream primer of the first target includes a target sequence binding region located at the 3' end.

[0054] The upstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region; the downstream primer of the second target includes a target sequence binding region located at the 3' end.

[0055] The first and second signal detection regions are designed not to be complementary to the target sequence.

[0056] Furthermore, all or part of the sequence of the first probe is the same as the first signal detection region, and all or part of the sequence of the first probe is the same as the second signal detection region, thereby enabling hybridization with the amplified product after amplification using the first primer set; the first probe carries a first detection group and a second detection group, and after hybridization, the first detection group and the second detection group can thus generate a signal change, and the 3' end of the first probe cannot be used as a primer for extension.

[0057] It is understandable that during the PCR amplification process, primer pairs targeting different targets specifically bind to their respective target sequences and extend to produce amplification products. Subsequently, the probe can hybridize with the amplification products, causing a change in the distance between the first detection group and the second detection group, resulting in a change in the signal, which can then be detected by the instrument.

[0058] After PCR amplification, melting curve analysis is performed. During temperature changes, the probe that hybridizes with the amplification product will melt at a certain temperature and separate from the product chain. At this time, the distance between the first and second detection groups changes, causing a signal change that can be detected by the instrument. By combining the type of the first detection group carried by the probe with the different melting temperatures of the probes binding to each target, multiple target detection can be achieved.

[0059] In a variation of the second aspect of the invention, the upstream primer of the first target includes a target sequence binding region located at the 3' end; the downstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region.

[0060] Furthermore, the upstream primer of the second target includes a target sequence binding region located at the 3' end; the downstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region.

[0061] In another variation of the second aspect of the present invention, the upstream primer of the first target includes a target sequence binding region located at the 3' end and a first signal detection region located upstream of the target sequence binding region; the downstream primer of the first target includes a target sequence binding region located at the 3' end.

[0062] Furthermore, the upstream primer of the second target includes a target sequence binding region located at the 3' end; the downstream primer of the second target includes a target sequence binding region located at the 3' end and a second signal detection region located upstream of the target sequence binding region.

[0063] In a preferred embodiment, the second aspect further comprises a second set of primers and a second probe.

[0064] The second primer set includes upstream and downstream primers for the third target, and upstream and downstream primers for the fourth target.

[0065] Wherein, the upstream or downstream primer of the third target includes a third signal detection region located upstream of the target sequence binding region; the upstream or downstream primer of the fourth target includes a fourth signal detection region located upstream of the target sequence binding region;

[0066] The third and fourth signal detection regions are designed not to be complementary to the target sequence.

[0067] Furthermore, all or part of the sequence of the second probe is the same as the third signal detection region, and all or part of the sequence of the second probe is the same as the fourth signal detection region, thereby enabling hybridization with the amplified product after amplification using the second primer set; the 3' end of the second probe cannot be used as a primer extension; the second probe carries a third detection group and a fourth detection group, which, after hybridization, can thus generate a signal change, and the signal generated by the second probe is different in type (e.g., wavelength) from the signal generated by the first probe.

[0068] It is understandable that by using the above method, the signal corresponding to the first probe can be detected and melting curve analysis can be performed in one detection channel to determine whether the target corresponding to the first primer set exists; at the same time, the signal corresponding to the second probe can be detected and melting curve analysis can be performed in another detection channel to determine whether the target corresponding to the second primer set exists.

[0069] Similarly, the combination of the present invention may further include a third probe, or even a fourth probe, so as to detect the target targeted by the corresponding third primer set and fourth primer set in their respective different detection channels.

[0070] In some embodiments, each primer set in the second aspect may contain upstream and downstream primer pairs for three, four or more targets, each upstream or downstream primer having its own signal detection region; in each primer set, the corresponding probe is capable of hybridizing with the amplification product of that primer set but not with the amplification products of other primer sets.

[0071] It is understandable that during the melting curve analysis process, different targets corresponding to each primer set are distinguished in each detection channel based on different melting temperatures.

[0072] Adjusting the melting temperature between the probe and the amplification product is within the capabilities of those skilled in the art. For example, the melting temperature of amplification products for different targets can be distinguished by the degree of mismatch between the complementary sequences of the probe and the signal detection region; specifically, the melting temperature is highest when the probe of the second aspect of the invention is perfectly matched with the complementary sequence of the signal detection region, and decreases when there are mismatched bases between the probe and the complementary sequence of the signal detection region.

[0073] In the second aspect of the method, melting curve analysis can be performed within the range of 45-85℃. In each detection channel, the melting temperature of the amplified product corresponding to each target differs by at least 2℃.

[0074] Furthermore, a second aspect of the present invention provides a combination for multiple target detection, comprising:

[0075] The first primer set includes an upstream and a downstream primer targeting a first target, and an upstream and a downstream primer targeting a second target; and

[0076] A first probe, whose sequence is wholly or partially identical to a first signal detection region and whose sequence is wholly or partially identical to a second signal detection region, is capable of hybridizing with the amplification product of the first primer set. The first probe carries a first detection group and a second detection group; after hybridization, the first and second detection groups generate signal changes, and the 3' end of the first probe cannot be used for primer extension.

[0077] The first signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the first target; the second signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the second target; the first and second signal detection regions are designed not to be complementary to the target sequence.

[0078] In some implementations, within each primer set of the first aspect combination, the target sequence binding region and the signal detection region located upstream therefrom may be spaced 0-10 nt apart.

[0079] In some embodiments, the combination of the present invention may further include amplification reagents for PCR reactions.

[0080] In some embodiments, the first or second detection group on the probe of the second aspect of the present invention is located at the 3' end.

[0081] In some embodiments, the 3' end of the probe of the second aspect of the present invention is closed. The closure can be performed using methods well known in the art, for example, by modifying the 3' end with a C3, C6, C9, or C12 interarm; or, for example, by modifying the 3' end with ddC.

[0082] In a specific embodiment, the first detection group on the probe of the second aspect of the present invention can be spaced 5-25 nt apart from the second detection group.

[0083] The methods and combinations of the present invention have the following beneficial effects:

[0084] This invention allows for simultaneous analysis of two dimensions—detection channels and melting temperature—in a single-tube reaction, thereby enabling the detection of the number of targets multiplied by the number of detection channels and the melting temperature characteristic.

[0085] Each detection channel of this invention uses only one probe, thereby greatly reducing the fluorescence background in the PCR reaction and improving the reaction sensitivity.

[0086] In the design of this invention, only the primers are complementary to the target sequence. Therefore, when the target sequence has many highly variable regions, such as viral or bacterial genomes, this invention has better inclusiveness and lower design difficulty.

[0087] This invention only requires a nucleic acid amplification analyzer to complete the test, without the need for additional downstream instruments or the need to divide the reaction using chips or multiple reaction tubes. This greatly reduces the cost of instruments and consumables and the complexity of operation. At the same time, it reduces the number of probes in the reagents, which greatly reduces the reagent cost and makes large-scale promotion possible.

[0088] This invention enables multiplex detection in a single reaction tube with minimal sample consumption, making it particularly suitable for detecting rare samples. It can significantly increase the concentration of sample added to the detection reaction and improve detection sensitivity. Furthermore, since it enables single-tube reactions, it provides the possibility for subsequent molecular point-of-care testing (POCT) detection.

[0089] The method of the present invention is completely sealed after the sample is added, and there is no need to open the cap for subsequent testing, which greatly reduces the possibility of contamination to the experimental environment.

[0090] This invention has very low requirements for the length of the target sequence, thereby improving the sensitivity of short nucleic acid detection. Attached Figure Description

[0091] Figure 1 The melting curve of ADV-positive targets in the FAM channel for multiple target detection in Example 2;

[0092] Figure 2 The melting curve of IBV-positive targets in the FAM channel for multiple target detection in Example 2;

[0093] Figure 3 The melting curve of MP-positive targets in the ROX channel for multiple target detection in Example 2;

[0094] Figure 4 The melting curve of SARS-CoV-2 positive multiple target detection in the ROX channel in Example 2 is shown.

[0095] Figure 5 The melting curve of RSV-positive multiple target detection in the Cy5 channel in Example 2 is shown.

[0096] Figure 6 The melting curve of IAV-positive targets in the Cy5 channel for multiple target detection in Example 2;

[0097] Figure 7 The results show that in Example 3, when ADV and IBV positive templates were added to the FAM channel, dual melting peaks of ADV and IBV appeared simultaneously in the reaction wells.

[0098] Figure 8 The results from Example 3 show that in the reaction wells where MP and SARS-CoV-2 positive templates were added to the ROX channel, dual melting peaks of MP and SARS-CoV-2 appeared simultaneously.

[0099] Figure 9 The results from Example 3 show that in the reaction wells where RSV and IAV positive templates were added to the Cy5 channel, dual melting peaks of RSV and IAV appeared simultaneously.

[0100] Figure 10 The melting curve of IBV-positive targets in the FAM channel for multiple target detection in Example 5;

[0101] Figure 11 The melting curve of IAV-positive targets in the FAM channel for multiple target detection in Example 5;

[0102] Figure 12The melting curve of ADV-positive targets in the ROX channel for multiple target detection in Example 5;

[0103] Figure 13 The melting curve of RSV-positive multiple target detection in the ROX channel in Example 5 is shown.

[0104] Figure 14 The melting curve of MP-positive targets in the Cy5 channel for multiple target detection in Example 5;

[0105] Figure 15 The melting curve of SARS-CoV-2 positive multiple target detection in the Cy5 channel in Example 5;

[0106] Figure 16 The results from Example 6 show that in the reaction wells where IAV and IBV positive templates were added to the FAM channel, dual melting peaks of IAV and IBV appeared simultaneously.

[0107] Figure 17 The results from Example 6 show that in the reaction wells where ADV and RSV positive templates were added to the ROX channel, dual melting peaks of ADV and RSV appeared simultaneously.

[0108] Figure 18 The results from Example 6 show that in the reaction wells where MP and SARS-CoV-2 positive templates were added to the Cy5 channel, dual melting peaks of MP and SARS-CoV-2 appeared simultaneously.

[0109] Figure 19 The melting curve of IBV-positive targets in the FAM channel for multiple target detection in Example 7;

[0110] Figure 20 The melting curve of IAV-positive targets in the FAM channel for multiple target detection in Example 7;

[0111] Figure 21 The melting curve of ADV-positive multiple target detection in the ROX channel in Example 7 is shown.

[0112] Figure 22 The melting curve of RSV-positive multiple target detection in the ROX channel in Example 7 is shown.

[0113] Figure 23 The melting curve of MP-positive targets in the Cy5 channel for multiple target detection in Example 7;

[0114] Figure 24 The melting curve of SARS-CoV-2 positive multiple target detection in the Cy5 channel in Example 7;

[0115] Figure 25The results from Example 8 show that in the reaction wells where IAV and IBV positive templates were added to the FAM channel, dual melting peaks of IAV and IBV appeared simultaneously.

[0116] Figure 26 The results from Example 8 show that in the reaction wells where ADV and RSV positive templates were added to the ROX channel, dual melting peaks of ADV and RSV appeared simultaneously.

[0117] Figure 27 The results from Example 8 show that in the reaction wells where MP and SARS-CoV-2 positive templates were added to the Cy5 channel, dual melting peaks of MP and SARS-CoV-2 appeared simultaneously.

[0118] Figure 28 The melting curve of IBV-positive targets in the FAM channel for multiple target detection in Example 9;

[0119] Figure 29 The melting curve of IAV-positive targets in the FAM channel for multiple target detection in Example 9;

[0120] Figure 30 The melting curve of ADV-positive multiple target detection in the ROX channel in Example 9 is shown.

[0121] Figure 31 The melting curve of RSV-positive multiple target detection in the ROX channel in Example 9 is shown.

[0122] Figure 32 The melting curve of RhV-positive targets in the Cy5 channel for multiple target detection in Example 9;

[0123] Figure 33 The melting curve of MP-positive targets in the Cy5 channel for multiple target detection in Example 9;

[0124] Figure 34 The melting curve of SARS-CoV-2 positive multiple target detection in the Cy5 channel in Example 9;

[0125] Figure 35 In Example 9, when RhV, MP, and SARS-CoV-2 positive templates were added to the Cy5 channel, three melting peaks of RhV, MP, and SARS-CoV-2 appeared simultaneously in the reaction wells.

[0126] Figure 36 The results of the multiple target detection in Example 9 show the amplification curve (top) and melting curve (bottom) of GAPDH-positive cells in the VIC channel. Detailed Implementation

[0127] The technical methods of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0128] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0129] This invention provides a method for multiplex nucleic acid detection, comprising the following steps:

[0130] The combination containing the first primer set and the first probe is mixed with a sample from the subject;

[0131] Amplify any target sequences that may be present in the sample;

[0132] Melting curve analysis of the amplified product was performed in the corresponding detection channel; and

[0133] Based on the results of the melting curve analysis, determine whether one or more of the targets exist.

[0134] The first primer set includes upstream and downstream primers for a first target, and upstream and downstream primers for a second target.

[0135] Wherein, the upstream or downstream primer of the first target includes a first signal detection region located upstream of the target sequence binding region;

[0136] Wherein, the upstream or downstream primer of the second target includes a second signal detection region located upstream of the target sequence binding region;

[0137] The first and second signal detection regions are designed not to be complementary to the target sequence.

[0138] Furthermore, all or part of the sequence of the first probe is the same as that of the first and second signal detection regions, thereby enabling it to form a double-stranded structure with the complementary sequences of the first and second signal detection regions, respectively; the first probe carries a first detection group and a second detection group, and after integration into the double-stranded structure, the first detection group and the second detection group can thus generate a change in signal.

[0139] It is understood that, for each target to be detected, the method of the present invention provides a corresponding primer pair, and each primer pair (upstream or downstream primer) provides a signal detection region, which does not pair complementaryly with the target sequence, but has a sequence that is wholly or partially the same as the probe of the present invention.

[0140] In this document, the term "nucleic acid" refers to a single-stranded and / or double-stranded polymer of nucleotide monomers, including but not limited to 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by phosphodiester bonds or nucleotide analogs. Nucleotide monomers in nucleic acids may be referred to as "nucleotide residues." Nucleic acids may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof, and may include nucleotide analogs. A nucleotide monomer unit may contain any of the nucleotides described herein, including but not limited to nucleotides, and / or nucleotide analogs (such as modified nucleotides). Nucleic acids typically range in size from a few nucleotide residues to several thousand nucleotide residues. "Oligonucleotide" generally refers to a relatively short (e.g., less than 80) polymer of nucleotides. Unless otherwise indicated, whenever a nucleic acid sequence is presented, it should be understood that the nucleotides are arranged in a 5' to 3' order from left to right. Unless otherwise indicated, "A" represents adenine, "C" represents cytosine, "G" represents guanine, "T" represents thymine, and "U" represents uracil.

[0141] According to the terminology commonly used in this field, the length of nucleic acids can be expressed in base pairs (abbreviated as "bp"), nucleotides (abbreviated as "nt"), or kilobases (abbreviated as "kb").

[0142] In this paper, "complementary base pairing" refers to the phenomenon where the correspondences of A and T, A and U, and G and C are linked by hydrogen bonds. Correspondingly, "mismatched bases" refers to all other pairing cases not specified in "complementary base pairing," such as A and C, A and G, T and G, or T and C mismatches.

[0143] In this paper, the term "primer" refers to an oligonucleotide that is capable of "initiating" DNA synthesis by a template-dependent DNA polymerase, i.e., the 3'-terminus of the oligonucleotide provides a free 3'-OH group, which can be linked to more "nucleotides" by the template-dependent DNA polymerase to establish a 3' to 5' phosphodiester bond, thereby using deoxynucleoside triphosphate and thus releasing pyrophosphate.

[0144] In this paper, the terms “target sequence,” “target nucleic acid,” “target nucleic acid sequence,” or “target” are used interchangeably and refer to a portion of a nucleic acid sequence to be amplified, detected, or amplified and detected, which may be annealed with primers under annealing or amplification conditions.

[0145] The term "hybridization" refers to the base-pairing interaction between two nucleic acids, which results in the formation of a double helix. It is understood that 100% complementarity across the entire length is not required for hybridization to occur.

[0146] In this document, the term "upstream primer," also known as the forward primer, refers to an oligonucleotide that extends continuously along the negative strand; the term "downstream primer," also known as the reverse primer, refers to an oligonucleotide that extends continuously along the positive strand. The positive strand, also called the sense strand or coding strand, is generally located at the top of the double-stranded DNA, oriented 5'-3' from left to right, and its base sequence is essentially the same as the gene's mRNA; the primer that binds to this strand is the reverse primer. The negative strand, also called the sense strand or non-coding strand, is complementary to the positive strand, and the primer that binds to this strand is the forward primer. It should be understood that when the designations of the sense and antisense strands are interchanged, the corresponding names of the forward and reverse primers can also be interchanged.

[0147] In this article, terms such as "upstream," "located / upstream of," and "upstream has..." refer to a portion of the same nucleic acid sequence closer to the 5' end than the referred region, in the context of describing a nucleic acid sequence. This could be, for example, immediately adjacent to the referred region or separated from it by one or more bases. Correspondingly, the terms "downstream," "located / downstream of," and "downstream has..." refer to a portion of the same nucleic acid sequence closer to the 3' end than the referred region, in the context of describing a nucleic acid sequence. This could be, for example, immediately adjacent to the referred region or separated from it by one or more bases. It should be understood that, unless otherwise stated, when the described nucleic acid is a double-stranded nucleic acid, the designation of "upstream" and "downstream" generally refers to the 5' and 3' ends of the positive strand.

[0148] In this paper, the term "probe" refers to a labeled oligonucleotide used to detect the presence of a target.

[0149] In this paper, hybridization can be achieved under stringent conditions, which can be any of low-stringent, medium-stringent, or high-stringent conditions. "Low-stringent conditions" include, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C; "medium-stringent conditions" include, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C; and "high-stringent conditions" include, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, higher temperatures are expected to yield more homologous polynucleotides, such as DNA. Although various factors affect the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, those skilled in the art can obtain similar stringency by appropriately selecting these elements.

[0150] In specific embodiments, the probe of the present invention may be selected from the group consisting of: oligonucleotides that are coiled due to molecular flexibility in the single-stranded state, oligonucleotides that can form hairpin structures in the single-stranded state, oligonucleotides that can form stem-loop structures in the single-stranded state, oligonucleotides that can form pseudo-knot structures in the single-stranded state, and oligonucleotides that can form triple helix structures in the single-stranded state.

[0151] In this article, "oligonucleotides that are coiled due to molecular flexibility in the single-stranded state" and "flexible probes" are used interchangeably. In the single-stranded state, the molecular flexibility causes the oligonucleotide to coil. After hybridization or annealing to form a double-stranded state, hydrogen bonding fixes it into a more rigid double-helix structure, causing a change in the distance between the labeled detection groups on the oligonucleotide / probe, thereby generating a detectable signal. For example, such oligonucleotides / probes can be obtained by referring to the description in US9845492.

[0152] The probe of the second aspect of this invention may also be called a hybridization probe.

[0153] In some embodiments, one of the first (third) detection group and the second (fourth) detection group of the present invention may be a fluorescent group, and the other may be a quenching group or other group that can generate a signal change with the fluorescent group through fluorescence resonance energy transfer.

[0154] In an exemplary embodiment, the group that generates a signal change through fluorescence resonance energy transfer can be Cy3 or Cy5, because they can generate a fluorescence signal change through FRET interaction.

[0155] In this invention, the first detection group may be selected from the group consisting of: FAM, HEX, VIC, ROX, Cy3, Cy5 and Cy5.5.

[0156] In this invention, the second detection group may be selected from the group consisting of: TAMRA, BHQ1, BHQ2, BHQ3, DABCYL, QXL and DDQI.

[0157] It is important to note that the probe of this invention can anneal and extend or hybridize with the amplification product of the corresponding primer set, thereby integrating the detection group on the probe into the resulting double strand. Compared with the single-stranded form, the double-stranded form of the probe causes a change (increase) in the distance between the detection groups, and this change in distance is not caused by the hydrolysis or release of the detection groups.

[0158] In some embodiments, the combination of the present invention may be a kit, which further includes amplification reagents.

[0159] In this invention, the term "amplification reagent" refers to reagents used for PCR, including but not limited to dNTPs, DNA polymerase, and some reagents that promote PCR reactions, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), etc.

[0160] In this invention, the components in the combination and kit can be individually packaged or pre-mixed.

[0161] In this invention, the purpose of "pre-denaturation" and "denaturation" is to break the hydrogen bonds between complementary base pairs on double-stranded DNA, thereby allowing the double strands to separate into two single strands. For example, single strands can be formed by heating a mixture containing double-stranded DNA, such as heating the mixture to 90°C, 92°C, 95°C, or 98°C to dissociate the double-stranded DNA. After dissociation, it is cooled to room temperature or below. Alternatively, single strand formation can also be achieved by changing the ionic strength of the solution (e.g., adding acids, bases, salts, etc.) to break the hydrogen bonds between the double-stranded DNA, or by using enzymes (e.g., helicases) to dissociate double-stranded DNA into single-stranded DNA.

[0162] In this invention, the Tm values ​​of the upstream and downstream primers can be 50℃-80℃, and the GC content can be 40%-80%.

[0163] In this invention, the Tm value of the probe can be 50℃-80℃, and the GC content can be 40%-80%.

[0164] In the method of this invention, the specific reaction conditions for the amplification step can be, for example: pre-denaturation at 90℃-96℃ for 5-15 minutes; denaturation at 90℃-95℃ for 10-60 seconds, followed by annealing and extension at 45℃-75℃ for 30-90 seconds, for a total of 3-10 cycles; or denaturation at 90℃-95℃ for 10-60 seconds, followed by annealing and extension at 45℃-75℃ for 30-90 seconds, for a total of 35-50 cycles. Alternatively, the specific reaction conditions for the amplification step can be: pre-denaturation at 90℃-96℃ for 5-15 minutes; denaturation at 90℃-95℃ for 10-60 seconds, followed by annealing and extension at 45℃-75℃ for 30-90 seconds, for a total of 35-50 cycles.

[0165] In one specific implementation, the specific reaction conditions for the amplification step can be: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 10 seconds, followed by annealing and extension at 61°C for 15 seconds, for a total of 10 cycles; or denaturation at 94°C for 10 seconds, followed by annealing and extension at 55°C for 15 seconds, for a total of 40 cycles.

[0166] In one specific implementation, the specific reaction conditions for amplification can be: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 10 seconds, followed by annealing and extension at 60°C for 15 seconds, for a total of 10 cycles; or denaturation at 94°C for 10 seconds, followed by annealing and extension at 56°C for 15 seconds, for a total of 40 cycles.

[0167] In a first aspect of the invention, the reaction conditions for melting curve analysis after amplification can be, for example, denaturation at 90°C-96°C for 10-60 seconds, annealing at 45°C-55°C for 60-120 seconds, and melting curve analysis at 50°C-95°C, with a heating rate of 0.02-0.1°C per second. Specifically, the reaction conditions for melting curve analysis can be: denaturation at 94°C for 10 seconds, annealing at 50°C for 90 seconds, and melting curve analysis at 65°C-95°C, with a heating rate of 0.05°C per second.

[0168] In a second aspect of the invention, the reaction conditions for melting curve analysis after amplification can be, for example: denaturation at 90°C-95°C for 10-60 seconds, annealing at 45°C-55°C for 60-120 seconds, and melting curve analysis at 45-85°C, with a heating rate of 0.02-0.1°C per second. Specifically, the reaction conditions for melting curve analysis can be: denaturation at 94°C for 10 seconds, annealing at 50°C for 120 seconds, and melting curve analysis at 50-90°C, with a heating rate of 0.03°C per second.

[0169] In a first aspect of the invention, when a signal detection region is included, the concentration of the upstream or downstream primer in the reaction system can be, for example, 30 nM-150 nM, preferably 30 nM-120 nM, more preferably 40 nM; otherwise, the concentration of the upstream or downstream primer in the reaction system can be, for example, 150 nM-1200 nM, preferably 150 nM-450 nM, more preferably 240 nM.

[0170] In a first aspect of the invention, the concentration of the probe in the reaction system can be, for example, 150 nM-1200 nM, preferably 150 nM-450 nM, and more preferably 240 nM.

[0171] In a second aspect of the invention, when a signal detection region is included, the concentration of the upstream or downstream primer in the reaction system can be, for example, 15nM-150nM, preferably 30nM-90nM, more preferably 45nM; otherwise, the concentration of the upstream or downstream primer in the reaction system can be, for example, 150nM-1500nM, preferably 300nM-600nM, more preferably 250nM.

[0172] In a second aspect of the invention, the probe in the reaction system can be, for example, 150 nM-1800 nM, preferably 300 nM-900 nM, and more preferably 250 nM.

[0173] In this invention, the terms "first," "second," "third," and "fourth," etc., are used for descriptive purposes only to distinguish the substances being defined, and do not in any way limit the order or importance of the substances.

[0174] In this invention, the sample to be tested can be selected from the group consisting of serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples, formalin-fixed paraffin-embedded tissue (FFPE) samples, urine samples, bacterial cultures, virus cultures, cell line cultures, artificially synthesized plasmid samples, and combinations thereof.

[0175] In some embodiments, the method of the present invention can be used for the detection of infectious pathogens. For example, it can be used to detect respiratory-associated pathogens, including but not limited to the detection of one or more pathogens selected from the following: influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae, and novel coronavirus (SARS-CoV-2).

[0176] It is understood that, when the target of the detection is RNA, the method of the present invention further includes a reverse transcription step of the RNA before the amplification step. Exemplary reverse transcription reaction conditions can be 40℃-60℃ for 1-30 minutes.

[0177] Example 1: Design of primer sets and flexible probes for detecting respiratory-associated pathogens

[0178] To verify the effectiveness of the combination of the present invention in detecting influenza A virus (IAV), influenza B virus (IBV), respiratory syncytial virus (RSV), adenovirus (ADV), mycoplasma pneumoniae (MP), and the novel coronavirus (SARS-CoV-2), primers and probes as shown in Table 1 below were designed.

[0179] Table 1

[0180]

[0181] Among them, upstream primer 1 and downstream primer 1 are specific primers designed for adenovirus target sequences; upstream primer 2 and downstream primer 2 are specific primers designed for influenza B virus target sequences; upstream primer 3 and downstream primer 3 are specific primers designed for mycoplasma pneumoniae target sequences; upstream primer 4 and downstream primer 4 are specific primers designed for novel coronavirus target sequences; upstream primer 5 and downstream primer 5 are specific primers designed for influenza A virus target sequences; and upstream primer 6 and downstream primer 6 are specific primers designed for respiratory syncytial virus target sequences.

[0182] The Tm values ​​of the amplification products after the above-mentioned flexible probes were amplified and extended are shown in Table 2 below.

[0183] Table 2

[0184]

[0185] Example 2: Single-sample detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0186] Sample preparation: In vitro transcribed RNA of each target sequence was used as a positive sample, and each target sequence was detected individually to verify the detection capability of a single virus infection. Pure water was used as a template-free control (NTC).

[0187] Reaction preparation:

[0188] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 3 below.

[0189] Table 3

[0190]

[0191] PCR amplification and melting curve analysis:

[0192] After sealing the PCR tubes, gently mix the samples, then briefly centrifuge and incubate at room temperature for 5 minutes. Place the PCR tubes again in a handheld centrifuge, briefly centrifuge, and then transfer to the tray of a quantitative PCR instrument (Suzhou Yarui, MA-6000). The program used was as follows: 50℃ reverse transcription for 10 minutes; 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 61℃ annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 94℃ denaturation for 10 seconds, 55℃ annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94℃ denaturation for 10 seconds, 50℃ annealing for 90 seconds, melting curve analysis at 65-95℃, with a heating rate of 0.05℃ per second.

[0193] In the absence of a target, the single-stranded flexible probe is coiled due to molecular flexibility. The distance between the fluorophore and quencher is relatively short, resulting in high fluorescence resonance energy transfer efficiency. However, when a target is present, the upstream and downstream primers specifically bind to the target sequence and extend to generate a pre-amplified template. This allows the flexible probe to pair with the pre-amplified template and extend, integrating the fluorophore and quencher into the double-stranded PCR amplification product. The change from single-stranded to double-stranded affects molecular flexibility, increasing the distance between the fluorophore and quencher, reducing fluorescence resonance energy transfer efficiency, and causing a change in the fluorescence signal that can be detected by the instrument. After PCR amplification, during melting curve analysis at a certain temperature, the double-stranded PCR amplification product melts, causing the product chain containing the fluorophore and quencher to become single-stranded. Molecular flexibility is restored, the distance between the fluorophore and quencher shortens, and fluorescence resonance energy transfer efficiency increases, causing another change in the fluorescence signal, which can be used for melting curve analysis.

[0194] Data Analysis:

[0195] Interpret the Tm values ​​of the melting curve. Figure 1-6 This is a melting curve of positive samples of various respiratory pathogens in multiple target detection.

[0196] Depend on Figures 1-6 It can be seen that the PCR reaction wells with ADV positive template formed a melting peak with a Tm value of 82.33℃ in the FAM channel; the PCR reaction wells with IBV positive template formed a melting peak with a Tm value of 76.87℃ in the FAM channel; the PCR reaction wells with MP positive template formed a melting peak with a Tm value of 85.23℃ in the ROX channel; the PCR reaction wells with SARS-CoV-2 positive template formed a melting peak with a Tm value of 81.41℃ in the ROX channel; the PCR reaction wells with RSV positive template formed a melting peak with a Tm value of 79.8℃ in the Cy5 channel; and the PCR reaction wells with IAV positive template formed a melting peak with a Tm value of 85.27℃ in the Cy5 channel.

[0197] Example 3: Multiplex detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0198] Sample preparation: In vitro transcribed RNA from each target sequence was used as a positive sample. Two target sequence templates from the same fluorescent channel in Table 2 were mixed and detected to verify the ability to detect viral co-infection. Ultrapure water was used as a template-free control (NTC).

[0199] Reaction preparation:

[0200] After the sample preparation is completed, the reaction system is configured according to the ratios described in Table 3.

[0201] PCR amplification and melting curve analysis:

[0202] After sealing the PCR tubes, gently mix the samples, then briefly centrifuge and incubate at room temperature for 5 minutes. Place the PCR tubes again in a handheld centrifuge, briefly centrifuge, and then transfer to the tray of a quantitative PCR instrument (Suzhou Yarui, MA-6000). The program used was as follows: 50℃ reverse transcription for 10 minutes; 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 10 seconds, 61℃ annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 94℃ denaturation for 10 seconds, 55℃ annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94℃ denaturation for 10 seconds, 50℃ annealing for 90 seconds, melting curve analysis at 65-95℃, with a heating rate of 0.05℃ per second.

[0203] Data Analysis:

[0204] The results are interpreted based on the Tm value of the melting curve.

[0205] Figure 7-9 This is a melting curve diagram of positive respiratory virus samples in multiplex target detection. Figure 7-9 It was observed that PCR reaction wells containing both ADV and IBV positive templates simultaneously exhibited dual melting peaks for ADV and IBV in the FAM channel; PCR reaction wells containing both MP and SARS-CoV-2 positive templates simultaneously exhibited dual melting peaks for MP and SARS-CoV-2 simultaneously in the ROX channel; and PCR reaction wells containing both RSV and IAV positive templates simultaneously exhibited dual melting peaks for RSV and IAV simultaneously in the Cy5 channel. Therefore, the combination of this invention demonstrates good specificity in detecting these six respiratory-associated pathogens, exhibits a uniform melting curve morphology, and clearly distinguishes the Tm values ​​of the two positive targets within the same channel.

[0206] Example 4: Design of primer sets and hybridization probes for detecting respiratory-associated pathogens

[0207] To verify the effectiveness of the combination of the present invention in detecting influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae, and novel coronavirus, primers and probes as shown in Table 4 below were designed.

[0208] Table 4

[0209]

[0210]

[0211] Among them, upstream primer 1' and downstream primer 1' are specific primers designed for the target sequence of influenza A virus; upstream primer 2' and downstream primer 2' are specific primers designed for the target sequence of influenza B virus; upstream primer 3' and downstream primer 3' are specific primers designed for the target sequence of respiratory syncytial virus; upstream primer 4' and downstream primer 4' are specific primers designed for the target sequence of adenovirus; upstream primer 5' and downstream primer 5' are specific primers designed for the target sequence of Mycoplasma pneumoniae; and upstream primer 6' and downstream primer 6' are specific primers designed for the target sequence of novel coronavirus.

[0212] The Tm values ​​of the hybridization probes are shown in Table 5 below.

[0213] Table 5

[0214]

[0215] Example 5: Single-sample detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0216] Sample preparation: In vitro transcribed RNA of each target sequence was used as a positive sample, and each target sequence was detected individually to verify the detection capability of a single virus infection. Pure water was used as a template-free control (NTC).

[0217] Reaction preparation:

[0218] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 6.

[0219] Table 6

[0220]

[0221]

[0222] PCR amplification and melting curve analysis:

[0223] After sealing the PCR tubes, gently mix the samples, briefly centrifuge, and then incubate at room temperature for 5 minutes. Place the PCR tubes again in a handheld centrifuge, briefly centrifuge, and then transfer to the tray of a quantitative PCR instrument (Suzhou Yarui, MA-6000). The program used was as follows: 50℃ reverse transcription for 10 minutes; 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 95℃ denaturation for 10 seconds, 56℃ annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94℃ denaturation for 10 seconds, 50℃ annealing for 120 seconds, and melting curve analysis from 50-90℃, with a heating rate of 0.03℃ per second.

[0224] When no target is present, the hybridization probe is in a single-stranded state. In this state, the distance between the fluorophore and the quencher is relatively short, resulting in high fluorescence resonance energy transfer efficiency. However, when the target is present, the upstream and downstream primers specifically bind to the target sequence and extend to generate an amplification template. This allows the hybridization probe to hybridize with the amplification template, causing a change in the distance between the fluorophore and the quencher, altering the fluorescence resonance energy transfer efficiency and thus changing the fluorescence signal, which can be detected by the instrument. After PCR amplification, during melting curve analysis, the hybridization probe hybridizing with the amplification product will unwind at a certain temperature and separate from the product chain. This changes the distance between the fluorophore and the quencher, altering the fluorescence resonance energy transfer efficiency again, leading to another change in the fluorescence signal, which can then be detected by the instrument and used for melting curve analysis.

[0225] Data Analysis:

[0226] The results are interpreted using the Tm value of the melting curve. Figure 10-15 This is a melting curve of positive respiratory virus samples in multiple target detection.

[0227] Depend on Figure 10-15 It can be seen that the PCR reaction wells with IBV positive template formed a melting peak with a Tm value of 59.46℃ in the FAM channel; the PCR reaction wells with IAV positive template formed a melting peak with a Tm value of 73.42℃ in the FAM channel; the PCR reaction wells with ADV positive template formed a melting peak with a Tm value of 64.49℃ in the ROX channel; the PCR reaction wells with RSV positive template formed a melting peak with a Tm value of 73.10℃ in the ROX channel; the PCR reaction wells with MP positive template formed a melting peak with a Tm value of 63.10℃ in the Cy5 channel; and the PCR reaction wells with SARS-CoV-2 positive template formed a melting peak with a Tm value of 74.48℃ in the Cy5 channel.

[0228] Example 6: Multiplex detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0229] Sample preparation: In vitro transcribed RNA from each target sequence was used as a positive sample. Two target sequence templates from the same fluorescent channel in Table 5 were mixed and detected to verify the ability to detect viral co-infection. TE buffer was used as a template-free control (NTC).

[0230] Reaction preparation:

[0231] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 6.

[0232] PCR amplification and melting curve analysis:

[0233] After sealing the PCR tubes, gently mix the samples, briefly centrifuge, and then incubate at room temperature for 5 minutes. Place the PCR tubes again in a handheld centrifuge, briefly centrifuge, and then transfer to the tray of a quantitative PCR instrument (Suzhou Yarui, MA-6000). The program used was as follows: 50℃ reverse transcription for 10 minutes; 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 95℃ denaturation for 10 seconds, 56℃ annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94℃ denaturation for 10 seconds, 50℃ annealing for 120 seconds, melting curve analysis from 50-90℃, with a heating rate of 0.03℃ per second.

[0234] Data Analysis:

[0235] The Tm value of the melting curve is used to interpret the results. Figure 16-18 This is a melting curve of positive respiratory virus samples in multiple target detection.

[0236] Depend on Figure 16-18 It was observed that PCR reaction wells containing both IBV and IAV positive templates simultaneously exhibited double melting peaks for IBV and IAV in the FAM channel; PCR reaction wells containing both ADV and RSV positive templates simultaneously exhibited double melting peaks for ADV and RSV simultaneously in the ROX channel; and PCR reaction wells containing both MP and SARS-CoV-2 positive templates simultaneously exhibited double melting peaks for MP and SARS-CoV-2 simultaneously in the Cy5 channel. Therefore, the combined detection of six respiratory-associated pathogens using this invention demonstrates good specificity, a clear melting curve morphology, and significant differentiation of the Tm values ​​of the two positive targets within the same channel.

[0237] Example 7: Single-sample detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0238] Sample preparation: In vitro transcribed RNA from each target sequence was used as a positive sample, and each target sequence was tested individually to verify the detection capability of a single virus infection. TE buffer was used as a template-free control (NTC).

[0239] Reaction preparation:

[0240] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 6.

[0241] PCR amplification and melting curve analysis:

[0242] After sealing the PCR tube, gently mix the sample, briefly centrifuge, and then incubate at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, briefly centrifuge, and then transfer to a real-time PCR instrument (Shanghai Hongshi). The sample was placed in a tray. The procedure used was as follows: 50℃ reverse transcription for 10 minutes; 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 95℃ denaturation for 10 seconds, 56℃ annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94℃ denaturation for 10 seconds, 50℃ annealing for 120 seconds, and melting curve analysis from 50 to 90℃, with a heating rate of 0.03℃ per second.

[0243] When no target is present, the hybridization probe is in a single-stranded state. In this state, the distance between the fluorophore and the quencher is relatively short, resulting in high fluorescence resonance energy transfer efficiency. However, when the target is present, the upstream and downstream primers specifically bind to the target sequence and extend to generate an amplification template. This allows the hybridization probe to hybridize with the amplification template, causing a change in the distance between the fluorophore and the quencher, altering the fluorescence resonance energy transfer efficiency and thus changing the fluorescence signal, which can be detected by the instrument. After PCR amplification, during melting curve analysis, the hybridization probe hybridizing with the amplification product will unwind at a certain temperature and separate from the product chain. This changes the distance between the fluorophore and the quencher, altering the fluorescence resonance energy transfer efficiency again, leading to another change in the fluorescence signal, which can then be detected by the instrument and used for melting curve analysis.

[0244] Data Analysis:

[0245] The results are interpreted using the Tm value of the melting curve. Figure 19-24 This is a melting curve of positive respiratory virus samples in multiple target detection.

[0246] Depend on Figure 19-24It can be seen that the PCR reaction wells with IBV positive template formed a melting peak with a Tm value of 60.82℃ in the FAM channel; the PCR reaction wells with IAV positive template formed a melting peak with a Tm value of 72.36℃ in the FAM channel; the PCR reaction wells with ADV positive template formed a melting peak with a Tm value of 63.94℃ in the ROX channel; the PCR reaction wells with RSV positive template formed a melting peak with a Tm value of 72.65℃ in the ROX channel; the PCR reaction wells with MP positive template formed a melting peak with a Tm value of 63.58℃ in the Cy5 channel; and the PCR reaction wells with SARS-CoV-2 positive template formed a melting peak with a Tm value of 74.18℃ in the Cy5 channel.

[0247] Example 8: Multiplex detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, mycoplasma pneumoniae, and novel coronavirus.

[0248] Sample preparation: In vitro transcribed RNA from each target sequence was used as a positive sample. Two target sequence templates from the same fluorescent channel in Table 5 were mixed and detected to verify the ability to detect viral co-infection. TE buffer was used as a template-free control (NTC).

[0249] Reaction preparation:

[0250] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 6.

[0251] PCR amplification and melting curve analysis:

[0252] After sealing the PCR tube, gently mix the sample, briefly centrifuge, and then incubate at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, briefly centrifuge, and then transfer to a real-time PCR instrument (Shanghai Hongshi). The sample was placed in a tray. The procedure used was as follows: 50°C, reverse transcription for 10 minutes; 95°C pre-denaturation for 3 minutes; 95°C denaturation for 10 seconds, 60°C annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 95°C denaturation for 10 seconds, 56°C annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94°C denaturation for 10 seconds, 50°C annealing for 120 seconds, melting curve analysis from 50°C to 90°C, with a heating rate of 0.03°C per second.

[0253] Data Analysis:

[0254] The Tm value of the melting curve is used to interpret the results. Figure 25-27 This is a melting curve of positive respiratory virus samples in multiple target detection.

[0255] Depend on Figure 25-27It was observed that in the FAM channel, wells containing IAV and IBV positive templates exhibited simultaneous double melting peaks for both IAV and IBV; in the ROX channel, wells containing ADV and RSV positive templates exhibited simultaneous double melting peaks for both ADV and RSV; and in the Cy5 channel, wells containing MP and SARS-CoV-2 positive templates exhibited simultaneous double melting peaks for both MP and SARS-CoV-2. Therefore, the combined detection of six respiratory-associated pathogens using this invention demonstrates good specificity, a clear melting curve morphology, and significant differentiation of the Tm values ​​of the two positive targets within the same channel.

[0256] Example 9: Multiplex detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, rhinovirus, mycoplasma pneumoniae, and novel coronavirus.

[0257] To verify the effectiveness of the combination of the present invention in detecting influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, rhinovirus (RhV), Mycoplasma pneumoniae, and novel coronavirus, primers and probes as shown in Table 7 were designed. Simultaneously, quality control was incorporated into the detection of respiratory-related pathogens to ensure the absence of systematic errors in the PCR process. The internal quality control consisted of a primer-probe pair designed targeting the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. This probe is a hydrolytic probe and therefore does not produce a melting curve.

[0258] Table 7

[0259]

[0260] The specific primers and probes for influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, and novel coronavirus are shown in Table 4; the downstream primers for Mycoplasma pneumoniae are also shown in Table 4. The remaining primers and probes are shown in Table 7. Among them, upstream primer 7' and downstream primer 7' are specific primers designed for rhinovirus target sequences; upstream primer 8', downstream primer 8', and hydrolysis probe 1 are specific primers and probes designed for the human glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH); upstream primer 9' is a specific primer designed for the Mycoplasma pneumoniae target sequence.

[0261] The Tm values ​​of the hybridization probes are shown in Table 8 below.

[0262] Table 8

[0263]

[0264]

[0265] Sample preparation: In vitro transcribed RNA of each target sequence was used as a positive sample, and each target sequence was detected individually to verify the detection capability of a single virus infection. Pure water was used as a template-free control (NTC).

[0266] Reaction preparation:

[0267] After the sample preparation is completed, the reaction system is prepared according to the ratios described in Table 9.

[0268] Table 9

[0269]

[0270] PCR amplification and melting curve analysis:

[0271] After sealing the PCR tube, gently mix the sample, briefly centrifuge, and then incubate at room temperature for 5 minutes. Place the PCR tube in a handheld centrifuge again, briefly centrifuge, and then transfer to a real-time PCR instrument (Shanghai Hongshi). The sample was placed in a tray. The procedure used was as follows: 50°C, reverse transcription for 10 minutes; 95°C pre-denaturation for 3 minutes; 95°C denaturation for 10 seconds, 60°C annealing and extension for 15 seconds, for a total of 10 cycles, without light exposure; 95°C denaturation for 10 seconds, 56°C annealing and extension for 15 seconds, for a total of 40 cycles, with light exposure; 94°C denaturation for 10 seconds, 50°C annealing for 120 seconds, melting curve analysis from 50°C to 90°C, with a heating rate of 0.03°C per second.

[0272] Data Analysis:

[0273] The Tm value of the melting curve is used to interpret the results. Figure 28-35 This is a melting curve of positive respiratory virus samples in multiple target detection.

[0274] Depend on Figure 28-35It can be seen that the PCR reaction wells with IBV positive template formed a melting peak with a Tm value of 61.27℃ in the FAM channel; the PCR reaction wells with IAV positive template formed a melting peak with a Tm value of 72.54℃ in the FAM channel; the PCR reaction wells with ADV positive template formed a melting peak with a Tm value of 65.62℃ in the ROX channel; the PCR reaction wells with RSV positive template formed a melting peak with a Tm value of 72.55℃ in the ROX channel; the PCR reaction wells with RhV positive template formed a melting peak with a Tm value of 62.47℃ in the Cy5 channel; the PCR reaction wells with MP positive template formed a melting peak with a Tm value of 68.47℃ in the Cy5 channel; and the PCR reaction wells with SARS-CoV-2 positive template formed a melting peak with a Tm value of 74.75℃ in the Cy5 channel. PCR reaction wells containing RhV, MP, and SARS-CoV-2 positive templates simultaneously exhibited three melting peaks for RhV, MP, and SARS-CoV-2 in the Cy5 channel. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 36 It can be seen that the reaction wells with added GAPDH positive template show a clear S-shaped amplification curve (upper side) in the VIC channel, but no melting peak (lower side) appears. sequence list <110> Mindray Bio-Medical Engineering Co., Ltd. <120> Multiplex nucleic acid detection methods, combinations and kits <130> 2020 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 acggatcaca gcgtaaagc 19 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 acggatcaca cacgatgaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 ccgtctcgca ttaacatatt 20 <210> 4 <211> 40 <212> DNA <213> Artificial sequence <400> 4 acggatcaca gcgtaaagct gcaacatgac caaggactgg 40 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <400> 5 atgtagaagc cctggtagc 19 <210> 6 <211> 39 <212> DNA <213> Artificial sequence <400> 6 acggatcaca gcgtaaagcc taagcagact cacagaact 39 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 atgagaactt gccataggtt 20 <210> 8 <211> 39 <212> DNA <213> Artificial sequence <400> 8 acggatcaca cacgatgaca taactcaagg cggacgatt 39 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <400> 9 ttaaccacgg cgttcagg 18 <210> 10 <211> 38 <212> DNA <213> Artificial sequence <400> 10 acggatcaca cacgatgaca tggacaacag cagacaac 38 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <400> 11 tccacacgta caaggtatct 20 <210> 12 <211> 40 <212> DNA <213> Artificial sequence <400> 12 ccgtctcgca ttaacatatt ataatcaggc acgagaacag 40 <210> 13 <211> 19 <212> DNA <213> Artificial sequence <400> 13 gactagcaac ctccatggc 19 <210> 14 <211> 42 <212> DNA <213> Artificial sequence <400> 14 ccgtctcgca ttaacatatt aagcaaccaa caacattcat tg 42 <210> 15 <211> 19 <212> DNA <213> Artificial sequence <400> 15 gcaattcagc atcacagac 19 <210> 16 <211> 41 <212> DNA <213> Artificial sequence <400> 16 ctccaccact tactgattat ctcaaagccg agatcgcaca g 41 <210> 17 <211> twenty three <212> DNA <213> Artificial sequence <400> 17 tccccttagt cagaggtgac agg 23 <210> 18 <211> 42 <212> DNA <213> Artificial sequence <400> 18 ctccacgact tacagattac ttgaagggtt tgagccatac tg 42 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <400> 19 ccctggggtt gaagggtaat c 21 <210> 20 <211> 45 <212> DNA <213> Artificial sequence <400> 20 acgacctatg aacgctctct cattaagcaa gaaaaggaaa agaag 45 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <400> twenty one tgcagatcca acacctaaca aa 22 <210> twenty two <211> 45 <212> DNA <213> Artificial sequence <400> twenty two acgacctatg cacgcactct ccaacctatg agcaggcagg tggtt 45 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <400> 23 tggtaaggtg acggctttgt agt 23 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <400> 24 caactaacca ggatcaacga gcaccccaac agtgaaacga 40 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 ggcacgagta aaacggcaaa 20 <210> 26 <211> 41 <212> DNA <213> Artificial Sequence <400> 26 caaccaacca gcatcaacat gaccgaaagg taagatggag a 41 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <400> 27 acaggcaaac tgagttggac g 21 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <400> 28 ctccaccact tactgatt 18 <210> 29 <211> 19 <212> DNA <213> Artificial sequence <400> 29 acgacctatg aacgctctc 19 <210> 30 <211> 18 <212> DNA <213> Artificial sequence <400> 30 caaccaacca gcatcaac 18 <210> 31 <211> 43 <212> DNA <213> Artificial sequence <400> 31 caaccaacca gcatgaaccg cttgaatatg ttgatatgct cca 43 <210> 32 <211> twenty three <212> DNA <213> Artificial sequence <400> 32 gcggataacg gtatctgttg ttt 23 <210> 33 <211> twenty one <212> DNA <213> Artificial sequence <400> 33 ggagcgagat ccctccaaaa t 21 <210> 34 <211> twenty three <212> DNA <213> Artificial sequence <400> 34 ggctgttgtc atacttctca tgg 23 <210> 35 <211> twenty one <212> DNA <213> Artificial sequence <400> 35 gcagggggga gccaaaaggg t 21 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <400> 36 caactaacca gcatcaacga gcaccccaac agtgaaacga 40

Claims

1. A method for multiplex nucleic acid detection, comprising the following steps: The combination containing the first primer set and the first oligonucleotide was mixed with a sample from the subject; Amplify any target sequences that may be present in the sample; Melting curve analysis of the amplified product was performed in the corresponding detection channel; and Based on the results of the melting curve analysis, determine whether one or more of the targets exist. The first primer set includes upstream and downstream primers for a first target, and upstream and downstream primers for a second target. The upstream or downstream primer of the first target consists of a target sequence binding region and a first signal detection region located upstream of it. The upstream or downstream primer of the second target consists of a target sequence binding region and a second signal detection region located upstream of it. The first and second signal detection regions are designed not to be complementary to the target sequence. Furthermore, the entire sequence of the first oligonucleotide is identical to that of the first and second signal detection regions, thereby enabling it to form a double-stranded structure with the complementary sequences of the first and second signal detection regions, respectively. The first oligonucleotide carries a first detection group and a second detection group; upon integration into the double-stranded structure, the first and second detection groups can thus generate signal changes. The first oligonucleotide is selected from the group consisting of: oligonucleotides that are coiled due to molecular flexibility in the single-stranded state, oligonucleotides that can form hairpin structures in the single-stranded state, oligonucleotides that can form stem-loop structures in the single-stranded state, oligonucleotides that can form pseudo-knot structures in the single-stranded state, and oligonucleotides that can form triple helix structures in the single-stranded state; and The first signal detection region and the second signal detection region are each independently separated from the target sequence binding region on the primer they are located in by 0-40 bases; The first signal detection area is the same as the second signal detection area.

2. The method according to claim 1, wherein, The combination further includes a second set of primers and a second oligonucleotide. The second primer set includes upstream and downstream primers for a third target, and upstream and downstream primers for a fourth target. The upstream or downstream primer of the third target consists of a target sequence binding region and a third signal detection region located upstream of it; the upstream or downstream primer of the fourth target consists of a target sequence binding region and a fourth signal detection region located upstream of it. The third and fourth signal detection regions are designed not to be complementary to the target sequence. Furthermore, all or part of the sequence of the second oligonucleotide is the same as the third and fourth signal detection regions, thereby enabling it to form a double-stranded structure with the complementary sequences of the third and fourth signal detection regions, respectively; the second oligonucleotide carries a third detection group and a fourth detection group, and after integration into the double-stranded structure, the third detection group and the fourth detection group can generate a signal change, and the signal generated by the second oligonucleotide corresponds to a different detection channel than the signal generated by the first oligonucleotide.

3. The method according to claim 2, wherein, The second oligonucleotide is selected from the group consisting of: oligonucleotides that are coiled due to molecular flexibility in the single-stranded state, oligonucleotides that can form hairpin structures in the single-stranded state, oligonucleotides that can form stem-loop structures in the single-stranded state, oligonucleotides that can form pseudo-knot structures in the single-stranded state, and oligonucleotides that can form triple helix structures in the single-stranded state.

4. The method according to any one of claims 1-3, wherein, The first detection group and the second detection group are 5-25 bases apart and are not located at the 3' end.

5. The method according to claim 2 or 3, wherein, The first and second detection groups are 5-25 bases apart and are not located at the 3' end; and the third and fourth detection groups are 5-25 bases apart and are not located at the 3' end.

6. The method according to claim 4, wherein, The first oligonucleotide can amplify the amplification product of the first primer set, but cannot amplify the amplification product of other primer sets.

7. The method according to claim 5, wherein, The first oligonucleotide can amplify the amplification product of the first primer set, but cannot amplify the amplification product of other primer sets; the second oligonucleotide can amplify the amplification product of the second primer set, but cannot amplify the amplification product of other primer sets.

8. The method according to claim 2 or 3, wherein, The third and fourth signal detection regions are each independently separated from the target sequence binding region on the primer by 0-40 bases.

9. The method according to claim 1, wherein, The interval is designed such that different amplification products of the first oligonucleotide have different melting temperatures.

10. The method according to claim 8, wherein, The intervals are designed such that different amplification products of the first oligonucleotide or the second oligonucleotide have different melting temperatures.

11. A combination for multiplex nucleic acid detection, comprising: The first primer set includes an upstream primer and a downstream primer for a first target, and an upstream primer and a downstream primer for a second target; and A first oligonucleotide, the entire sequence of which is identical to the first and second signal detection regions, and capable of forming a double-stranded structure with the complementary sequences of the first and second signal detection regions, respectively; the first oligonucleotide carries a first detection group and a second detection group, which can generate a signal change after integration into the double-stranded structure. The first signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the first target, and the second signal detection region is located upstream of the target sequence binding region of the upstream or downstream primer for the second target; the first and second signal detection regions are designed not to pair complementaryly with the target sequence. The first oligonucleotide is selected from the group consisting of: oligonucleotides that are coiled due to molecular flexibility in the single-stranded state, oligonucleotides that can form hairpin structures in the single-stranded state, oligonucleotides that can form stem-loop structures in the single-stranded state, oligonucleotides that can form pseudo-knot structures in the single-stranded state, and oligonucleotides that can form triple helix structures in the single-stranded state; and The first signal detection region and the second signal detection region are each independently separated from the target sequence binding region on the primer they are located in by 0-40 bases; The first signal detection area is the same as the second signal detection area.

12. The combination according to claim 11, wherein, The combination further includes a second set of primers and a second oligonucleotide. The second primer set includes upstream and downstream primers for a third target, and upstream and downstream primers for a fourth target. The upstream or downstream primer of the third target consists of a target sequence binding region and a third signal detection region located upstream of it; the upstream or downstream primer of the fourth target consists of a target sequence binding region and a fourth signal detection region located upstream of it. The third and fourth signal detection regions are designed not to be complementary to the target sequence. Furthermore, all or part of the sequence of the second oligonucleotide is the same as the third and fourth signal detection regions, and can form a double-stranded structure with the complementary sequences of the third and fourth signal detection regions, respectively; the second oligonucleotide carries a third detection group and a fourth detection group, which can generate a detectable signal change after integration into the double-stranded structure; and the signal generated by the second oligonucleotide is of a different type than the signal generated by the first oligonucleotide.

13. The combination according to claim 12, wherein, The second oligonucleotide is selected from the group consisting of: oligonucleotides that are coiled due to molecular flexibility in the single-stranded state, oligonucleotides that can form hairpin structures in the single-stranded state, oligonucleotides that can form stem-loop structures in the single-stranded state, oligonucleotides that can form pseudo-knot structures in the single-stranded state, and oligonucleotides that can form triple helix structures in the single-stranded state.

14. The combination according to any one of claims 11-13, wherein, The first oligonucleotide can amplify the amplification product of the first primer set, but cannot amplify the amplification product of other primer sets.

15. The combination according to claim 12 or 13, wherein, The first oligonucleotide can amplify the amplification product of the first primer set, but cannot amplify the amplification product of other primer sets; the second oligonucleotide can amplify the amplification product of the second primer set, but cannot amplify the amplification product of other primer sets.

16. A kit comprising a primer set and oligonucleotides as defined in any one of claims 1-10.

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