CN110273013B

CN110273013B - Method for detecting respiratory tract pathogen

Info

Publication number: CN110273013B
Application number: CN201810204483.5A
Authority: CN
Inventors: 廖逸群; 李庆阁; 许晔; 许海坡; 林明芳
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2018-03-13
Filing date: 2018-03-13
Publication date: 2022-11-01
Anticipated expiration: 2038-03-13
Also published as: CN110273013A

Abstract

The present application provides a method of detecting a smoking tract pathogen that is capable of simultaneously detecting the presence or level of multiple smoking tract pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract) in a sample. In addition, provided herein are a set of probes and kits comprising one or more of the same, which can be used to practice the methods of the invention. In addition, the present application provides a kit that is capable of simultaneously detecting the presence or level of multiple respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract) in a sample in one round of reaction.

Description

Method for detecting respiratory pathogens

Technical Field

The present application relates to multiplex detection of nucleic acid molecules. In particular, the present application provides a method of detecting respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract) that can simultaneously detect the presence or level of multiple (e.g., 2,5, 10, 15, 19, or more) gut pathogens in a sample. In addition, provided herein are probe sets, and kits comprising one or more of the probe sets, which can be used to practice the methods of the invention. In addition, the present application provides a kit that is capable of simultaneously detecting the presence or level of multiple (e.g., 2,5, 10, 15, 19 or more) respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract) in a sample in a single round of reaction.

Background

Respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract) are of a wide variety and are difficult to identify by conventional methods. Currently, methods for detecting bacteria and fungi that infect the respiratory tract mainly include culture identification, immunoserological analysis, and nucleic acid detection. Compared with culture identification and immune serological analysis, the nucleic acid detection has the obvious advantages of rapid detection, simple and convenient operation, good specificity and the like. Therefore, a large number of documents and patents at home and abroad have reported that the species of bacteria and fungi infecting the respiratory tract are identified by a nucleic acid detection method. On the basis, most nucleic acid detection methods adopt a multiplex PCR amplification technology to improve the efficiency and sensitivity of the detection method. Real-time PCR is a common method for detecting nucleic acid, and has simple operation and wide application. And, by using multiple real-time PCR, a plurality of target sequences can be simultaneously detected in a single reaction tube, which not only improves the detection efficiency, but also reduces the cost.

In real-time PCR methods, target sequences can be detected by using fluorescently labeled oligonucleotide probes. In general, a fluorescently labeled probe specifically binds to a target sequence between two primers used for PCR amplification to avoid interfering signals caused by non-specific amplification of primer dimers, improving the specificity of the detection result. For multiplex real-time PCR, when multiple oligonucleotide probes specific for a target sequence are used, each oligonucleotide probe can be labeled with a different fluorophore, whereby the target sequence specifically recognized by each probe can be detected by detecting the unique fluorescent signal carried by each probe. In the real-time PCR method, the target sequence can be detected by two modes, namely, a real-time detection mode and a post-amplification melting curve analysis (also referred to as post-PCR MCA mode). In the real-time detection mode, the detection of the target sequence and the PCR amplification are performed simultaneously, without performing additional steps. Therefore, the real-time detection mode is simple and direct. However, the maximum number of target sequences that can be detected in this mode in a single round of detection is limited by the number of fluorescent detection channels of the real-time PCR instrument, and generally does not exceed 6. In the MCA mode, an additional step after PCR amplification is required, namely analysis of the melting point of the duplex formed by the probe and the target sequence. The MCA pattern may identify or distinguish target sequences by fluorescent color and/or melting point. Thus, the MCA model, while relatively cumbersome (i.e., adds an additional step), increases the number of maximal target sequences that can be detected in a single round of detection.

However, multiplex real-time PCR based on fluorescent probe detection also presents some problems. First, the preparation of fluorescent probes involves complex chemical modification and purification processes, which are much more costly than non-labeled probes. Therefore, the use of a plurality of fluorescent probes leads to an increase in detection cost. Secondly, in the multiplex real-time PCR method, the coexistence of a plurality of fluorescent probes increases the background fluorescence of the reaction system, which in turn may cause a decrease in detection sensitivity. Therefore, there is a need for improvements in multiplex real-time PCR methods in order to detect as many target sequences as possible with as few fluorescent probes as possible.

Faltin et al (Clinical Chemistry 2012,58 (11): 1546-1556) describe a real-time PCR detection method based on "mediator probes". In the conventional real-time PCR detection method, a fluorescent probe specific to each target sequence is used. However, in the method reported by Faltin et al, two probes need to be used for each target sequence: one mediator probe specific for the target sequence that is not fluorescently labeled, and another fluorescently labeled probe (fluorescent probe) that does not bind to the target sequence; wherein the mediator probe consists of a target specific sequence at the 3 '-end and a tag sequence (mediator) at the 5' -end; the fluorescent probe is composed of a 3 '-end single-chain sequence containing a mediator hybridization site and a 5' -end sequence which contains a quenching group and a fluorescent group and has a hairpin structure, wherein the quenching group and the fluorescent group are both positioned on the arms of the hairpin structure and are close to each other, so that fluorescence quenching occurs. During PCR, the mediator probe is bound to the target sequence through the target-specific sequence, and the tag sequence (mediator) at its 5' -end remains in a single-stranded free state; subsequently, the mediator probe is subjected to enzyme digestion under the action of DNA polymerase with 5 '-nuclease activity, and the mediator with 3' -OH is released. Subsequently, the released mediator binds to the mediator hybridization site in the fluorescent probe and is extended by polymerase, resulting in the cleavage or displacement of the sequence labeled with the quencher, which in turn causes the separation of the fluorophore and the quencher, resulting in an increase in fluorescence intensity.

The method described by Faltin et al is characterized in that the generation of the fluorescence signal is dependent on two probes: a mediator probe and a fluorescent probe; wherein the mediator probe is used as a hybridization probe and is not fluorescently labeled per se; the fluorescent probe is used to generate a fluorescent signal that specifically binds to the mediator, but not to the target sequence. In this method, a fluorescent probe can be used as a universal probe. For example, when detecting multiple target sequences using single real-time PCR, PCR reactions can be performed separately using multiple mediator probes, each carrying a unique target-specific sequence but containing the same mediator sequence, and one and the same fluorescent probe. In addition, for multiplex real-time PCR, multiple mediator sub-probes and one fluorescent probe with the same mediator sub-sequence can also be used to achieve screening of multiple target sequences when there is no need to identify or distinguish each target sequence. Compared with the conventional real-time PCR method, the method of Faltin et al can use a common fluorescent probe to detect a plurality of target sequences without synthesizing a unique fluorescent probe for each target sequence, which significantly reduces the detection cost.

However, the Faltin et al approach also suffers from significant drawbacks. In particular, when the method of Faltin et al is used to perform multiplex real-time PCR where each target sequence needs to be distinguished, it is necessary to design a mediator probe carrying a target-specific sequence and a corresponding fluorescent probe carrying a unique fluorescent signal for each target sequence. In this case, the Faltin et al method requires the use of double the number of probes, as compared to conventional multiplex real-time PCR using a single probe for each target sequence. Accordingly, the whole reaction system becomes more complicated and the detection cost becomes higher. For example, faltin et al disclose a dual PCR method for simultaneously detecting HPV18 and the human ACTB gene, in which 2 mediator probes and 2 fluorescent probes are used; in contrast, the conventional dual real-time PCR method requires only 2 fluorescent probes. Similar examples are also found in Wadle S et al (Biotechniques 2016,61 (3): 123-8), which describes a quintuple PCR system using a total of 5 mediator probes and 5 fluorescent reporter probes. In contrast, the conventional quintuple real-time PCR method requires only 5 fluorescent probes. In this case, the Faltin et al method is more complicated and costly than the conventional multiplex real-time PCR.

US 2013/0109588 A1 discloses a real-time PCR assay useful for melting curve analysis, which, like the method of fantin et al, achieves detection of a target sequence by means of two probes (PTO probe, which corresponds to a mediator probe; and CTO probe, which corresponds to a fluorescent probe). Accordingly, the method of US 2013/0109588 A1 has similar advantages and disadvantages as the method of fantin et al. In particular, when the method described in this patent application is used to perform multiplex real-time PCR, which requires distinguishing each target sequence, it is necessary to design one PTO probe and one CTO probe for each target sequence, respectively; i.e., a double number of probes is used. For example, this patent application describes a dual real-time PCR for simultaneous detection of Neisseria gonorrhoeae and Staphylococcus aureus, using 2 PTO probes and 2 CTO probes. In this case, the method of US 2013/0109588 A1 is more complicated and costly than the conventional multiplex real-time PCR using a single probe for each target sequence.

US 2014/0057264 A1 discloses another real-time PCR method using two probes. In this method, the fluorescence signal is generated by cleavage of the labeled probe, and therefore, this method can be used only in the real-time detection mode, but not in the MCA mode. Furthermore, similar to the method of Faltin et al, when the method described in US 2014/0057264 A1 is used to perform multiplex real-time PCR requiring discrimination of each target sequence, two probes need to be designed for each target sequence separately, which results in a more complicated reaction system and high cost.

US 2015/0072887 A1 discloses a real-time PCR assay useful for melting curve analysis, which enables detection of target sequences through 3 probes. However, when the method described in this patent application is used to perform multiplex real-time PCR requiring discrimination of each target sequence, 3 probes need to be designed for each target sequence separately, which results in a more complicated reaction system and high cost. Similar real-time PCR assays using 3 probes are also disclosed in US 2015/0167060 A1 and US 2016/0060690 A1. However, these methods are similar to the methods disclosed in US 2015/0072887 A1, and are more complex and costly than the conventional real-time PCR method when used to perform multiplex real-time PCR that requires distinguishing each target sequence.

In general, improved real-time PCR methods using two or three probes (e.g., fantin et al) have significant advantages over traditional real-time PCR methods when performing single real-time PCR or multiplex real-time PCR without the need to identify or distinguish between each target sequence: that is, multiple mediator probes carrying the same mediator sequence but different target-specific sequences and a common fluorescent probe can be used, thereby significantly reducing the detection cost. However, when performing multiplex real-time PCR, which requires the differentiation of each target sequence, these improved real-time PCR methods require the use of double or even triple probes, which are more complicated and costly than conventional real-time PCR methods.

In the present application, the inventors developed a novel real-time PCR assay method that can distinguish and identify each target sequence in a sample with a simpler reaction system and at a lower detection cost. Based on this, the inventors of the present application developed a more rapid, simple, sensitive, specific, stable, reliable, high-throughput nucleic acid detection method and kit that can be used to detect a variety of respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract).

Disclosure of Invention

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the nucleic acid chemistry laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

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 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 term "mediator probe" refers to a single-stranded nucleic acid molecule containing a mediator sequence and a targeting sequence (i.e., target-specific sequences) in the 5 'to 3' direction. In the present application, the mediator sequence does not contain a sequence complementary to the target nucleic acid sequence, and the target-specific sequence contains a sequence complementary to the target nucleic acid sequence. Thus, under conditions that allow nucleic acid hybridization, annealing, or amplification, the mediator sub-probe hybridizes or anneals to a target nucleic acid sequence through the target-specific sequence (i.e., forms a double-stranded structure), and the mediator sub-sequence in the mediator sub-probe does not hybridize to the target nucleic acid sequence but is in an episomal state (i.e., maintains a single-stranded structure).

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, the targeting or target-specific sequence hybridizes or anneals only to a particular target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.

As used herein, the term "mediator sequence" refers to a stretch of oligonucleotide sequence in the mediator probe that is not complementary to the target nucleic acid sequence, which is located upstream (5' to) the target-specific sequence. In the present application, a unique mediator probe having a unique mediator subsequence (in other words, the mediator subsequences in all the mediator probes used are different from each other) is designed or provided for each target nucleic acid sequence; thus, each target nucleic acid sequence corresponds to a unique mediator probe (unique mediator sequence). Thus, by detecting the unique mediator sequence, the target nucleic acid sequence corresponding thereto can be detected.

As used herein, the term "upstream oligonucleotide 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 mediator probe.

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. For example, in the present application, the target-specific sequences in the upstream oligonucleotide sequence and the mediator probe each comprise a sequence that is complementary (e.g., substantially complementary or fully complementary) to the target nucleic acid sequence. Thus, the target-specific sequences in the upstream oligonucleotide sequences and mediator probes will selectively/specifically hybridize or anneal to the target nucleic acid sequences under conditions that allow for nucleic acid hybridization, annealing, or amplification. 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. For example, in the present application, the mediator sequence includes a sequence that is not complementary to the target nucleic acid sequence. Thus, under conditions that allow nucleic acid hybridization, annealing, or amplification, the mediator sequence does not hybridize or anneal to the target nucleic acid sequence, cannot form a duplex, but is in a free state (i.e., remains a single-stranded structure).

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, low stringency hybridization conditions can be used when the length of the two nucleic acid molecules that are complementary is relatively short and/or the GC content is relatively low. High stringency hybridization conditions can be used when the two nucleic acid molecules that are complementary are relatively long in length and/or relatively high in GC content. 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 for nucleic acid amplification" has the meaning commonly understood by those skilled in the art and refers to conditions that allow a nucleic acid polymerase (e.g., a DNA polymerase) to synthesize one nucleic acid strand as a template for another nucleic acid strand 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 the working conditions of a nucleic acid polymerase (e.g., a DNA polymerase).

As used herein, the expression "conditions that allow a nucleic acid polymerase to perform an extension reaction" 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 extend one nucleic acid strand as a template for another nucleic acid strand (e.g., a primer or a probe), 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, and ionic strength of the hybridization buffer, and the like. 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 "condition that allows the nucleic acid polymerase to perform the extension reaction" is preferably a working condition of the nucleic acid polymerase (e.g., DNA polymerase). In the present application, the expressions "conditions allowing a nucleic acid polymerase to perform an extension reaction" and "conditions allowing nucleic acid extension" have the same meaning and are used interchangeably.

As used herein, the expression "conditions which allow cleavage of the mediator probe" refers to conditions which allow an enzyme having 5' nuclease activity to cleave the mediator probe hybridized to the target nucleic acid sequence and release a nucleic acid fragment comprising the mediator sequence or a portion thereof. In the method of the invention, the conditions which allow cleavage of the mediator probe are preferably working conditions for an enzyme having 5' nuclease activity. For example, when the enzyme having 5 'nuclease activity used is a nucleic acid polymerase having 5' nuclease activity, the conditions that allow cleavage of the mediator probe may be the operating conditions of the nucleic acid polymerase.

The working conditions for the various enzymes can be determined by the person skilled in the art by conventional methods and can generally involve the following factors: temperature, pH of the buffer, composition, concentration, ionic strength, and the like. Alternatively, conditions recommended by the manufacturer of the enzyme may be used.

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 "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 a 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".

As used herein, the term "fluorescent probe" refers to a piece of oligonucleotide that carries a fluorophore and is capable of generating a fluorescent signal. In the present application, a fluorescent probe is used as a detection probe.

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, by performing derivative analysis on the obtained curve, a curve (i.e., melting curve of the duplex) is obtained with the rate of change of signal intensity as ordinate and the temperature as abscissa. The peak in the melting curve is the melting peak and the corresponding temperature is the melting point (T) of the duplex_mA value). 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 bisT of chain body_mThe presence and identity of a 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.

In the present application, the inventors developed a novel real-time PCR assay method that can distinguish and identify a plurality of target sequences in a sample with a simpler reaction system and at a lower detection cost. On the basis of the method, the inventor of the application develops a method and a kit which are more rapid, simple, sensitive, specific, stable and reliable and can simultaneously detect a plurality of respiratory pathogens (such as bacteria, chlamydia, mycoplasma, rickettsia and fungi which can infect respiratory tracts). For example, the methods and kits of the invention can simultaneously detect 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract).

Detection method

Accordingly, in one aspect, the present invention provides a method of detecting the presence of at least two targets in a sample, wherein the at least two targets are each independently a respiratory pathogen and the respiratory pathogen is selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract, and the method comprises the steps of:

(1) Contacting the sample with a first upstream oligonucleotide sequence, a first mediator probe, a second upstream oligonucleotide sequence, and a second mediator probe under conditions that allow nucleic acid hybridization,

(i) The first upstream oligonucleotide sequence comprises a sequence complementary to a first target nucleic acid sequence; and, the first mediator probe comprises, in the 5 'to 3' direction, a first mediator subsequence comprising a sequence that is not complementary to the first target nucleic acid sequence and a first target-specific sequence comprising a sequence that is complementary to the first target nucleic acid sequence; and, when hybridized to the first target nucleic acid sequence, the first upstream oligonucleotide sequence is located upstream of the first target-specific sequence; wherein the first target nucleic acid sequence is specific for a first target, preferably a genomic sequence of a first respiratory pathogen, or a specific fragment thereof;

(ii) The second upstream oligonucleotide sequence comprises a sequence complementary to a second target nucleic acid sequence; and, the second mediator sub-probe comprises in the 5 'to 3' direction a second mediator sub-sequence and a second target-specific sequence, wherein the second mediator sub-sequence comprises a sequence that is not complementary to a second target nucleic acid sequence, and the second target-specific sequence comprises a sequence that is complementary to the second target nucleic acid sequence; and, when hybridized to a second target nucleic acid sequence, a second upstream oligonucleotide sequence is located upstream of the second target-specific sequence; wherein the second target nucleic acid sequence is specific for a second target, preferably a genomic sequence of a second respiratory pathogen, or a specific fragment thereof; and the number of the first and second electrodes,

(iii) The first intermediate subsequence is different from the second intermediate subsequence; and the number of the first and second electrodes,

(2) Contacting the product of step (1) with an enzyme having 5' nuclease activity under conditions that allow cleavage of the mediator probe;

(3) Contacting the product of step (2) with a detection probe comprising, in the 3 'to 5' direction, a first capture sequence complementary to the first mediator sequence or a portion thereof, a second capture sequence complementary to the second mediator sequence or a portion thereof, and a template sequence (templating sequence) under conditions permitting nucleic acid hybridization; and the number of the first and second electrodes,

the detection probe is marked with a reporter group and a quenching group, wherein the reporter group can emit signals, and the quenching group can absorb or quench the signals emitted by the reporter group; 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;

(4) Contacting the product of step (3) with a nucleic acid polymerase under conditions which allow the nucleic acid polymerase to undergo an extension reaction;

(5) Analyzing a melting curve of the product obtained in the step (4); and determining whether the first and second target nucleic acid sequences are present in the sample based on the results of the melting curve analysis, thereby determining whether the first and second targets are present in the sample.

In the methods of the invention, since the first target nucleic acid sequence is specific for the first target and the second target nucleic acid sequence is specific for the second target, respectively, the presence of the first target and the second target can be determined by detecting the presence of the first target nucleic acid sequence and the second target nucleic acid sequence.

As used herein, the expression "a target nucleic acid sequence is specific for a target (e.g., a respiratory pathogen)" means that the target nucleic acid sequence is unique to the target (e.g., the respiratory pathogen) and is not present in other targets (e.g., a host of the respiratory pathogen or other pathogens). In other words, the target nucleic acid sequence is only detectable in the target (e.g., the respiratory pathogen), and thus the presence of the target nucleic acid sequence is indicative of the presence of the target, and vice versa. A typical example of such a target nucleic acid sequence includes, but is not limited to, the genomic sequence of the pathogen or a specific fragment thereof. As used herein, the expression "specific fragment of a genomic sequence of a pathogen" has a similar meaning, i.e. the fragment is characteristic of the pathogen or its genome. Such specific fragments can be non-coding sequences (e.g., sequences that do not encode any RNA or protein), or coding sequences (e.g., sequences capable of being transcribed or translated), or a combination of both, so long as they are pathogen-specific.

Whether a nucleic acid sequence or a fragment is target-specific can be determined by various well-known methods. For example, it may be determined whether the nucleic acid sequence is specific to a target by performing a Blast search of the nucleic acid sequence in a public database (e.g., the NCBI database).

In step (1) of the method of the invention, since the first upstream oligonucleotide sequence comprises a sequence complementary to the first target nucleic acid sequence and the first target-specific sequence comprises a sequence complementary to the first target nucleic acid sequence, both the first upstream oligonucleotide sequence and the first mediator probe hybridize to the first target nucleic acid sequence when present. Similarly, since the second upstream oligonucleotide sequence comprises a sequence complementary to a second target nucleic acid sequence and the second target-specific sequence comprises a sequence complementary to the second target nucleic acid sequence, both the second upstream oligonucleotide sequence and the second mediator probe hybridize to the second target nucleic acid sequence when present.

In step (2) of the method of the invention, the first upstream oligonucleotide sequence and the first mediator probe both hybridize to a first target nucleic acid sequence when present. Further, since the first mediator subsequence comprises a sequence that is not complementary to the first target nucleic acid sequence, the first mediator subsequence in the first mediator probe is in an episomal state and does not hybridize to the first target nucleic acid sequence. In this case, under the action of the enzyme having 5' nuclease activity, the first mediator sequence or a portion thereof is cleaved from the first mediator probe hybridized with the first target nucleic acid sequence by the presence of the first upstream oligonucleotide sequence or an extension product thereof, to form a first mediator fragment. Similarly, when the second target nucleic acid sequence is present, both the second upstream oligonucleotide sequence and the second mediator probe hybridize to the second target nucleic acid sequence, and the second mediator sequence in the second mediator probe is in a free state and does not hybridize to the second target nucleic acid sequence. In this case, the second mediator sequence or a portion thereof is cleaved from the second mediator probe hybridized to the second target nucleic acid sequence by the presence of the second upstream oligonucleotide sequence or its extension product under the action of the enzyme having 5' nuclease activity to form a second mediator fragment.

In step (3) of the method of the invention, when the first mediator fragment is present, the first mediator fragment hybridises to the detection probe in that the first mediator fragment comprises a first mediator subsequence or portion thereof and the detection probe comprises a first capture sequence complementary to the first mediator subsequence or portion thereof. Similarly, when a second mediator segment is present, it hybridizes to the detection probe as it comprises a second mediator subsequence, or portion thereof, and the detection probe comprises a second capture sequence that is complementary to the second mediator subsequence, or portion thereof.

In step (4) of the method of the invention, when a first mediator fragment is present, the nucleic acid polymerase will extend the first mediator fragment, using the detection probe as a template, to form a first duplex, since the first mediator fragment hybridises to the detection probe and the detection probe comprises an additional sequence (e.g. a template sequence). Similarly, when a second mediator segment is present, the nucleic acid polymerase will extend the second mediator segment to form a second duplex using the detection probe as a template, since the second mediator segment hybridizes to the detection probe and the detection probe comprises additional sequences (e.g., a template sequence).

In step (5) of the method of the present invention, when the first duplex is present, a melting peak corresponding to the first duplex can be detected. Thus, the presence or absence of the first target nucleic acid sequence in the sample can be determined by the presence or absence of a melting peak corresponding to the first duplex. For example, determining the presence or absence of a first target nucleic acid sequence in the sample when a melting peak corresponding to the first duplex is detected or not detected; further, since the first target nucleic acid sequence is specific for the first target, the presence or absence of the first target in the sample can be determined. Similarly, the presence or absence of the second target nucleic acid sequence in the sample can be determined by the presence or absence of a melting peak corresponding to the second duplex. For example, determining the presence or absence of a second target nucleic acid sequence in the sample when a melting peak corresponding to a second duplex is detected or not detected; further, since the second target nucleic acid sequence is specific for the second target, the presence or absence of the second target in the sample can be determined.

In particular, in the method of the present invention, since the first mediator sub-sequence and the second mediator sub-sequence used are different, the first mediator fragment and the second mediator fragment formed have different sequences and hybridize to different positions of the detection probe. Thus, the first duplex comprising the extension product of the first mediator segment and the detection probe is also different in structure (sequence) from the second duplex comprising the extension product of the second mediator segment and the detection probe. Accordingly, the first duplex will have a different melting point (T) than the second duplex_mA value). Therefore, in the melting curve analysis, the first duplex shows a melting peak different from that of the second duplex. Thus, by detecting the melting peak of the first duplex or the second duplex, the presence of the first target nucleic acid sequence or the second target nucleic acid sequence in the sample can be determined.

In addition, since the sequences of the first mediator sequence, the second mediator sequence, and the detection probe are known or predetermined, the melting points (T) of the first duplex and the second duplex can be calculated in advance_mValue). Thus, the melting point (T) of the first or second double chain is detected by melting curve analysis_mValue), the presence of the first target nucleic acid sequence or the second target nucleic acid sequence in the sample can be determined.

Based on the same principle as above, the method of the present invention can be used to simultaneously detect more target nucleic acid sequences by designing more mediator probes, and thus, for example, can be used to detect more targets (e.g., more respiratory pathogens). Thus, in certain preferred embodiments, in step (1), the sample is contacted with a third upstream oligonucleotide sequence and a third mediator probe under conditions that allow nucleic acid hybridization in addition to the first upstream oligonucleotide sequence, the first mediator probe, the second upstream oligonucleotide sequence, and the second mediator probe, wherein,

the third upstream oligonucleotide sequence comprises a sequence complementary to a third target nucleic acid sequence; and, the third mediator sub-probe comprises, in the 5 'to 3' direction, a third mediator sub-sequence and a third target-specific sequence, wherein the third mediator sub-sequence comprises a sequence that is not complementary to a third target nucleic acid sequence, and the third target-specific sequence comprises a sequence that is complementary to a third target nucleic acid sequence; wherein the third target nucleic acid sequence is specific for a third target, wherein the third target is a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract); preferably, the third target nucleic acid sequence is a genomic sequence of a third respiratory pathogen, or a specific fragment thereof;

and, when hybridized to a third target nucleic acid sequence, the third upstream oligonucleotide sequence is upstream of the third target-specific sequence; and, the third intermediary subsequence is different from the first and second intermediary subsequences;

and, in step (3), the detection probe used further comprises a third capture sequence complementary to a third mediator sequence, or a portion thereof, downstream of the template sequence.

In such embodiments, in step (1), the third upstream oligonucleotide sequence and the third mediator probe hybridize to a third target nucleic acid sequence when present. Further, in step (2), when a third target nucleic acid sequence is present, the third mediator subsequence or portion thereof is cleaved from the third mediator probe hybridized with the third target nucleic acid sequence by the presence of the third upstream oligonucleotide sequence or extension product thereof to form a third mediator fragment. Further, in steps (3) and (4), when a third mediator fragment is present, it hybridizes to the detection probe and the nucleic acid polymerase will extend the third mediator fragment using the detection probe as a template to form a third duplex. Further, in step (5), when a melting peak corresponding to the third duplex is detected or not detected, it is determined that the third target nucleic acid sequence is present or absent in the sample. Still further, since the third target nucleic acid sequence is specific for the third target, the presence or absence of the third target in the sample can be determined.

Similarly, in the method of the invention, the first mediator fragment, the second mediator fragment and the third mediator fragment are formed to have different sequences and hybridize to different positions of the detection probe, due to the difference in the first, second and third mediator sequences used. Thus, the first duplex comprising the extension product of the first mediator fragment and the detection probe, the second duplex comprising the extension product of the second mediator fragment and the detection probe, the third duplex comprising the extension product of the third mediator fragment and the detection probe differ from each other in structure (sequence). Accordingly, the first, second and third duplexes have melting points (T) different from each other_mValue). Thus, in melt curve analysis, the first, second and third duplexes exhibit three melt peaks that are distinguishable from each other. Thus, by detecting melting peaks of the first, second and third duplexes, the presence of the first, second and third target nucleic acid sequences in the sample can be determined.

In addition, since the sequences of the first, second, and third mediator sequences and the detection probe are known or predetermined, the melting points (T) of each of the first, second, and third duplexes can be pre-calculated_mValue). Thus, by detecting the melting point (T) of the duplex having the first, second or third duplex in a melting curve analysis_mValue), the presence of the first, second, or third target nucleic acid sequence in the sample can be determined.

In certain preferred embodiments, in step (1), the sample is contacted with a fourth upstream oligonucleotide sequence and a fourth mediator probe in addition to the first upstream oligonucleotide sequence, the first mediator probe, the second upstream oligonucleotide sequence, the second mediator probe, the third upstream oligonucleotide sequence, and the third mediator probe, wherein,

said fourth upstream oligonucleotide sequence comprises a sequence complementary to a fourth target nucleic acid sequence; and, the fourth mediator probe comprises, in the 5 'to 3' direction, a fourth mediator subsequence comprising a sequence that is not complementary to a fourth target nucleic acid sequence and a fourth target-specific sequence comprising a sequence that is complementary to a fourth target nucleic acid sequence; wherein the fourth target nucleic acid sequence is specific for a fourth target, wherein the fourth target is a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract); preferably, the fourth target nucleic acid sequence is a genomic sequence of a fourth respiratory tract pathogen, or a specific fragment thereof;

and, when hybridized to a fourth target nucleic acid sequence, the fourth upstream oligonucleotide sequence is located upstream of the fourth target-specific sequence; and, said fourth intermediate subsequence is different from said first, second and third intermediate subsequences;

and, in step (3), the detection probe used further comprises a fourth capture sequence complementary to a fourth mediator sequence, or a portion thereof, downstream of the template sequence.

In such embodiments, in step (1), when a fourth target nucleic acid sequence is present, the fourth upstream oligonucleotide sequence and the fourth mediator probe hybridize to the fourth target nucleic acid sequence. Further, in step (2), when a fourth target nucleic acid sequence is present, a fourth mediator sequence or a portion thereof is cleaved from the fourth mediator probe hybridized with the fourth target nucleic acid sequence by the presence of the fourth upstream oligonucleotide sequence or an extension product thereof, to form a fourth mediator fragment. Further, in steps (3) and (4), when a fourth mediator fragment is present, it hybridizes to the detection probe, and the nucleic acid polymerase will extend the fourth mediator fragment using the detection probe as a template to form a fourth duplex. Further, in step (5), when a melting peak corresponding to the fourth duplex is detected or not detected, it is determined that the fourth target nucleic acid sequence is present or absent in the sample. Still further, since the fourth target nucleic acid sequence is specific for the fourth target, the presence or absence of the fourth target in the sample can be determined.

Similarly, in the method of the present invention, the first mediator segment, the second mediator segment, the third mediator segment and the fourth mediator segment are formed to have different sequences and hybridize to different positions of the detection probe, since the first, second, third and fourth mediator sequences used are different. Thus, the first duplex comprising the extension product of the first mediator fragment and the detection probe, the second duplex comprising the extension product of the second mediator fragment and the detection probe, the third duplex comprising the extension product of the third mediator fragment and the detection probe, the fourth duplex comprising the extension product of the fourth mediator fragment and the detection probe differ from each other in structure (sequence). Accordingly, the first, second, third and fourth duplexes have different melting points (T) from each other_mValue). Thus, in melt curve analysis, the first, second, third and fourth duplexes exhibit four melt peaks that are distinguishable from each other. Thus, by detecting melting peaks of the first, second, third and fourth duplexes, the presence of the first, second, third and fourth target nucleic acid sequences in the sample can be determined.

In addition, since the sequences of the first, second, third and fourth mediator sequences and the detection probe are known or predetermined, the melting points (T) of each of the first, second, third and fourth duplexes can be calculated in advance_mA value). Thus, by detecting the melting point (T) of the duplex having the first, second, third or fourth duplex in a melting curve analysis_mValue) of the first, second, third or fourth target nucleic acid sequence in the sample.

Similarly, more upstream oligonucleotide sequences and more mediator probes may be used to carry out the methods of the invention. For example, in certain embodiments, the methods of the invention can be practiced using at least 5 upstream oligonucleotide sequences, at least 5 mediator probes, and a detection probe, wherein,

each upstream oligonucleotide sequence comprises a sequence complementary to a target nucleic acid sequence; and the number of the first and second electrodes,

each mediator probe comprises a mediator sequence and a target specific sequence from 5 'to 3', wherein the mediator sequence comprises a sequence that is not complementary to a target nucleic acid sequence, and the target specific sequence comprises a sequence that is complementary to a target nucleic acid sequence; thus, when a target nucleic acid sequence is present, both the upstream oligonucleotide sequence and the mediator probe corresponding to the target nucleic acid sequence are capable of hybridizing to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence of the mediator probe; and, all the mediator subsequences contained in the mediator probe are different from each other; and, each target nucleic acid sequence is specific for one target; and each target is, independently, a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract), and,

the detection probe comprises a template sequence and a plurality of sequences which are positioned at the downstream of the template sequence and are respectively complementary with the medium subsequence or the part thereof in each medium sub-probe. In such embodiments, the methods of the invention can be used to simultaneously detect at least 5 target nucleic acid sequences.

In certain embodiments, the methods of the invention can employ at least 6 upstream oligonucleotide sequences, at least 6 mediator probes, and one detection probe; preferably, at least 7 upstream oligonucleotide sequences, at least 7 mediator probes and a detection probe; preferably, at least 8 upstream oligonucleotide sequences, at least 8 mediator probes and a detection probe; preferably, at least 9 upstream oligonucleotide sequences, at least 9 mediator probes and one detection probe; preferably, at least 10 upstream oligonucleotide sequences, at least 10 mediator probes and a detection probe; preferably, at least 12 upstream oligonucleotide sequences, at least 12 mediator probes and a detection probe; preferably, at least 15 upstream oligonucleotide sequences, at least 15 mediator probes and a detection probe; preferably, at least 20 upstream oligonucleotide sequences, at least 20 mediator probes and one detection probe; wherein the upstream oligonucleotide sequence, mediator probe and detection probe are as defined above. In such embodiments, the methods of the invention can be used to simultaneously detect at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 19, or at least 20 target nucleic acid sequences or targets; wherein each target is independently a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi capable of infecting the respiratory tract).

Thus, in certain embodiments, the invention provides a method of detecting the presence of n targets in a sample, wherein n is an integer ≧ 2 (e.g., n is an integer of 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more), and each target is independently a respiratory pathogen selected from the group consisting of a bacterium, a chlamydia, a mycoplasma, a rickettsia, and a fungus capable of infecting the respiratory tract, and the method includes the steps of:

(1) For each target to be detected, determining at least one target nucleic acid sequence specific for the target; then, for each target nucleic acid sequence, providing an upstream oligonucleotide sequence and a mediator probe; wherein the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, the mediator probe comprises, in the 5 'to 3' direction, a mediator subsequence comprising a sequence that is not complementary to the target nucleic acid sequence and a target-specific sequence comprising a sequence that is complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is upstream of the target-specific sequence; and, all the mediator subsequences contained in the mediator sub-probes are different from each other;

and, contacting the sample with the provided upstream oligonucleotide sequences and mediator probes under conditions that allow nucleic acid hybridization;

(3) Contacting the product of step (2) with a detection probe comprising, in the 3 'to 5' direction, a capture sequence complementary to each of the mediator sequences or parts thereof, and a template sequence (templating sequence), under conditions permitting nucleic acid hybridization; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement;

(4) Contacting the product of step (3) with a nucleic acid polymerase under conditions that allow the nucleic acid polymerase to perform an extension reaction;

(5) Performing melting curve analysis on the product obtained in the step (4); and determining whether each target nucleic acid sequence is present in the sample based on the results of the melting curve analysis, and further determining whether a target corresponding to each target nucleic acid sequence is present in the sample.

In step (1) of such embodiments, when a target nucleic acid sequence is present, both the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence (i.e., the upstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence) and the mediator probe corresponding to the target nucleic acid sequence (i.e., the mediator probe whose target-specific sequence comprises a sequence complementary to the target nucleic acid sequence) hybridize to the target nucleic acid sequence.

Further, in step (2) of such embodiments, when a target nucleic acid sequence is present, the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence and the mediator sub-probe are hybridized with the target nucleic acid sequence, but the mediator sub-sequence in the mediator sub-probe is in a free state and is not hybridized with the target nucleic acid sequence. In this case, under the action of the enzyme having 5' nuclease activity, the mediator sequence or a part thereof in the mediator probe (the mediator probe corresponding to the target nucleic acid sequence) is cleaved from the mediator probe by the presence of the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence or an extension product thereof, to form a mediator fragment corresponding to the target nucleic acid sequence.

Further, in steps (3) and (4) of such embodiments, when a mediator fragment corresponding to a certain target nucleic acid sequence is present, the mediator fragment hybridizes to the detection probe, and the nucleic acid polymerase will extend the mediator fragment using the detection probe as a template to form a duplex corresponding to the target nucleic acid sequence. Further, in step (5) of such embodiments, when a melting peak of a duplex corresponding to a certain target nucleic acid sequence is detected or not detected, the presence or absence of the target nucleic acid sequence in the sample is determined, and thereby the presence or absence of a target corresponding to the target nucleic acid sequence in the sample is determined.

In particular, in such embodiments, since all mediator probes used contain different mediator sequences from each other, each mediator fragment formed has a different sequence and hybridizes to a different position of the detection probe. Thus, each duplex consisting of the extension product of the mediator fragment and the detection probe has a structure (sequence) that is different from each other. Accordingly, each of the duplexes has a melting point (T) different from each other_mA value). Thus, in melting curve analysis, each duplex shows a melting peak that is different from each other. Thus, by detecting the melting peak of a certain duplex, the presence of the target nucleic acid sequence corresponding to the duplex in the sample can be determined.

In addition, since the sequence of each mediator subsequence, and of the detection probe, is known or predetermined, the respective melting point (T) of each duplex can be pre-calculated_mValue). Thus, by detecting the melting point (T) of a duplex in a melting curve analysis_mValue) to determine the presence of a target nucleic acid sequence corresponding to the duplex in the sample.

Having briefly summarized the basic principles of the method of the present invention, reference will now be made in detail to the steps of the method of the present invention, which are illustrated and exemplified.

With respect to steps (1) and (2)

In the methods of the invention, target nucleic acid sequences (e.g., first or second target nucleic acid sequences; if present) specific for the target in the sample are first hybridized to the corresponding upstream oligonucleotide sequence (e.g., first or second upstream oligonucleotide sequence) and the corresponding mediator probe (e.g., first or second mediator probe).

In the method of the present invention, the sample may be any sample to be detected. For example, in certain preferred embodiments, the sample comprises or is DNA (e.g., genomic DNA or cDNA). In certain preferred embodiments, the sample comprises or is RNA (e.g., mRNA). In certain preferred embodiments, the sample comprises or is a mixture of nucleic acids (e.g., a mixture of DNA, a mixture of RNA, or a mixture of DNA and RNA). In certain preferred embodiments, the sample to be tested is a sample obtained from a subject, e.g., nasal secretions, nasal or pharyngeal swabs, alveolar lavage, sputum, and the like. In certain preferred embodiments, the subject is a mammal, e.g., a primate, e.g., a human.

In the method of the present invention, the target nucleic acid sequence to be detected is not limited to its sequence composition or length. For example, the target nucleic acid sequence can be a DNA (e.g., genomic DNA or cDNA) or an RNA molecule (e.g., mRNA). In addition, the target nucleic acid sequence to be detected may be single-stranded or double-stranded.

When the sample or target nucleic acid sequence to be detected is mRNA, preferably, prior to performing the method of the present invention, a reverse transcription reaction is performed to obtain cDNA complementary to the mRNA. For a detailed description of the reverse transcription reaction, see, for example, joseph Sam-brook, et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (2001).

The sample or target nucleic acid sequence to be detected can be obtained from any source. In the methods of the invention, the sample to be detected is a sample containing or suspected of containing the target. As used herein, the term "target" is a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract); including, but not limited to, staphylococcus epidermidis (Staphylococcus epidermidis), chlamydia pneumoniae (Chlamydia pneumoniae), staphylococcus aureus (Staphylococcus aureus), mycoplasma pneumoniae (Mycoplasma pneumoniae), candida albicans (Candida albicans), bordetella pertussis (Bordetella pertussis), rickettsia (Rickettsia), streptococcus pneumoniae (Streptococcus pneumoniae), haemophilus influenzae (Haemophilus influenzae), moraxella catarrhalis (Moraxella catarrhalis), acinetobacter baumannii (Acinetobacter baumannii), klebsiella pneumoniae (Klebsiella pneumoniae), cryptococcus (Cryptococcus), escherichia coli (Escherichia coli), streptococcus pneumoniae (Leptococcus pneumoniae), pseudomonas aeruginosa (Pseudomonas aeruginosa), and combinations thereof. It will be readily appreciated that in the method of the invention the target is not limited to the type described but may be any bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract, such as Aspergillus flavus, candida tropicalis, candida glabrata, enterobacter cloacae, enterococcus, proteus mirabilis, pseudomonas maltophilia, bordetella parapertussis, chlamydia psittaci. The sample or target nucleic acid sequence to be detected may also be any form of nucleic acid sequence, such as genomic sequences, artificially isolated or fragmented sequences, synthetic sequences, and the like.

In certain embodiments of the invention, the at least one respiratory pathogen is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia, moraxella catarrhalis, cryptococcus, legionella pneumophila, aspergillus flavus, candida tropicalis, candida glabrata, bordetella parapertussis, and chlamydia psittaci. In certain embodiments of the invention, the at least one respiratory pathogen is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia, moraxella catarrhalis, cryptococcus and legionella pneumophila.

In certain embodiments of the invention, the mediator probe 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 mediator probe comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the mediator probe comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the mediator 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 the method of the present invention, the mediator sub-probes are not limited by their length. For example, the length of the mediator probe may be 15 to 1000nt, such as 15 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 100nt,100 to 200nt,200 to 300nt,300 to 400nt,400 to 500nt,500 to 600nt,600 to 700nt,700 to 800nt,800 to 900nt, and 900 to 1000nt. For example, the length of the mediator probe may be 15 to 150nt, such as 15 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 100nt,100 to 110nt,110 to 120nt,120 to 130nt,130 to 140nt, and 140 to 150nt. The target-specific sequence in the mediator probe may be of any length as long as it is capable of specifically hybridizing to the target nucleic acid sequence. For example, the length of the target-specific sequence in the mediator probe may be 10 to 500nt, such as 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 100nt,100 to 150nt,150 to 200nt,200 to 250nt,250 to 300nt,300 to 350nt,350 to 400nt,400 to 450nt, and 450 to 500nt. For example, the length of the target-specific sequence in the mediator probe may be 10-140nt, such as 10-20nt,20-30nt,30-40nt,40-50nt,50-60nt,60-70nt,70-80nt,80-90nt,90-100nt,100-110nt,110-120nt,120-130nt,130-140nt. The mediator sequence in the mediator probe may be of any length as long as it is capable of specifically hybridizing to and extending the detection probe. For example, the length of the medium subsequence in the medium probe may be 5 to 140nt, such as 5 to 10nt,8 to 50nt,8 to 15nt,15 to 20nt,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 100nt,100 to 110nt,110 to 120nt,120 to 130nt, and 130 to 140nt. In certain preferred embodiments, the target-specific sequence in the mediator probe is 10-100nt (e.g., 10-90nt,10-80nt,10-50nt,10-40nt,10-30nt,10-20 nt) in length, and the mediator sequence is 5-100nt (e.g., 10-90nt,10-80nt,10-50nt,10-40nt,10-30nt,10-20 nt) in length.

In certain preferred embodiments, the mediator probe has a 3' -OH terminus. In certain preferred embodiments, the 3' -end of the mediator probe is blocked to inhibit extension thereof. The 3' -end of a nucleic acid (e.g., a mediator probe) can be blocked by various methods. For example, the 3 '-end of the mediator probe may be blocked by modifying the 3' -OH of the last nucleotide of the mediator probe. In certain embodiments, the 3 '-end of the mediator probe may be blocked by adding a chemical moiety (e.g., biotin or alkyl) to the 3' -OH of the last nucleotide of the mediator probe. In certain embodiments, the 3 '-end of the mediator probe may be blocked by removing the 3' -OH of the last nucleotide of the mediator probe, or replacing the last nucleotide with a dideoxynucleotide.

In certain embodiments of the invention, the upstream oligonucleotide sequence 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 upstream oligonucleotide sequence comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the upstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the upstream oligonucleotide sequence 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 the method of the present invention, the upstream oligonucleotide sequence is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence. For example, the length of the upstream oligonucleotide sequence may be 15 to 150nt, such as 15 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 100nt,100 to 110nt,110 to 120nt,120 to 130nt,130 to 140nt, and 140 to 150nt.

In the method of the present invention, conditions allowing nucleic acid hybridization can be routinely determined by one skilled in the art. For example, suitable hybridization conditions can be determined based on the target nucleic acid sequence to be detected, the upstream oligonucleotide sequence used, and the target-specific sequence in the mediator probe. In certain embodiments of the invention, the conditions that allow nucleic acid hybridization are stringent conditions such that the target-specific sequence in the upstream oligonucleotide sequence and the mediator sub-probe hybridize to the corresponding target nucleic acid sequence by base-complementary pairing, and the mediator sub-sequence in the mediator sub-probe does not hybridize to the target nucleic acid sequence. In certain preferred embodiments, the sample is contacted with various upstream oligonucleotide sequences and various mediator probes under high stringency conditions.

In the methods of the invention, after contacting the sample with the various upstream oligonucleotide sequences and the various mediator probes, it is desirable to induce cleavage of the mediator probes to release fragments containing the mediator sequences or portions thereof (i.e., mediator fragments). In general, cleavage of a mediator probe hybridized to a target nucleic acid sequence can be induced using an enzyme having 5' nuclease activity, using an upstream oligonucleotide sequence hybridized to the target nucleic acid sequence or an extension product thereof. Specifically, in step (1), when the mediator probe is contacted with the target nucleic acid sequence, the mediator probe comprises a target-specific sequence that hybridizes to the target nucleic acid sequence and forms a double-stranded structure, while the mediator probe does not hybridize to the target nucleic acid sequence and maintains a single-stranded structure. Thus, such oligonucleotides comprising a double-stranded structure and a single-stranded structure can be cleaved with an enzyme having 5' nuclease activity, and fragments having a single-stranded structure are released.

It will be readily appreciated that in the method of the invention, the upstream oligonucleotide sequence and the mediator probe will hybridize to the same strand of the target nucleic acid sequence under conditions which permit nucleic acid hybridization, and the upstream oligonucleotide sequence is located upstream of the mediator probe, thereby inducing cleavage of the mediator probe. In certain embodiments of the invention, cleavage of the mediator probe may be induced in two ways: (A) A manner of extension independent of the upstream oligonucleotide sequence; and (B) a mode of extension dependent on the upstream oligonucleotide sequence. In particular, if the upstream oligonucleotide sequence and mediator probe are sufficiently close together that an enzyme having 5' nuclease activity is able to induce cleavage of the mediator probe after hybridization of the upstream oligonucleotide sequence and mediator probe to the target nucleic acid sequence, the enzyme will bind to the upstream oligonucleotide sequence and cleave the mediator probe without performing an extension reaction (i.e., mode a). Conversely, if the upstream oligonucleotide sequence is distal to the mediator probe after hybridization to the target nucleic acid sequence, then a nucleic acid polymerase is used to catalyze extension of the upstream oligonucleotide sequence using the target nucleic acid sequence as a template, followed by an enzyme with 5' nuclease activity that binds to the extension product of the upstream oligonucleotide sequence and cleaves the mediator probe (i.e., mode B).

Thus, in certain preferred embodiments, the upstream oligonucleotide sequence is located in upstream proximity to the mediator probe after hybridization to the target nucleic acid sequence. In such embodiments, the upstream oligonucleotide sequence directly induces the enzyme cleavage mediator probe with 5' nuclease activity without the need for an extension reaction. Thus, in such embodiments, the upstream oligonucleotide sequence is an upstream probe specific for the target nucleic acid sequence that induces cleavage of the mediator probe in an extension-independent manner. As used herein, the term "adjacent" is intended to mean that two nucleic acid sequences are adjacent to each other, forming a gap. In certain preferred embodiments, the two adjacent nucleic acid sequences (e.g., the upstream oligonucleotide sequence and the mediator probe) are separated by no more than 30nt, such as no more than 20nt, such as no more than 15nt, such as no more than 10nt, such as no more than 5nt, such as 4nt,3nt,2nt, and 1nt.

In certain preferred embodiments, the upstream oligonucleotide sequence has a sequence that partially overlaps the target-specific sequence of the mediator probe after hybridization to the target nucleic acid sequence. In such embodiments, the upstream oligonucleotide sequence directly induces the enzyme cleavage mediator probe with 5' nuclease activity without performing an extension reaction. Thus, in such embodiments, the upstream oligonucleotide sequence is an upstream probe specific for the target nucleic acid sequence that induces cleavage of the mediator probe in an extension-independent manner. In certain preferred embodiments, the partially overlapping sequences are 1 to 10nt in length, e.g., 1 to 5nt, or 1 to 3nt.

In certain preferred embodiments, the upstream oligonucleotide sequence is located at the upstream distal end of the mediator probe after hybridization to the target nucleic acid sequence. In such embodiments, the upstream oligonucleotide sequence is extended by a nucleic acid polymerase, and the resulting extension product induces an enzyme cleavage mediator probe having 5' nuclease activity. Thus, in such embodiments, the upstream oligonucleotide sequence is a primer specific for the target nucleic acid sequence that is used to initiate the extension reaction and induce cleavage of the mediator probe in an extension-dependent manner. As used herein, the term "distal" is intended to mean that two nucleic acid sequences are distant from each other, e.g., at least 30nt, at least 50nt, at least 80nt, at least 100nt or longer.

Thus, in certain preferred embodiments, the upstream oligonucleotide sequence is a primer specific for the target nucleic acid sequence or a probe specific for the target nucleic acid sequence. The primers are suitable for inducing cleavage of the mediator probe in an extension-dependent manner. The probe is adapted to induce cleavage of the mediator sub-probe in an extension-independent manner.

Various methods of using an upstream oligonucleotide to induce cleavage of a downstream oligonucleotide (downstream probe) are known to those skilled in the art and can be used in the present invention. For a detailed description of such methods see, for example, U.S. Pat. Nos. 5,210,015,5,487,972,5,691,142,5,994,069 and 7,381,532, and U.S. application US 2008/0241838.

In certain embodiments, the cleavage site on the mediator probe is located at the junction of the mediator sequence and the target-specific sequence (i.e., the junction of the sequence that hybridizes to the target nucleic acid and the sequence that does not hybridize to the target nucleic acid). In such embodiments, cleavage of the mediator probe by the enzyme will release a fragment comprising the entire mediator sequence. In certain embodiments, the cleavage site on the mediator probe is located within the 3' -terminal region of the mediator sequence (i.e., upstream of the 3' -terminus of the mediator sequence, and for example, a few nucleotides, such as 1-3 nucleotides, from the 3' -terminus of the mediator sequence). In such embodiments, cleavage of the mediator probe by the enzyme will release a fragment comprising a portion (the 5' -end portion) of the mediator sequence. Thus, in certain embodiments of the invention, the vector subsequence comprises the entire vector subsequence, or a portion (5 '-end portion) of the vector subsequence, e.g., at least 5nt, at least 8nt, at least 10nt, at least 20nt, at least 30nt, at least 40nt, at least 50nt, e.g., 5-50nt,5-10nt,10-20nt,20-30nt,30-40nt,40-50nt, comprising the 5' -end of the vector subsequence.

In the present application, the method of the invention can be carried out using various enzymes having 5' nuclease activity. In certain preferred embodiments, the enzyme having 5 'nuclease activity is an enzyme having 5' exonuclease activity. In certain preferred embodiments, the enzyme having 5' nuclease activity is a nucleic acid polymerase (e.g., a DNA polymerase, particularly a thermostable DNA polymerase) having 5' nuclease activity (e.g., 5' exonuclease activity). In certain embodiments, the use of a nucleic acid polymerase having 5' nuclease activity is particularly advantageous because the polymerase is capable of catalyzing extension of the upstream oligonucleotide sequence with both the target nucleic acid sequence as a template and inducing cleavage of the mediator probe.

In certain preferred embodiments, the DNA polymerase having 5' nuclease activity is a thermostable DNA polymerase obtainable from various bacterial species, e.g., thermus aquaticus (Taq), thermus thermophilus (Tth), thermus filiformis, thermus flavus, thermococcus literalis, thermus antandaili, thermus caldophlus, thermus brachiophilus, thermus flavus, thermus igniterrae, thermus lacteus, thermus oshima, thermus ruber, thermus rubens, thermus scotoductus, thermus silvanus, thermus thermophilus, thermus marimaritima maritima, thermus neocolitana, thermosiphoa aricana, thermococcus litoralis, thermococcus barossi, thermococcus gorgonius, thermotogamaritima, thermotoga neocolitana, thermosiphorhoafricans, pyrococcus woesei, pyrococcus gorgonius, pyrococcus crassi, pyrococcus abaysi, pyrococcus occi, pyrodium occullum, aquifex aeoliticum, azifex purpureus and Azifex aeolieus. Particularly preferably, the DNA polymerase having 5' nuclease activity is Taq polymerase.

Alternatively, in step (2), two different enzymes may be used: nucleic acid polymerases and enzymes having 5' nuclease activity. In such embodiments, the nucleic acid polymerase is used to catalyze extension of the upstream oligonucleotide sequence with the target nucleic acid sequence as a template, and the enzyme having 5' nuclease activity binds to the extension product of the upstream oligonucleotide sequence and catalyzes cleavage of the mediator probe.

In certain preferred embodiments, in steps (1) and/or (2), the sample is also contacted with a downstream oligonucleotide sequence (or downstream primer) specific for the target nucleic acid sequence. In certain embodiments, the use of a nucleic acid polymerase and a downstream oligonucleotide sequence (or downstream primer) is particularly advantageous. In particular, the nucleic acid polymerase can generate additional target nucleic acid sequences using the target nucleic acid sequence as a template and the upstream and downstream oligonucleotide sequences as primers, thereby increasing the sensitivity of the methods of the invention.

Thus, in certain preferred embodiments, in step (1), in addition to the upstream oligonucleotide sequence and mediator probe defined above, a downstream oligonucleotide sequence is provided for each target nucleic acid sequence to be detected; wherein the downstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the downstream oligonucleotide sequence is located downstream of the target-specific sequence;

the sample is then contacted with the provided upstream oligonucleotide sequences, mediator probes, and downstream oligonucleotide sequences under conditions that allow nucleic acid hybridization.

In such embodiments, the upstream and downstream oligonucleotide sequences serve as upstream and downstream primers, respectively, for amplification of the target nucleic acid sequence. Thus, it is readily understood that the upstream and downstream oligonucleotide sequences are targeted to different ones of the two complementary strands, respectively. Thus, when the target nucleic acid sequence is a double-stranded molecule, the upstream and downstream oligonucleotide sequences are complementary to different strands (sense and antisense strands) of the target nucleic acid sequence, respectively; when the target nucleic acid sequence is a single-stranded molecule, the upstream oligonucleotide sequence and the downstream oligonucleotide sequence are respectively complementary with the target nucleic acid sequence and a complementary sequence thereof, so that the amplification of the target nucleic acid sequence can be realized. However, in the present application, for the sake of simplicity, when describing the relationship between the upstream oligonucleotide sequence/the downstream oligonucleotide sequence and the target nucleic acid sequence, they are collectively referred to as "complementary to the target nucleic acid sequence", and the sense strand and the antisense strand of the target nucleic acid sequence are not distinguished in detail, and the target nucleic acid sequence and its complementary sequence are not distinguished in detail. However, the complementary and positional relationship of the upstream/downstream oligonucleotide sequences to the target nucleic acid sequence can be properly understood by those skilled in the art.

For example, when the methods of the invention are used to detect first and second target nucleic acid sequences specific for first and second targets, respectively, first and second downstream oligonucleotide sequences may be provided which comprise sequences complementary to the first and second target nucleic acid sequences, respectively. Similarly, a third downstream oligonucleotide sequence may be provided for a third target nucleic acid sequence specific for a third target, comprising a sequence complementary to the third target nucleic acid sequence. A fourth downstream oligonucleotide sequence may also be provided for a fourth target nucleic acid sequence specific for a fourth target, comprising a sequence complementary to the fourth target nucleic acid sequence.

Further, in certain preferred embodiments, in step (2), the product of step (1) is contacted with a nucleic acid polymerase (particularly preferably, a nucleic acid polymerase having 5' nuclease activity). In a further preferred embodiment, the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow for nucleic acid amplification. In such embodiments, the nucleic acid polymerase will amplify the target nucleic acid sequence using the upstream and downstream oligonucleotides as primers. And, during amplification of the target nucleic acid, the nucleic acid polymerase induces cleavage of the mediator probe hybridized to the target nucleic acid sequence by its own 5' nuclease activity, thereby releasing the mediator fragment comprising the mediator sequence or a portion thereof. The methods of the invention can be carried out using a variety of nucleic acid polymerases having 5' nuclease activity, particularly those described above. In the present application, it is particularly preferred that the nucleic acid polymerase used is a template-dependent nucleic acid polymerase (e.g., a template-dependent DNA polymerase).

In certain embodiments of the invention, the downstream oligonucleotide sequence 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 downstream oligonucleotide sequence comprises or consists of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides). In certain preferred embodiments, the downstream oligonucleotide sequence comprises modified nucleotides, such as modified deoxyribonucleotides or ribonucleotides, such as 5-methylcytosine or 5-hydroxymethylcytosine. In certain preferred embodiments, the downstream oligonucleotide sequence 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 the method of the present invention, the downstream oligonucleotide sequence is not limited by its length as long as it can specifically hybridize to the target nucleic acid sequence. For example, the downstream oligonucleotide sequence may be 15 to 150nt in length, such as 15 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 100nt,100 to 110nt,110 to 120nt,120 to 130nt,130 to 140nt,140 to 150nt.

In certain preferred embodiments, the target nucleic acid sequence is amplified in a symmetric amplification manner. In such embodiments, amplification is performed using equal amounts of upstream and downstream oligonucleotide sequences for a target nucleic acid sequence. In certain preferred embodiments, the target nucleic acid sequence is amplified in an asymmetric amplification manner. In such embodiments, amplification is performed using unequal amounts of upstream and downstream oligonucleotide sequences for a particular target nucleic acid sequence. In certain embodiments, the upstream oligonucleotide sequence is in excess (e.g., at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, e.g., 1-10-fold excess) relative to the downstream oligonucleotide sequence. In certain embodiments, the downstream oligonucleotide sequence is in excess (e.g., at least 1-fold, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, e.g., 1-10-fold excess) relative to the upstream oligonucleotide sequence.

In certain preferred embodiments, the target nucleic acid sequence is amplified in a three-step process. In such embodiments, each round of nucleic acid amplification requires three steps: the nucleic acid denaturation is performed at a first temperature, the nucleic acid annealing is performed at a second temperature, and the nucleic acid extension is performed at a third temperature. In certain preferred embodiments, the target nucleic acid sequence is amplified in a two-step process. In such embodiments, each round of nucleic acid amplification requires two steps: the nucleic acid denaturation is performed at a first temperature, and the nucleic acid annealing and extension are performed at a second temperature. The temperature suitable for performing nucleic acid denaturation, nucleic acid annealing, and nucleic acid extension can be readily determined by one skilled in the art 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 mediator probes used generally correspond one-to-one to the target nucleic acid sequence. In other words, a unique mediator probe is provided for each target nucleic acid sequence to be detected. However, it will be readily appreciated that there need not be a one-to-one correspondence between the upstream oligonucleotide sequences, the downstream oligonucleotide sequences and the target nucleic acid sequences. For example, in some cases, the sample tested is a DNA library, and one or both ends of all fragments in the library comprise the same linker. In this case, the same upstream oligonucleotide sequence may be used for extension, or the same upstream oligonucleotide sequence and/or downstream oligonucleotide sequence may be used for amplification and thereby inducing cleavage of the mediator probe. Thus, in the methods of the invention, the same or different upstream oligonucleotide sequences may be used for different target nucleic acid sequences; and/or, the same or different downstream oligonucleotide sequences may be used. For example, the first, second, third and fourth upstream oligonucleotide sequences may be the same or different. The first, second, third and fourth downstream oligonucleotide sequences may also be the same or different.

Furthermore, when a Nucleic acid polymerase having 5' nuclease activity is used in step (2), a HANDS strategy can also be employed to increase the efficiency of Nucleic acid amplification (see, for example, nucleic Acids Research,1997,25 (16): 3235-3241). For example, in certain preferred embodiments, an identical oligonucleotide sequence can be introduced at the 5' end of all upstream and downstream oligonucleotide sequences, and amplification can be performed using universal primers complementary to the identical oligonucleotide sequence (preferably in an amount generally much greater than the upstream and downstream oligonucleotide sequences).

Thus, in certain preferred embodiments, in step (1), all of the upstream oligonucleotide sequences (e.g., first, second, third and fourth upstream oligonucleotide sequences) and the downstream oligonucleotide sequences (e.g., first, second, third and fourth downstream oligonucleotide sequences) provided have an identical oligonucleotide sequence at the 5' end, and a universal primer is also provided, the universal primer having a sequence complementary to the identical oligonucleotide sequence; the sample is then contacted with the provided upstream oligonucleotide sequences, mediator probes, downstream oligonucleotide sequences and universal primers under conditions that allow nucleic acid hybridization. In certain preferred embodiments, the identical oligonucleotide sequences are 8-50nt in length, e.g., 8-15nt,15-20nt,20-30nt,30-40nt, or 40-50nt. Accordingly, the universal primer may be 8-50nt in length, such as 8-15nt,15-20nt,20-30nt,30-40nt, or 40-50nt. Subsequently, in certain preferred embodiments, in step (2), the product of step (1) is contacted with a nucleic acid polymerase (particularly preferably, a nucleic acid polymerase having 5' nuclease activity). In a further preferred embodiment, the product of step (1) is contacted with a nucleic acid polymerase having 5' nuclease activity under conditions that allow for nucleic acid amplification. In such embodiments, the nucleic acid polymerase will perform a preliminary amplification of the target nucleic acid sequence using the upstream and downstream oligonucleotides as primers to obtain a preliminary amplified product; subsequently, the preliminarily amplified product is subjected to re-amplification using the universal primer. And, throughout the amplification process, the nucleic acid polymerase cleaves the mediator probe hybridized to the target nucleic acid sequence or the product of the preliminary amplification by its own 5' nuclease activity, thereby releasing the mediator fragment comprising the mediator sequence or a portion thereof.

In certain embodiments of the invention, the universal primer 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 comprises or consists of a natural nucleotide (e.g., a deoxyribonucleotide or a ribonucleotide). In certain preferred embodiments, the universal primer 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 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 the method of the present invention, the universal primer is not limited by its length as long as it can specifically hybridize to the same oligonucleotide sequences contained in the upstream and downstream oligonucleotide sequences. For example, the universal primer can be 8-50nt, such as 8-15nt,15-20nt,20-30nt,30-40nt, or 40-50nt in length.

With respect to steps (3) and (4)

In step (2), the mediator probe hybridized to the target nucleic acid sequence is cleaved by an enzyme having 5' nuclease activity, releasing a mediator fragment containing the mediator sequence or a portion thereof, which is then hybridized to the detection probe in step (3). In the present application, the detection probe comprises, in the 3 'to 5' direction, a capture sequence complementary to each of the mediator sequences or portions thereof, and a template sequence. Thus, in step (4), the detection probe is used as a template for extension of the mediator fragment under the action of the nucleic acid polymerase; and the vector fragment serves as a primer for initiating the extension reaction; and after the extension reaction is finished, the extension product of the mediator fragment is hybridized with the detection probe to form a nucleic acid duplex.

In the methods of the invention, the detection probe comprises a plurality of capture sequences that are complementary to the plurality of mediator sequences or portions thereof (e.g., a first capture sequence that is complementary to a first mediator sequence or portion thereof, a second capture sequence that is complementary to a second mediator sequence or portion thereof, a third capture sequence that is complementary to a third mediator sequence or portion thereof, and/or a fourth capture sequence that is complementary to a fourth mediator sequence or portion thereof). It will be readily appreciated that the individual capture sequences may be arranged in any order. For example, the first capture sequence may be located upstream (5 'end) or downstream (3' end) of the second capture sequence. For example, the detection probe may comprise, in order from 3 'to 5', a first capture sequence and a second capture sequence; alternatively, the second capture sequence and the first capture sequence. Similarly, the detection probes can comprise additional capture sequences (e.g., first, second, third, fourth capture sequences) in any order.

Furthermore, the individual capture sequences may be arranged in any manner. For example, the individual capture sequences can be arranged in an adjacent manner or in a spaced-apart manner with a linker sequence. For example, the first capture sequence may be arranged adjacent to the second capture sequence; alternatively, the two may be separated by a linker sequence (also referred to herein simply as a "linker"); alternatively, there may be an overlap between the two. Similarly, the detection probes can comprise additional capture sequences (e.g., first, second, third, fourth capture sequences) in any arrangement.

In some cases, it is particularly advantageous to arrange the individual capture sequences in an overlapping manner. In such embodiments, the plurality of media subsequences can be designed such that different media subsequences comprise overlapping sequences. For example, the first and second intermediate subsequences may be designed such that the 3 'end portion of the first intermediate subsequence has the same sequence as the 5' end portion of the second intermediate subsequence. Accordingly, in the detection probe, the 5 'end portion of the first capture sequence complementary to the first mediator sequence has the same sequence as the 3' end portion of the second capture sequence complementary to the second mediator sequence. Thus, the detection probe may comprise the first capture sequence and the second capture sequence in a 3 'to 5' orientation, and both may be arranged in an overlapping manner. In this case, the overlapping sequence is the same sequence or a portion thereof that is common to the first and second capture sequences. By arranging the capture sequences in an overlapping manner, the detection probe can be made to comprise more capture sequences within a predetermined length, thereby allowing hybridization to more mediator subsections. In other words, by arranging the capture sequences in an overlapping manner, a single detection probe may be used in combination with more mediator sub-probes.

As described above, in the methods of the invention, a single detection probe is used in combination with at least 2 (e.g., 3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) mediator sub-probes. Thus, in certain preferred embodiments, a single detection probe is used in excess (e.g., at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold excess) relative to a single mediator sub-probe. Such embodiments are advantageous in certain circumstances because the entire reaction system contains enough detection probes to hybridize with the released mediator fragments, mediate extension of the mediator fragments, and form duplexes.

As described above, a media sub-fragment may contain the entire media sub-sequence or a portion thereof. When the mediator fragment comprises the entire mediator subsequence, the detection probe preferably comprises a sequence complementary to the mediator subsequence. When the mediator fragment comprises a portion (5 '-end portion) of the mediator subsequence, the detection probe preferably comprises a sequence complementary to the portion (5' -end portion) of the mediator subsequence, or alternatively, a sequence complementary to the entire mediator subsequence. In certain preferred embodiments, the detection probe comprises a sequence complementary to the mediator sequence. Such detection probes are particularly advantageous in certain cases because they are capable of hybridizing to both vector subsegments containing the entire vector subsequence, and vector subsegments containing portions (5' -end portions) of the vector subsequence. However, it will be appreciated that the detection probe may also comprise a sequence that is complementary to only a portion (e.g., the 3' -end portion) of the mediator segment, so long as the detection probe is capable of stably hybridizing to the mediator segment and initiating the extension reaction.

Furthermore, the detection probe may comprise additional sequences at the 3' end (i.e., downstream of the capture sequence) in addition to the capture sequence and the template sequence. The additional sequences typically comprise sequences that are not complementary to the mediator sub-fragments and do not participate in hybridization with the mediator sub-fragments.

According to the invention, the template sequence in the detection probe may comprise any sequence and is located upstream (5' to) the respective capture sequence and thus may be used as a template for extending the media fragment. In certain preferred embodiments, the template sequence comprises a sequence that is not complementary to the mediator probe (mediator sequence and target-specific sequence). Such a template sequence is particularly advantageous in certain circumstances because it can improve the specificity of hybridisation of the mediator fragment to the detection probe, avoiding hybridisation of the mediator fragment to undesired positions and thus avoiding the production of undesired duplexes.

In certain embodiments of the invention, the detection probes 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 nucleotides), 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 the method of the present invention, the detection probe is not limited by its length. For example, the length of the detection probe may be 15 to 1000nt, such as 15 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 100nt,100 to 200nt,200 to 300nt,300 to 400nt,400 to 500nt,500 to 600nt,600 to 700nt,700 to 800nt,800 to 900nt, and 900 to 1000nt. The capture sequence in the detection probe can be of any length so long as it is capable of specifically hybridizing to the mediator fragments. For example, the length of the capture sequence in the detection probe may be 10 to 500nt, such as 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 100nt,100 to 150nt,150 to 200nt,200 to 250nt,250 to 300nt,300 to 350nt,350 to 400nt,400 to 450nt, and 450 to 500nt. The template sequence in the detection probe may be of any length as long as it can serve as a template for extension of the vector fragment. For example, the length of the template sequence in the detection probe may be 1 to 900nt, such as 1 to 5nt,5 to 10nt,10 to 2nt, 20 to 30nt,30 to 40nt,40 to 50nt,50 to 60nt,60 to 70nt,70 to 80nt,80 to 90nt,90 to 100nt,100 to 200nt,200 to 300nt,300 to 400nt,400 to 500nt,500 to 600nt,600 to 700nt,700 to 800nt,800 to 900nt. In certain preferred embodiments, the capture sequence in the detection probe is 10-200nt (e.g., 10-190nt,10-180nt,10-150nt,10-140nt,10-130nt,10-120nt,10-100nt,10-90nt,10-80nt,10-50nt,10-40nt,10-30nt,10-20 nt) in length, and the template sequence is 5-200nt (e.g., 10-190nt,10-180nt,10-150nt,10-140nt,10-130nt,10-120nt,10-100nt,10-90nt,10-80nt,10-50nt,10-40nt,10-30nt,10-20nt in length).

In certain preferred embodiments, the detection probe has a 3' -OH terminus. In certain preferred embodiments, the 3' -end of the detection probe is blocked to inhibit extension thereof. The 3' -end of a nucleic acid (e.g., detection probe) can be blocked by various methods. For example, the 3 '-end of the detection probe can be blocked by modifying the 3' -OH of the last nucleotide of the detection probe. In certain embodiments, 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. In certain embodiments, the 3 '-end of the detection probe can be blocked by removing the 3' -OH of the last nucleotide of the detection probe, or replacing the last nucleotide with a dideoxynucleotide.

In the method of the invention, the mediator fragment is hybridized to the detection probe and thereby initiates an extension reaction of the nucleic acid polymerase. Although the uncleaved mediator probe is also capable of hybridizing to the detection probe via the mediator subsequence, the mediator probe further comprises a target-specific sequence that is downstream of the mediator subsequence and that does not hybridize to the detection probe (i.e., is in a free state), such that the nucleic acid polymerase cannot extend the mediator probe hybridized to the detection probe.

As described above, the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group is capable of emitting a signal and the quencher group is capable of absorbing or quenching the signal emitted by the reporter group; and 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 certain preferred embodiments, the detection probe is 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,70-80nt. 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 20nt. In certain preferred embodiments, the reporter and quencher are at least 5nt, at least 10nt, at least 15nt, or at least 20nt apart.

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 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, 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 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, 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 the methods of the invention, the reporter and quencher groups can be any suitable group or molecule known in the art, specific examples of which include, but are not limited to, cy2^TM(506),YO-PRO^TM-l(509),YOYO^TM-l(509),Calcein(517),FITC(518),FluorX^TM(519),Alexa^TM(520),Rhodamine 110(520),Oregon Green^TM500(522),Oregon Green^TM 488(524),RiboGreen^TM(525),Rhodamine Green^TM(527),Rhodamine 123(529),Magnesium Green^TM(531),Calcium Green^TM(533),TO-PRO^TM-l(533),TOTOl(533),JOE(548),BODIPY530/550(550),Dil(565),BODIPY TMR(568),BODIPY558/568(568),BODIPY564/570(570),Cy3^TM(570),Alexa^TM546(570),TRITC(572),Magnesium Orange^TM(575),Phycoerythrin R&B(575),Rhodamine Phalloidin(575),Calcium Orange^TM(576),PyroninY(580),Rhodamine B(580),TAMRA(582),Rhodamine Red^TM(590),Cy3.5^TM(596),ROX(608),Calcium Crimson^TM(615),Alexa^TM594(615),Texas Red(615),Nile Red(628),YO-PRO^TM-3(631),YOYO^TM-3(631),R-phycocyanin(642),C-Phycocyanin(648),TO-PRO^TM-3(660),T0T03(660),DiD DilC(5)(665),Cy5^TM(670) Thiadiacarbycanine (671), cy5.5 (694), HEX (556), TET (536), biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), fluoroescein-C3 (520), pulsar 650 (566), quasar 570 (667), quasar 670 (705), and Quasar 705 (610). The numbers in parentheses indicate the maximum emission wavelength in nm.

In addition, various suitable pairs of reporter and quencher groups are known in the art, see, e.g., pesce et al, editors, fluorescence Spectroscopy (Marcel Dekker, new York, 1971); white et al, fluorescence Analysis A Practical Approach (Marcel Dekker, new York, 1970); berlman, handbook of Fluorescence Spectra of Aromatic Molecules,2nd Edition (Academic Press, new York, 1971); griffiths, color AND Consistition of organic Molecules (Academic Press, new York, 1976); bishop, editor, indicators (Peigimon Press, oxford, 1972); haughland, handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, eugene, 1992); pringsheim, fluoressence and Phosphorescence (Interscience Publishers, new York, 1949); haughland, R.P., handbook of Fluorescent Probes and Research Chemicals,6th Edition (Molecular Probes, eugene, oreg., 1996); U.S. Pat. nos. 3,996,345 and 4,351,760.

In certain preferred embodiments, the reporter is a fluorophore. In such embodiments, the signal emitted by the reporter is fluorescence, and the quencher is a molecule or group capable of absorbing/quenching the fluorescence (e.g., another fluorescent molecule capable of absorbing the fluorescence, or a quencher capable of quenching the fluorescence). In certain preferred embodiments, the fluorescent group includes, but is not limited to, various fluorescent molecules, such as 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 like. In certain preferred embodiments, the quenching group includes, but is not limited to, various quenchers, such as DABCYL, BHQ (e.g., BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA, and the like.

In the methods of the invention, the detection probe may also be modified, for example, to be resistant to nuclease activity (e.g., 5' nuclease activity, e.g., 5' to 3' exonuclease activity). For example, modifications that resist 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 may be introduced into the backbone of the detection probe.

In the methods of the invention, the detection probe may be linear or may 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,6-12nt.

Furthermore, in the method of the present invention, "hybridization", "nucleic acid hybridization" and "conditions allowing nucleic acid hybridization" in step (3) may be as defined above.

Performing step (4) using the product of step (3) and a nucleic acid polymerase. In step (4), the nucleic acid polymerase will extend the fragment of the mediator hybridised to the detection probe using the detection probe as a template under conditions which allow the nucleic acid polymerase to perform an extension reaction and thereby form a duplex.

As described in detail above, each mediator probe comprises a unique mediator sequence and, under the action of an enzyme having 5' nuclease activity, releases a mediator fragment comprising the unique mediator sequence or a portion thereof. Each mediator sub-fragment then hybridizes to a different location of the detection probe (i.e., a capture sequence complementary to the corresponding mediator sub-sequence or portion thereof), is extended by the nucleic acid polymerase, and forms a duplex with the detection probe. Thus, for each mediator probe, when its corresponding target sequence is present, a unique duplex will be generated in step (4) that comprises the detection probe (as one strand) and the extension product of the mediator fragment corresponding to that mediator probe (as the other strand). Thus, each of the duplexes produced in step (4) has a structure (sequence) different from each other, and thus has a T different from each other_mAnd show melting peaks different from each other in melting curve analysis.

In certain preferred embodiments, the nucleic acid polymerase used in step (4) is a template-dependent nucleic acid polymerase (e.g., a DNA polymerase, particularly a thermostable DNA polymerase). In certain preferred embodiments, the nucleic acid polymerase is a thermostable DNA polymerase, which is obtainable from various bacterial species, e.g., thermus aquaticus (Taq), thermus thermophiles (Tth), thermus filiformis, thermus flavus, thermococcus litera, thermus antarildanii, thermus caldophlus, thermus chalarphilus, thermus flavus, thermus agniterae, thermus lacteus, thermus oshimai, thermus ruber, thermus rubens, thermus scotoductus, thermus silvanus, thermus thermophilus, thermotoga maritima, thermotoga neapolitana, thermosipho africanus, thermococcus litoralis, thermococcus barossi, thermococcus gorgonius, thermotoga maritima, thermotoga neapolia, thermotoga neoolitana, thermosoarubicananus, pyrococcus wooseei, pyrococcus horikoshi, pyrococcus abyssi, pyrodium occullum, aquifexpyrphilus and Aquifex aeolieus. Particularly preferably, the template-dependent nucleic acid polymerase is Taq polymerase.

In certain preferred embodiments, the enzyme having 5 'nuclease activity used in step (2) is a nucleic acid polymerase having 5' nuclease activity and is the same as the nucleic acid polymerase used in step (4). In certain preferred embodiments, the enzyme having 5' nuclease activity used in step (2) is different from the nucleic acid polymerase used in step (4).

For example, in certain embodiments, in step (2), a first nucleic acid polymerase is used to catalyze extension of the upstream oligonucleotide sequence and an enzyme with 5' nuclease activity is used to catalyze cleavage of the mediator probe, followed by a second nucleic acid polymerase in step (4) to catalyze extension of the mediator fragment. In certain embodiments, in step (2), a first nucleic acid polymerase having 5' nuclease activity is used to catalyze the extension of the upstream oligonucleotide sequence and cleavage of the mediator probe, followed by a second nucleic acid polymerase used to catalyze the extension of the mediator fragment in step (4). However, it is particularly preferred to use the same enzyme in steps (2) and (4). For example, a template-dependent nucleic acid polymerase having 5' nuclease activity (e.g., a DNA polymerase, particularly a thermostable DNA polymerase) may be used to catalyze the extension of the upstream oligonucleotide sequence and cleavage of the mediator probe in step (2), and to catalyze the extension of the mediator fragment in step (4).

In the method of the present invention, one or more of steps (1) to (4) may be repeatedly performed as necessary. In certain preferred embodiments, steps (1) - (2) are repeated one or more times, and prior to each repetition, a step of nucleic acid denaturation is performed. It will be readily appreciated that the repetition of steps (1) - (2) may produce more media fragments for subsequent steps (i.e., steps (3) - (5)). Thus, in certain preferred embodiments, the process of the invention is carried out by the following scheme: repeating steps (1) - (2) one or more times, and prior to each repetition, performing a nucleic acid denaturation step; followed by steps (3) - (5).

In certain preferred embodiments, steps (1) - (4) are repeated one or more times, and prior to each repetition, a step of nucleic acid denaturation is performed. It will be readily appreciated that repetition of steps (1) - (4) may result in more duplexes of extension products comprising the detection probe and the mediator fragment for use in subsequent steps (i.e., step (5)). Thus, in certain preferred embodiments, the process of the invention is carried out by the following scheme: repeating steps (1) - (4) one or more times, and prior to each repetition, performing a nucleic acid denaturation step; step (5) is then performed.

In certain preferred embodiments, steps (1) - (4) of the method of the invention may be carried out by a protocol comprising the following steps (a) - (f):

(a) Providing a detection probe and, for each target nucleic acid sequence to be detected, an upstream oligonucleotide sequence, a mediator probe and a downstream oligonucleotide sequence; and, optionally, providing a universal primer; wherein the detection probe, mediator probe, upstream oligonucleotide sequence, downstream oligonucleotide sequence and universal primer are as defined above;

(b) Mixing the sample to be tested with the provided detection probe, upstream oligonucleotide sequence, mediator probe and downstream oligonucleotide sequence, and a template-dependent nucleic acid polymerase having 5' nuclease activity (e.g., a DNA polymerase, particularly a thermostable DNA polymerase); and optionally, adding a universal primer;

(c) Incubating the product of the previous step under conditions that allow denaturation of the nucleic acids;

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

(e) Incubating the product of the previous step under conditions that allow for extension of the nucleic acid; and

(f) Optionally, repeating steps (c) - (e) one or more times.

In such embodiments, in step (c), all nucleic acid molecules in the sample will dissociate into a single stranded state; subsequently, in step (d), complementary nucleic acid molecules (e.g., extension products of the upstream oligonucleotide sequence and the target nucleic acid sequence or the downstream oligonucleotide sequence, extension products of the downstream oligonucleotide sequence and the target nucleic acid sequence or the upstream oligonucleotide sequence, mediator probes and the target nucleic acid sequence or amplification products thereof, mediator probes or mediator fragments resulting from cleavage of the mediator probes and detection probes, universal primers and the upstream/downstream oligonucleotide sequence or the extension products of the upstream/downstream oligonucleotide sequence) will anneal or hybridize together to form a duplex; subsequently, in step (e), the template-dependent nucleic acid polymerase having 5 'nuclease activity will extend the upstream/downstream oligonucleotide sequences hybridized to the target nucleic acid sequence, cleave the free 5' end of the mediator probe hybridized to the target nucleic acid sequence, extend the mediator fragment hybridized to the detection probe, and extend the universal primer hybridized to the extension product of the upstream/downstream oligonucleotide sequences. Thus, by cycling through steps (c) - (e), amplification of the target nucleic acid sequence, cleavage of the mediator probe, and formation of a duplex containing the extension product of the detection probe and the mediator fragment can be achieved, thereby completing steps (1) - (4) of the method of the present invention.

It will be readily appreciated that the nucleic acid polymerase does not extend the mediator probe hybridized to the detection probe, since the target-specific sequence at the 3' end of the mediator probe is not hybridized to the detection probe, and is free. Furthermore, it is preferable that the 3' end of the mediator probe is blocked, so that undesired extension of the mediator probe, for example, extension of the mediator probe hybridized to a target nucleic acid sequence or a detection probe, can be prevented.

The incubation time and temperature of step (c) can be routinely determined by one skilled in the art. In certain preferred embodiments, in step (c), the product of step (b) is incubated at a temperature of 80-105 ℃ (e.g., 80-85 ℃,85-90 ℃,90-95 ℃,91 ℃,92 ℃,93 ℃,94 ℃,95 ℃,96 ℃,97 ℃,98 ℃,99 ℃,100 ℃,101 ℃,102 ℃,103 ℃,104 ℃, or 105 ℃) to thereby denature the nucleic acid. In certain preferred embodiments, in step (c), the product of step (b) is incubated for 10s to 5min, e.g., 10 to 20s,20 to 40s,40 to 60s,1 to 2min, or 2 to 5min.

The incubation time and temperature of step (d) can be routinely determined by one skilled in the art. In certain preferred embodiments, in step (d), the product of step (c) is incubated at a temperature of 35-70 ℃ (e.g., 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 preferred embodiments, in step (d), the product of step (c) is incubated for 10s to 5min, e.g., 10 to 20s,20 to 40s,40 to 60s,1 to 2min, or 2 to 5min.

The incubation time and temperature of step (e) can be routinely determined by one skilled in the art. In certain preferred embodiments, in step (e), the product of step (d) is incubated at a temperature of 35-85 ℃ (e.g., 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 preferred embodiments, in step (e), the product of step (d) is incubated for 10s to 30min, e.g., 10 to 20s,20 to 40s,40 to 60s,1 to 2min,2 to 5min,5 to 10min,10 to 20min, or 20 to 30min.

In certain embodiments, steps (d) and (e) may be performed at different temperatures, i.e., annealing and extension of the nucleic acid is performed at different temperatures. In certain embodiments, steps (d) and (e) may be performed at the same temperature, i.e., annealing and extension of the nucleic acid may be performed at the same temperature. In this case, steps (d) and (e) may be combined into one step.

In the method of the invention, steps (c) - (e) may be 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 some cases, multiple repetitions of steps (c) - (e) are advantageous because they enable amplification of the target nucleic acid sequence, increasing the sensitivity of detection. However, it will be readily appreciated that the conditions used in steps (c) - (e) for each cycle need not be the same when steps (c) - (e) are repeated one or more times. For example, one condition may be used to perform steps (c) - (e) of the first 5 cycles, followed by another condition to perform steps (c) - (e) of the remaining cycles.

Step (5)

In step (5) of the method according to the invention, the product of step (4) is subjected to a melting curve analysis; and determining whether each of the target nucleic acid sequences is present in the sample based on the results of the melting curve analysis.

As discussed above, melting curve analysis can be performed by using a detection probe labeled with a reporter group and a quencher group.

In certain embodiments, the product of step (4) may be subjected to a gradual temperature increase and the signal emitted by the reporter group on the detection probe monitored in real time to obtain a plot of the signal intensity of the product of step (4) as a function of temperature. For example, the product of step (4) can be gradually warmed from a temperature of 45 ℃ or less (e.g., no more than 45 ℃, no more than 40 ℃, no more than 35 ℃, no more than 30 ℃, no more than 25 ℃) to a temperature of 75 ℃ or more (e.g., at least 75 ℃, at least 80 ℃, at least 85 ℃, at least 90 ℃, at least 95 ℃) and the signal emitted by the reporter group on the detection probe can be monitored in real time to obtain a curve of the intensity of the signal from the reporter group as a function of temperature. The rate of temperature rise may be routinely determined by one skilled in the art. For example, the rate of temperature rise may be: heating at 0.01-1 deg.C per step (e.g., 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, e.g., 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.C, 0.9 deg.C, or 1.0.0 deg.C), and maintaining at 0.5-15s per step (e.g., 0.5-1s,1-2s,2-3s,3-4s,4-5s,5-10s,10-15 s); or raising the temperature at 0.01-1 deg.C (e.g., 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, e.g., 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.8 deg.C, 0.9 deg.C, or 1.0 deg.C) per second.

In certain embodiments, the product of step (4) may be gradually cooled and the signal from the reporter group on the detection probe monitored in real time to obtain a plot of the signal intensity of the product of step (4) as a function of temperature. For example, the product of step (4) can be gradually cooled from a temperature of 75 ℃ or more (e.g., at least 75 ℃, at least 80 ℃, at least 85 ℃, at least 90 ℃, at least 95 ℃) to a temperature of 45 ℃ or less (e.g., not more than 45 ℃, not more than 40 ℃, not more than 35 ℃, not more than 30 ℃, not more than 25 ℃) and the signal emitted by the reporter on the detection probe can be monitored in real time to obtain a curve of the intensity of the signal from the reporter as a function of temperature. The rate of temperature reduction may be routinely determined by those skilled in the art. For example, the rate of cooling may be: cooling at 0.01-1 deg.C (e.g., 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, e.g., 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.C, 0.9 deg.C, or 1.0.0.0 deg.C per step, and maintaining at 0.5-15s (e.g., 0.5-1s,1-2s,2-3s,3-4s,4-5s,5-10s,10-15 s); or reducing the temperature by 0.01-1 deg.C per second (e.g., 0.01-0.05 deg.C, 0.05-0.1 deg.C, 0.1-0.5 deg.C, 0.5-1 deg.C, 0.04-0.4 deg.C, e.g., 0.01 deg.C, 0.02 deg.C, 0.03 deg.C, 0.04 deg.C, 0.05 deg.C, 0.06 deg.C, 0.07 deg.C, 0.08 deg.C, 0.09 deg.C, 0.1 deg.C, 0.2 deg.C, 0.3 deg.C, 0.4 deg.C, 0.5 deg.C, 0.6 deg.C, 0.7 deg.C, 0.8 deg.C, 0.9 deg.C, or 1.0 deg.C).

Subsequently, the obtained curve may be derived to obtain a melting curve of the product of step (4). From the melting peak (melting point) in the melting curve, the presence of a media sub-segment corresponding to the melting peak (melting point) can be determined. Subsequently, by the correspondence of the mediator sequence in the mediator fragment to the target nucleic acid sequence, the presence of the target nucleic acid sequence corresponding to the mediator fragment, and thus the presence of the target corresponding to the target nucleic acid sequence, can be determined.

For example, when the results of the melting curve analysis show the presence or absence of a melting peak corresponding to a first duplex comprising the detection probe and the first mediator fragment extension product, the presence or absence of the first target nucleic acid sequence/first target in the sample can be determined. Similarly, when the results of the melting curve analysis show the presence or absence of a melting peak corresponding to a second duplex comprising a detection probe and a second mediator fragment extension product, the presence or absence of a second target nucleic acid sequence/second target in the sample can be determined. When the results of the melting curve analysis show the presence or absence of a melting peak corresponding to a third duplex comprising the detection probe and a third mediator fragment extension product, the presence or absence of a third target nucleic acid sequence/third target in the sample can be determined. The presence or absence of a fourth target nucleic acid sequence/fourth target in the sample may be determined when the results of the melting curve analysis show the presence or absence of a melting peak corresponding to a fourth duplex comprising the detection probe and a fourth mediator fragment extension product. Thus, the methods of the invention allow for the simultaneous detection (multiplexed detection) of at least two (e.g., 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) target nucleic acid sequences/targets by using one detection probe and at least two (e.g., 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mediator probes.

Without being bound by theory, the resolution or accuracy of the melting curve analysis may reach 0.5 ℃ or higher. In other words, melting curve analysis can distinguish two melting peaks whose melting points differ by only 0.5 ℃ or less (e.g., 0.1 ℃,0.2 ℃, 0.3 ℃, 0.4 ℃, 0.5 ℃). Thus, in certain embodiments of the methods of the invention, the difference in melting point between any two duplexes (e.g., a first duplex and a second duplex) can be at least 0.5 ℃ (e.g., by designing the sequences of the first mediator subsequence, the second mediator subsequence, and the detection probe) such that the any two duplexes (e.g., the first duplex and the second duplex) can be distinguished and distinguished by melting curve analysis. However, for the purpose of facilitating differentiation and discrimination, a greater difference in melting point of the two duplexes (e.g., the first duplex and the second duplex) may be preferred in some circumstances. Thus, in certain embodiments of the methods of the invention, the difference in melting point between two duplexes (e.g., a first duplex and a second duplex) can be any desired value (e.g., at least 0.5 ℃, at least 1 ℃, at least 2 ℃, at least 3 ℃, at least 4 ℃, at least 5 ℃, at least 8 ℃, at least 10 ℃, at least 15 ℃, or at least 20 ℃) so long as the difference in melting point can be distinguished and distinguished by melt curve analysis.

Simultaneous use of one or more detection probes

In the methods described above, multiplex detection of multiple target nucleic acid sequences/targets is achieved using one detection probe. However, it will be readily appreciated that the method of the invention is not limited to the number of detection probes used. The methods of the invention can use one or more detection probes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or more detection probes). Also, based on the same principle as described above, at least two or more kinds (e.g., 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more kinds of detection probes) of mediator probes can be designed for each detection probe, whereby the method of the present invention can be used to simultaneously detect the presence of a plurality of target nucleic acid sequences/targets, and the maximum number of target nucleic acid sequences/targets that can be simultaneously detected by the method of the present invention far exceeds the number of detection probes used, and is equal to the sum of the number of mediator probes designed for each detection probe (i.e., the number of all mediator probes used). Furthermore, it is readily understood that one or more mediator probes may be designed for each target nucleic acid sequence/target. Thus, the actual number of target nucleic acid sequences/targets that can be simultaneously detected by the methods of the invention can be equal to or less than the number of all mediator probes used, while still being greater than the number of detection probes used.

Thus, in certain embodiments, the invention provides a method of detecting the presence of n target nucleic acid sequences/targets in a sample, wherein n is an integer greater than or equal to 2 (e.g., n is an integer of 2,3,4,5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or greater), and each target is independently a respiratory pathogen selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi that can infect the respiratory tract, and the method comprises the steps of:

(1) For each target to be detected, determining at least one target nucleic acid sequence specific for the target; then, for each target nucleic acid sequence, providing an upstream oligonucleotide sequence and a mediator probe; wherein the upstream oligonucleotide sequence comprises a sequence complementary to the target nucleic acid sequence; and, the mediator probe comprises, in the 5 'to 3' direction, a mediator subsequence comprising a sequence that is not complementary to the target nucleic acid sequence and a target-specific sequence comprising a sequence that is complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is upstream of the target-specific sequence; and, all the mediator subsequences contained in the mediator probe are different from each other;

(3) Providing m detection probes and contacting the product of step (2) with the m detection probes under conditions that allow nucleic acid hybridization,

m is an integer less than n and greater than 0, and

each detection probe independently comprises, in the 3 'to 5' direction, one or more capture sequences complementary to one or more mediator sequences or portions thereof, and a template sequence (mapping sequence); and, the m detection probes comprise a plurality (e.g., at least n) of capture sequences that are complementary to the mediator subsequence, or portion thereof, of each of the mediator sub-probes provided in step (1), respectively; and the number of the first and second electrodes,

each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and each detection probe emits a signal when hybridized to its complement that is different from a signal when not hybridized to its complement; and also,

Further, in step (2) of such embodiments, when a target nucleic acid sequence is present, the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence and the mediator probe hybridize to the target nucleic acid sequence, but the mediator sequence in the mediator probe is in a free state and does not hybridize to the target nucleic acid sequence. In this case, under the action of the enzyme having 5' nuclease activity, the mediator sequence or a portion thereof in the mediator probe (corresponding to the target nucleic acid sequence) is cleaved from the mediator probe by the presence of the upstream oligonucleotide sequence corresponding to the target nucleic acid sequence or an extension product thereof, to form a mediator fragment corresponding to the target nucleic acid sequence.

Further, in steps (3) and (4) of such embodiments, when a mediator fragment corresponding to a certain target nucleic acid sequence is present, the mediator fragment hybridizes to a complementary detection probe (i.e., a detection probe containing a capture sequence complementary to the mediator sequence or a portion thereof in the mediator fragment), and the nucleic acid polymerase will extend the mediator fragment using the complementary detection probe as a template to form a duplex corresponding to the target nucleic acid sequence.

It will be readily appreciated that in the method of the invention, the m detection probes comprise a plurality of capture sequences, the collection of which encompasses the complement of the mediator sub-sequences or part thereof of all of the mediator sub-probes provided in step (1), whereby the m detection probes or the plurality of capture sequences are capable of "capturing" mediator fragments cleaved from any mediator sub-probe. That is, any mediator fragment that is cleaved from the mediator probe is capable of hybridizing to at least one detection probe or at least one capture sequence.

Further, in step (5) of such embodiments, when a melting peak of a duplex corresponding to a certain target nucleic acid sequence is detected or not detected, the presence or absence of the target nucleic acid sequence in the sample is determined, and thereby the presence or absence of a target corresponding to the target nucleic acid sequence in the sample is determined.

In certain embodiments, in step (1), for each target to be detected, one (or more) target nucleic acid sequence(s) specific for that target is/are determined, and accordingly, n (or more) mediator probes are provided, each for one target nucleic acid sequence; subsequently, in step (3), the m detection probes comprise n (or more) capture sequences that are complementary to the mediator subsequences or portions, respectively, of the n (or more) mediator subsequences provided in step (1); thus, any one of the mediator fragments produced in step (2) is capable of hybridizing to at least one detection probe comprising a capture sequence complementary to a mediator sequence or part thereof in that mediator fragment and forming a duplex for subsequent extension and detection. In certain exemplary embodiments, the m detection probes comprise n capture sequences that are complementary to the mediator sequences or portions of the n mediator probes, respectively.

In certain preferred embodiments, the m detection probes do not comprise the same capture sequence as each other. In this case, for each mediator probe, there is one and only one detection probe (which contains a capture sequence complementary to the mediator subsequence in the mediator probe) that hybridizes to the mediator fragment from the mediator probe and, after the extension reaction, only one duplex is generated. Subsequently, by detecting the presence of the duplex in step (5), the presence of the target nucleic acid sequence corresponding to the mediator probe can be determined.

In certain preferred embodiments, the m detection probes may comprise the same capture sequence as each other. In this case, for each mediator probe, there may be one or more detection probes (which all comprise a capture sequence complementary to the mediator sequence in the mediator probe) that hybridize to the mediator fragment from the mediator probe and, after the extension reaction, generate one or more duplexes. Subsequently, by detecting the presence of the one or more duplexes in step (5), the presence of the target nucleic acid sequence corresponding to the mediator probe can be determined.

In step (5) of such embodiments, the duplexes may be distinguished and distinguished by their melting points and/or a reporter group in the detection probe. In certain preferred embodiments, the m detection probes comprise the same reporter group. In this case, the product of step (4) may be subjected to melting curve analysis, and the presence of a certain duplex may be determined from the melting peak (melting point) in the melting curve, and the presence of a target nucleic acid sequence corresponding to the duplex may be determined. In certain preferred embodiments, the m detection probes comprise different reporter groups than each other. In this case, when the product of step (4) is subjected to melting curve analysis, the signal of each reporter group can be monitored separately in real time, thereby obtaining a plurality of melting curves each corresponding to the signal of one reporter group. The presence of a duplex, and hence the target nucleic acid sequence corresponding to that duplex, can then be determined based on the signal type of the reporter and the melting peak (melting point) in the melting curve.

In certain exemplary embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10 detection probes can be used (i.e., m is an integer of ≧ 1, ≧ 2, ≧ 3, ≧ 4, ≧ 5, ≧ 6, ≧ 8, ≧ 10). In certain exemplary embodiments, 1-10 detection probes (i.e., m is an integer from 1-10; e.g., m is 1, 2,3,4,5, 6,7,8,9, or 10) can be used. Further preferably, the detection probes used are each labeled with a different reporter group.

For example, in certain exemplary embodiments, the methods of the present invention may use first and second detection probes that are labeled with a first reporter group and a second reporter group, respectively. Thus, in step (5), the change in the signal of the first reporter group and the second reporter group with temperature is monitored in real time, respectively, to obtain a first melting curve and a second melting curve. Subsequently, from the melting peak in the first (or second) melting curve, the presence of the duplex comprising the first (or second) detection probe can be determined, and thereby the presence of the target nucleic acid sequence corresponding to the mediator fragment hybridized to the first (or second) detection probe can be determined.

In certain exemplary embodiments, the methods of the invention employ at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, or at least 10 detection probes; and, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes. Thus, the methods of the invention can achieve simultaneous detection (multiplex detection) of multiple target nucleic acid sequences, where the maximum number of detectable target nucleic acid sequences is equal to the number of mediator sub-probes used.

For example, in certain exemplary embodiments, the methods of the invention achieve simultaneous detection of 2-6 (e.g., 2,3,4,5, or 6) targets using 1 detection probe and 2-6 (e.g., 2,3,4,5, or 6) mediator sub-probes. In certain exemplary embodiments, the methods of the invention use 2 detection probes and 3-12 (e.g., 3,4,5,6,7,8,9, 10, 11, 12) mediator probes to achieve simultaneous detection of 3-12 targets. In certain exemplary embodiments, the methods of the invention achieve simultaneous detection of 4-18 (e.g., 5-10) targets using 3 detection probes and 4-18 (e.g., 5-10) mediator sub-probes. In certain exemplary embodiments, the methods of the invention use 4 detection probes and 5-24 (e.g., 6-12) mediator sub-probes, enabling simultaneous detection of 5-24 (e.g., 6-12) targets. In certain exemplary embodiments, the methods of the invention use 5 detection probes and 6-30 (e.g., 8-15) mediator sub-probes, enabling simultaneous detection of 6-30 (e.g., 8-15) targets. In certain exemplary embodiments, the methods of the invention use 6 detection probes and 7-36 (e.g., 10-18) mediator sub-probes, enabling simultaneous detection of 7-36 (e.g., 10-18) targets. In certain exemplary embodiments, the methods of the invention achieve simultaneous detection of 8-42 (e.g., 12-20, e.g., 19) targets using 7 detection probes and 8-42 (e.g., 12-20) mediator sub-probes.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: the detection probe shown as SEQ ID NO. 2, the detection probe shown as SEQ ID NO. 12, the detection probe shown as SEQ ID NO. 22, the detection probe shown as SEQ ID NO. 32, the detection probe shown as SEQ ID NO. 42, the detection probe shown as SEQ ID NO. 52, the detection probe shown as SEQ ID NO. 62, or any combination thereof.

In certain exemplary embodiments, the mediator probe used in the methods of the present invention comprises: the vector sub-probe shown in SEQ ID NO. 5, the vector sub-probe shown in SEQ ID NO. 8, the vector sub-probe shown in SEQ ID NO. 11, the vector sub-probe shown in SEQ ID NO. 15, the vector sub-probe shown in SEQ ID NO. 18, the vector sub-probe shown in SEQ ID NO. 21, the vector sub-probe shown in SEQ ID NO. 25, the vector sub-probe shown in SEQ ID NO. 28, the vector sub-probe shown in SEQ ID NO. 31, the vector sub-probe shown in SEQ ID NO. 35, the vector sub-probe shown in SEQ ID NO. 38, the vector sub-probe shown in SEQ ID NO. 41, the vector sub-probe shown in SEQ ID NO. 45, the vector sub-probe shown in SEQ ID NO. 48, the vector sub-probe shown in SEQ ID NO. 51, the vector sub-probe shown in SEQ ID NO. 55, the vector sub-probe shown in SEQ ID NO. 58, the vector sub-probe shown in SEQ ID NO. 61, the vector sub-probe shown in SEQ ID NO. 65, the vector sub-probe shown in SEQ ID NO. 68, any combination thereof, or any combination thereof.

In certain exemplary embodiments, the upstream oligonucleotides used in the methods of the invention comprise: the upstream oligonucleotide shown as SEQ ID NO. 3, the upstream oligonucleotide shown as SEQ ID NO. 6, the upstream oligonucleotide shown as SEQ ID NO. 9, the upstream oligonucleotide shown as SEQ ID NO. 13, the upstream oligonucleotide shown as SEQ ID NO. 16, the upstream oligonucleotide shown as SEQ ID NO. 19, the upstream oligonucleotide shown as SEQ ID NO. 23, the upstream oligonucleotide shown as SEQ ID NO. 26, the upstream oligonucleotide shown as SEQ ID NO. 29, the upstream oligonucleotide shown as SEQ ID NO. 33, the upstream oligonucleotide shown as SEQ ID NO. 36, the upstream oligonucleotide shown as SEQ ID NO. 39, the upstream oligonucleotide shown as SEQ ID NO. 43, the upstream oligonucleotide shown as SEQ ID NO. 46, the upstream oligonucleotide shown as SEQ ID NO. 49, the upstream oligonucleotide shown as SEQ ID NO. 53, the upstream oligonucleotide shown as SEQ ID NO. 56, the upstream oligonucleotide shown as SEQ ID NO. 63, the upstream oligonucleotide shown as SEQ ID NO. 59, or any combination thereof.

In certain exemplary embodiments, the methods of the invention also use a downstream oligonucleotide, and the downstream oligonucleotide used comprises: the downstream oligonucleotide shown as SEQ ID NO. 4, the downstream oligonucleotide shown as SEQ ID NO. 7, the downstream oligonucleotide shown as SEQ ID NO. 10, the downstream oligonucleotide shown as SEQ ID NO. 14, the downstream oligonucleotide shown as SEQ ID NO. 17, the downstream oligonucleotide shown as SEQ ID NO. 20, the downstream oligonucleotide shown as SEQ ID NO. 24, the downstream oligonucleotide shown as SEQ ID NO. 27, the downstream oligonucleotide shown as SEQ ID NO. 30, the downstream oligonucleotide shown as SEQ ID NO. 34, the downstream oligonucleotide shown as SEQ ID NO. 37, the downstream oligonucleotide shown as SEQ ID NO. 40, the downstream oligonucleotide shown as SEQ ID NO. 44, the downstream oligonucleotide shown as SEQ ID NO. 47, the downstream oligonucleotide shown as SEQ ID NO. 50, the downstream oligonucleotide shown as SEQ ID NO. 54, the downstream oligonucleotide shown as SEQ ID NO. 57, the downstream oligonucleotide shown as SEQ ID NO. 60, or any combination thereof.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 2, and the mediator probe comprises: 5, 8 and 11 as shown in SEQ ID NO. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 3 upstream oligonucleotides as shown in

SEQ ID NO

3, 6 and 9, respectively. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 4, 7 and 10 shown in SEQ ID NO of 3 downstream oligonucleotides. Such embodiments are useful, for example, for detecting rickettsia, streptococcus pneumoniae, and haemophilus influenzae.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 12 and the mediator probe comprises: and 3 medium sub-probes shown in

SEQ ID NO

15, 18 and 21. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 13, 16 and 19, respectively, as shown in SEQ ID NO. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 14, 17 and 20 shown in SEQ ID NO:3 downstream oligonucleotides. Such embodiments are useful, for example, for detecting moraxella catarrhalis, acinetobacter baumannii, and klebsiella pneumoniae.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 22, and the used mediator probe comprises: and 3 kinds of vector sub-probes shown in SEQ ID NO 25, 28 and 31. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 3 upstream oligonucleotides as shown in SEQ ID NO 23, 26 and 29, respectively. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 24, 27 and 30, respectively, as shown in SEQ ID NO. Such embodiments are useful, for example, for detecting staphylococcus epidermidis, chlamydia pneumoniae, and staphylococcus aureus.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 32, and the mediator probe comprises: 35, 38 and 41 of the 3 mediator probes respectively. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 33, 36 and 39, respectively. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 34, 37 and 40 of the nucleic acid sequences shown in SEQ ID NO. Such embodiments are useful, for example, for detecting mycoplasma pneumoniae, candida albicans, and bordetella pertussis.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 42 and the mediator probe comprises: and 3 medium sub-probes shown in SEQ ID NO 45, 48 and 51. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 43, 46 and 49, respectively, as shown in SEQ ID NO. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 44, 47 and 50 of the shown in SEQ ID NO. Such embodiments are useful, for example, for detecting E.coli, legionella pneumophila, and Cryptococcus.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 52, and the mediator probe comprises: and 3 mediator probes shown in SEQ ID NO 55, 58 and 61, respectively. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 53, 56 and 59 in SEQ ID NO:3 upstream oligonucleotides. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 54, 57 and 60, respectively. Such embodiments are useful, for example, in staphylococcus haemolyticus, pseudomonas aeruginosa, and streptococcus pyogenes.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: the detection probe shown as SEQ ID NO. 62, and the medium sub-probe used comprises: 2 mediator sub-probes as shown in SEQ ID NO 65 and 68, respectively. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 63 and 66, respectively. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 2 downstream oligonucleotides as shown in SEQ ID NO 64 and 67, respectively. Such embodiments may be used, for example, to detect human ribonuclease P (used as a control) and/or Aspergillus fumigatus.

In certain exemplary embodiments, the detection probes used in the methods of the invention comprise: 2, 12, 22, 32, 42, 52 and 62, respectively, and the mediator sub-probes used comprise: 5, 8, 11, 15, 18, 21, 25, 28, 31, 35, 38, 41, 45, 48, 51, 55, 58, 61, 65 and 68 of the medium sub-probes shown in SEQ ID NO. Preferably, the upstream oligonucleotide used in the method of the invention comprises: 3, 6, 9, 13, 16, 19, 23, 26, 29, 33, 36, 39, 43, 46, 49, 53, 56, 59, 63 and 66 of the SEQ ID NO. More preferably, the method of the invention also uses a downstream oligonucleotide, and the downstream oligonucleotide used comprises: 4, 7, 10, 14, 17, 20, 24, 27, 30, 34, 37, 40, 44, 47, 50, 54, 57, 60 and 64, respectively. More preferably, the method also uses a universal primer (e.g., a universal primer as shown in SEQ ID NO: 1). Such embodiments can be used, for example, to detect staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus haemolyticus, or any combination thereof.

It will also be readily appreciated that various technical features described in detail for a method using one detection probe are equally applicable to a method using two or more detection probes. For example, the various detailed descriptions above for the sample to be detected, the target nucleic acid sequence, the mediator probe, the upstream oligonucleotide sequence, the downstream oligonucleotide sequence, the universal primer, the detection probe, the conditions that allow nucleic acid hybridization, the conditions that allow cleavage of the mediator probe, the enzyme with 5' nuclease activity, the conditions that allow the nucleic acid polymerase to perform an extension reaction, the nucleic acid polymerase, melting curve analysis, repetition of steps, and the like, can be applied to methods that use two or more detection probes. Thus, in certain preferred embodiments, the methods of the invention using two or more detection probes may involve any one or more of the features, or any combination of the features, as described in detail above.

For example, as described above, one or more of steps (1) - (4) may be repeated as desired. In certain preferred embodiments, steps (1) - (2) are repeated one or more times, and prior to each repetition, a step of nucleic acid denaturation is performed. It will be readily appreciated that repetition of steps (1) - (2) may result in more media sub-segments for subsequent steps (i.e., steps (3) - (5)). Thus, in certain preferred embodiments, the process of the invention is carried out by the following scheme: repeating steps (1) - (2) one or more times, and before each repetition, performing a step of nucleic acid denaturation; followed by steps (3) - (5).

In certain preferred embodiments, steps (1) - (4) are repeated one or more times, and prior to each repetition, a step of nucleic acid denaturation is performed. It will be readily appreciated that repetition of steps (1) - (4) may produce more duplexes of extension products comprising the detection probe and the mediator fragment for use in subsequent steps (i.e., step (5)). Thus, in certain preferred embodiments, the process of the invention is carried out by the following scheme: repeating steps (1) - (4) one or more times, and prior to each repetition, performing a nucleic acid denaturation step; step (5) is then performed.

(a) Providing m detection probes, and for each target nucleic acid sequence to be detected, providing an upstream oligonucleotide sequence, a mediator probe and a downstream oligonucleotide sequence; and, optionally, providing a universal primer; wherein the detection probe, mediator probe, upstream oligonucleotide sequence, downstream oligonucleotide sequence and universal primer are as defined above;

(f) Optionally, repeating steps (c) - (e) one or more times.

With respect to steps (a) - (f), these have been described in detail above.

Optional step (6) and quantitative/semi-quantitative detection

The method of the invention can be used for qualitative detection of the target, and can also be used for quantitative or semi-quantitative detection of the target level. It will be readily appreciated that as the amount of a target in a sample increases, the amount of its specific target nucleic acid sequence increases, and accordingly, the number of mediator probes that hybridize to the target nucleic acid sequence in step (1) increases; furthermore, the more mediator sub-probes are cleaved in step (2), the more mediator fragments are released; furthermore, in steps (3) and (4), the more the fragment of the mediator that hybridizes to the detection probe, the more duplexes that are produced by the extension reaction; furthermore, in step (5), the more duplexes that can be subjected to melting curve analysis, the stronger the signal generated, and the higher the height of the melting peak obtained. Thus, by the relative height of the melting peaks, the content/level of the corresponding target in the sample can be judged (quantitative or semi-quantitative detection). Thus, the methods of the invention can be used not only to detect the presence of two or more targets in a sample, but also to detect the levels of the two or more targets in a sample.

Thus, in certain preferred embodiments, the method of the present invention further comprises the steps of:

(6) From the results of the melting curve analysis (in particular, the peak heights of the melting peaks in the melting curve), the levels of the targets corresponding to the respective melting peaks are determined.

Probe needleKit and kit

In another aspect, the present invention provides a probe set (probe set) comprising a detection probe and at least two mediator sub-probes, wherein,

each of said mediator probes independently comprises in the 5 'to 3' direction a mediator subsequence comprising a sequence complementary to a target nucleic acid sequence specific to a target or a control sequence and a target-specific sequence comprising a sequence not complementary to said target nucleic acid sequence or control sequence, and each target independently is a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract), and the mediator subsequences comprised by all mediator probes are different from each other; and

the detection probe comprises, in the 3 'to 5' direction, a capture sequence complementary to each mediator sequence or a portion thereof, and a template sequence (templating sequence); and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and wherein the detection probe emits a signal when hybridized to its complement that is different from a signal when not hybridized to its complement.

In certain preferred embodiments, the set of probes comprises 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, at least 12, at least 15, or at least 20 mediator probes.

It will be readily appreciated that such a set of probes 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 mediator and detection probes are equally applicable to the mediator and detection probes in the probe set. Thus, in certain preferred embodiments, the set of probes comprises a mediator probe as defined above. In certain preferred embodiments, the set of probes comprises detection probes as defined above.

In certain preferred embodiments, all mediator probes each target a different target nucleic acid sequence. In certain preferred embodiments, all of the mediator sequences contained in the mediator probes are different from each other; and, all mediator probes contain target-specific sequences that are different from each other. In certain preferred embodiments, the different target nucleic acid sequences may each be specific for the same or different targets. In certain preferred embodiments, at least one mediator probe (or the target-specific sequence it comprises) targets a control sequence. In certain preferred embodiments, the control sequence is a host-specific sequence, such as a human-specific sequence. In certain preferred embodiments, the control sequence is the gene sequence of human ribonuclease P.

In certain preferred embodiments, the panel of probes comprises 2,3,4,5, 6,7,8,9, 10 or more mediator probes. Preferably, the mediator probes (or target-specific sequences they comprise) target specific nucleic acid sequences of 2,3,4,5, 6,7,8,9, 10 or more targets. Each target is independently a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract). In the present application, respiratory pathogens include, but are not limited to, staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus haemolyticus, aspergillus flavus, candida tropicalis, candida glabrata, enterobacter cloacae, enterococcus mirabilis, pseudomonas maltophilia, bordetella parapertussis, chlamydia psittaci, or any combination thereof. Preferably, the targets are each independently selected from staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus haemolyticus, or any combination thereof.

In certain embodiments of the invention, the at least one respiratory pathogen is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia, moraxella catarrhalis, cryptococcus, legionella pneumophila, aspergillus flavus, candida tropicalis, candida glabrata, bordetella parapertussis, chlamydia psittaci. In certain embodiments of the invention, the at least one respiratory pathogen is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia, moraxella catarrhalis, cryptococcus and legionella pneumophila.

In certain preferred embodiments, the probe set comprises 1 detection probe, and 2-6 (e.g., 2,3,4,5, or 6) mediator probes. Thus, the probe set can be used to detect 2-6 (e.g., 2,3,4,5, or 6) targets simultaneously.

In certain preferred embodiments, the set of probes further comprises an upstream oligonucleotide sequence as defined above. For example, an upstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence can be provided for each target nucleic acid sequence and mediator probe; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is located upstream of the target-specific sequence of the mediator probe.

In certain preferred embodiments, the set of probes further comprises a downstream oligonucleotide sequence as defined above. For example, a downstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence can be provided for each target nucleic acid sequence and mediator probe; and, when hybridized to the target nucleic acid sequence, the downstream oligonucleotide sequence is located downstream of the target-specific sequence of the mediator probe.

In certain preferred embodiments, the probe set further comprises a universal primer as defined above. For example, in certain preferred embodiments, the upstream oligonucleotide sequence and the downstream oligonucleotide sequence comprise an identical oligonucleotide sequence 5' to each other; thus, the probe set may further comprise a universal primer having a sequence complementary to the same oligonucleotide sequence.

In certain preferred embodiments, the set of probes further comprises an upstream oligonucleotide sequence and a downstream oligonucleotide sequence as defined above. In certain preferred embodiments, the probe set further comprises an upstream oligonucleotide sequence, a downstream oligonucleotide sequence and a universal primer as defined above.

In certain exemplary embodiments, the set of probes comprises detection probes selected from the group consisting of: the detection probe shown as SEQ ID NO. 2, the detection probe shown as SEQ ID NO. 12, the detection probe shown as SEQ ID NO. 22, the detection probe shown as SEQ ID NO. 32, the detection probe shown as SEQ ID NO. 42, the detection probe shown as SEQ ID NO. 52 and the detection probe shown as SEQ ID NO. 62.

In certain exemplary embodiments, the set of probes comprises a mediator probe selected from the group consisting of: the mediator probe shown in SEQ ID NO. 5, the mediator probe shown in SEQ ID NO. 8, the mediator probe shown in SEQ ID NO. 11, the mediator probe shown in SEQ ID NO. 15, the mediator probe shown in SEQ ID NO. 18, the mediator probe shown in SEQ ID NO. 21, the mediator probe shown in SEQ ID NO. 25, the mediator probe shown in SEQ ID NO. 28, the mediator probe shown in SEQ ID NO. 31, the mediator probe shown in SEQ ID NO. 35, the mediator probe shown in SEQ ID NO. 38, the mediator probe shown in SEQ ID NO. 41, the mediator probe shown in SEQ ID NO. 45, the mediator probe shown in SEQ ID NO. 48, the mediator probe shown in SEQ ID NO. 51, the mediator probe shown in SEQ ID NO. 55, the mediator probe shown in SEQ ID NO. 58, the mediator probe shown in SEQ ID NO. 61, the mediator probe shown in SEQ ID NO. 65, the mediator probe shown in SEQ ID NO. 68, or any combination thereof.

In certain exemplary embodiments, the set of probes further comprises an upstream oligonucleotide selected from the group consisting of: the upstream oligonucleotide shown in SEQ ID NO. 3, the upstream oligonucleotide shown in SEQ ID NO. 6, the upstream oligonucleotide shown in SEQ ID NO. 9, the upstream oligonucleotide shown in SEQ ID NO. 13, the upstream oligonucleotide shown in SEQ ID NO. 16, the upstream oligonucleotide shown in SEQ ID NO. 19, the upstream oligonucleotide shown in SEQ ID NO. 23, the upstream oligonucleotide shown in SEQ ID NO. 26, the upstream oligonucleotide shown in SEQ ID NO. 29, the upstream oligonucleotide shown in SEQ ID NO. 33, the upstream oligonucleotide shown in SEQ ID NO. 36, the upstream oligonucleotide shown in SEQ ID NO. 39, the upstream oligonucleotide shown in SEQ ID NO. 43, the upstream oligonucleotide shown in SEQ ID NO. 46, the upstream oligonucleotide shown in SEQ ID NO. 49, the upstream oligonucleotide shown in SEQ ID NO. 53, the upstream oligonucleotide shown in SEQ ID NO. 56, the upstream oligonucleotide shown in SEQ ID NO. 59, or any combination thereof.

In certain exemplary embodiments, the set of probes further comprises a downstream oligonucleotide selected from the group consisting of: the downstream oligonucleotide shown in SEQ ID NO. 4, the downstream oligonucleotide shown in SEQ ID NO. 7, the downstream oligonucleotide shown in SEQ ID NO. 10, the downstream oligonucleotide shown in SEQ ID NO. 14, the downstream oligonucleotide shown in SEQ ID NO. 17, the downstream oligonucleotide shown in SEQ ID NO. 20, the downstream oligonucleotide shown in SEQ ID NO. 24, the downstream oligonucleotide shown in SEQ ID NO. 27, the downstream oligonucleotide shown in SEQ ID NO. 30, the downstream oligonucleotide shown in SEQ ID NO. 34, the downstream oligonucleotide shown in SEQ ID NO. 37, the downstream oligonucleotide shown in SEQ ID NO. 40, the downstream oligonucleotide shown in SEQ ID NO. 44, the downstream oligonucleotide shown in SEQ ID NO. 47, the downstream oligonucleotide shown in SEQ ID NO. 50, the downstream oligonucleotide shown in SEQ ID NO. 54, the downstream oligonucleotide shown in SEQ ID NO. 57, the downstream oligonucleotide shown in SEQ ID NO. 60, or any combination thereof.

In certain exemplary embodiments, the set of probes (hereinafter referred to simply as the first set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 2, and 3 medium sub-probes shown as SEQ ID NO. 5, 8 and 11 respectively. Preferably, the first set of probes further comprises: 3 upstream oligonucleotides as shown in

SEQ ID NO

3, 6 and 9, respectively. More preferably, the first set of probes further comprises: 4, 7 and 10 shown in SEQ ID NO of 3 downstream oligonucleotides. Such probe sets are useful, for example, for detecting rickettsia, streptococcus pneumoniae, and haemophilus influenzae.

In certain exemplary embodiments, the set of probes (hereinafter referred to simply as the second set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 12, and 3 kinds of vector sub-probes shown as SEQ ID NO. 15, 18 and 21 respectively. Preferably, the second set of probes further comprises: 13, 16 and 19, respectively, as shown in SEQ ID NO:3 upstream oligonucleotides. More preferably, the second set of probes further comprises: 14, 17 and 20, respectively, as shown in SEQ ID NO. Such probe sets can be used, for example, to detect Moraxella catarrhalis, acinetobacter baumannii, and Klebsiella pneumoniae.

In certain exemplary embodiments, the set of probes (hereinafter referred to simply as the third set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 22, and 3 medium sub-probes shown as SEQ ID NO. 25, 28 and 31 respectively. Preferably, the third set of probes further comprises: 23, 26 and 29 respectively as shown in SEQ ID NO. More preferably, the third set of probes further comprises: 24, 27 and 30 of the shown in SEQ ID NO:3 downstream oligonucleotides. Such probe sets may be used, for example, to detect staphylococcus epidermidis, chlamydia pneumoniae and staphylococcus aureus.

In certain exemplary embodiments, the set of probes (hereinafter referred to simply as the fourth set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 32, and 3 medium sub-probes shown as SEQ ID NO. 35, 38 and 41 respectively. Preferably, the fourth set of probes further comprises: 33, 36 and 39, respectively. More preferably, the fourth set of probes further comprises: 34, 37 and 40 of the nucleic acid sequences shown in SEQ ID NO. Such probe sets may be used, for example, to detect mycoplasma pneumoniae, candida albicans, and bordetella pertussis.

In certain exemplary embodiments, the set of probes (hereinafter referred to simply as the fifth set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 42, and 3 kinds of vector sub-probes shown as SEQ ID NO. 45, 48 and 51 respectively. Preferably, the fifth set of probes further comprises: 43, 46 and 49 of the upstream oligonucleotides. More preferably, the fifth set of probes further comprises: 44, 47 and 50 of the shown in SEQ ID NO. Such probe sets are useful, for example, for detecting E.coli, legionella pneumophila, and Cryptococcus.

In certain exemplary embodiments, the set of probes (hereinafter referred to as the sixth set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 52, and 3 medium sub-probes shown as SEQ ID NO. 55, 58 and 61 respectively. Preferably, the sixth set of probes further comprises: 53, 56 and 59 as shown in SEQ ID NO:3 upstream oligonucleotides. More preferably, the sixth set of probes further comprises: 54, 57 and 60, respectively. Such a set of probes may be used, for example, with staphylococcus haemolyticus, pseudomonas aeruginosa and streptococcus pyogenes.

In certain exemplary embodiments, the set of probes (hereinafter referred to as the seventh set of probes for ease of distinction and description) comprises: a detection probe shown as SEQ ID NO. 62, and 2 medium sub-probes shown as SEQ ID NO. 65 and 68 respectively. Preferably, the seventh set of probes further comprises: 63 and 66, respectively. More preferably, the seventh probe set further comprises: 2 downstream oligonucleotides as shown in SEQ ID NO 64 and 67, respectively. Such a probe set can be used, for example, for the detection of human ribonuclease P (used as a control) and/or Aspergillus fumigatus.

In certain preferred embodiments, a probe set of the invention further comprises a universal primer (e.g., a universal primer as shown in SEQ ID NO: 1). For example, the first, second, third, fourth, fifth, sixth and/or seventh probe set described above may comprise universal primers as set forth in SEQ ID NO. 1.

In another aspect, the invention provides a kit comprising one or more sets of probes as defined above.

In certain preferred 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 probe sets.

In certain preferred embodiments, all of the mediator sequences in the kit are each targeted to a different target nucleic acid sequence. In certain preferred embodiments, all of the mediator sequences contained in the mediator probes in the kit are different from each other. In certain preferred embodiments, all of the mediator probes in the kit comprise target-specific sequences that are different from each other.

In certain preferred embodiments, all of the detection probes in the kit comprise the same reporter group. In certain preferred embodiments, all of the detection probes in the kit are each independently labeled with the same or different reporter groups. In certain preferred embodiments, all of the detection probes in the kit comprise a reporter group that is different from each other.

In certain preferred embodiments, the kit comprises 1-7 probe sets. Preferably, all detection probes in the kit comprise reporter groups that are the same or different from each other. Further preferably, all of the mediator probes in the kit comprise different mediator sequences from each other, and all of the mediator probes in the kit comprise different target-specific sequences from each other.

In certain preferred embodiments, the kit comprises one or more of the first to seventh probe sets described above, e.g., 1, 2,3,4,5, 6, or 7.

The present application also provides a kit comprising m detection probes and n mediator sub-probes, wherein n is an integer ≧ 2 (e.g., n is an integer of 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), m is an integer less than n and greater than 0, and,

each mediator sub-probe independently comprises in the 5 'to 3' direction a mediator sub-sequence and a target specific sequence, the target specific sequence comprising a sequence complementary to a target nucleic acid sequence or a control sequence specific for a target, the mediator sub-sequence comprising a sequence that is not complementary to the target nucleic acid sequence or the control sequence, and each target independently is a respiratory pathogen (i.e., selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract), and the mediator sub-sequences comprised by all mediator sub-probes are different from each other; and

each detection probe independently comprises, in the 3 'to 5' direction, one or more capture sequences complementary to one or more mediator sequences or portions thereof, and a template sequence (templating sequence); and, the m detection probes comprise a plurality (e.g., at least n) of capture sequences that are complementary to the mediator sequences, or portions thereof, of each mediator probe, respectively; and the number of the first and second electrodes,

each detection probe is independently labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and, each detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement.

In an exemplary embodiment of the invention, the m detection probes comprise a plurality of capture sequences, the collection of said plurality of capture sequences covering the complement of the mediator subsequence or part thereof of all the mediator sub-probes provided in step (1), whereby said m detection probes or said plurality of capture sequences are capable of "capturing" mediator fragments cleaved from any mediator sub-probe. That is, any mediator fragment that is cleaved from the mediator probe is capable of hybridizing to at least one detection probe or at least one capture sequence.

In certain exemplary embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10 detection probes (i.e., m is an integer of ≧ 1, ≧ 2, ≧ 3, ≧ 4, ≧ 5, ≧ 6, ≧ 8, ≧ 10). In certain exemplary embodiments, the kit comprises 1-10 detection probes (i.e., m is an integer from 1-10; e.g., m is 1, 2,3,4,5, 6,7,8,9, or 10). Further preferably, the detection probes are each labeled with the same or different reporter groups.

In certain exemplary embodiments, the kit comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, or at least 10 detection probes; and, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 mediator probes. Thus, the kit can be used for the simultaneous detection of multiple target nucleic acid sequences/targets, where the maximum number of detectable target nucleic acid sequences/targets is equal to the number of mediator probes used.

For example, in certain exemplary embodiments, the kit comprises 1 detection probe and 2-6 (e.g., 2,3,4,5, or 6) mediator probes, and can be used to simultaneously detect 2-6 (e.g., 2,3,4,5, or 6) targets. In certain exemplary embodiments, the kit comprises 2 detection probes and 3-12 (e.g., 3,4,5,6,7,8,9, 10, 11, 12) mediator probes, which can be used to simultaneously detect 3-12 targets. In certain exemplary embodiments, the kit comprises 3 detection probes and 4-18 (e.g., 5-10) mediator probes, and can be used for simultaneous detection of 4-18 (e.g., 5-10) targets. In certain exemplary embodiments, the kit comprises 4 detection probes and 5-24 (e.g., 6-12) mediator probes, and can be used to simultaneously detect 5-24 (e.g., 6-12) targets. In certain exemplary embodiments, the kit comprises 5 detection probes and 6-30 (e.g., 8-15) mediator probes, and can be used for simultaneous detection of 6-30 (e.g., 8-15) targets. In certain exemplary embodiments, the kit comprises 6 detection probes and 7-36 (e.g., 10-18) mediator probes, and can be used for simultaneous detection of 7-36 (e.g., 10-18) targets. In certain exemplary embodiments, the kit comprises 7 detection probes and 8-42 (e.g., 12-20) mediator probes, and can be used to simultaneously detect 8-42 (e.g., 12-20, e.g., 19) targets.

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 detailed above for the mediator and detection probes are equally applicable to the mediator and detection probes in the kit. Also, such kits may further comprise other reagents necessary to carry out the methods of the invention.

For example, in certain preferred embodiments, the kit may further comprise an upstream oligonucleotide sequence, a downstream oligonucleotide sequence, a universal primer, an enzyme having 5' nuclease activity, a nucleic acid polymerase, or any combination thereof, as defined above. In certain preferred embodiments, the kit may further comprise reagents for performing nucleic acid hybridization, reagents for performing mediator probe cleavage, reagents for performing nucleic acid extension, reagents for performing nucleic acid amplification, reagents for performing reverse transcription, or any of the sameAnd (4) combining. Such reagents can be routinely determined by one of skill in the art and include, but are not limited to, working buffers for enzymes (e.g., nucleic acid polymerases), dNTPs, water, ions containing (e.g., mg)²⁺) A Single Strand DNA-Binding Protein (SSB), or any combination thereof. For example, reagents for performing reverse transcription include, but are not limited to, reverse transcriptase working buffer, oligo d (T), dNTPs, nuclease-free water, RNase inhibitors, or any combination thereof.

Use of probe set

The present application also relates to the use of a set of probes as defined above for the preparation of a kit for detecting the presence or level of said target (respiratory pathogen) in a sample or for diagnosing whether a subject is infected with said target (respiratory pathogen).

It will be readily appreciated that the set of probes or kit 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 target, the probe set, the kit, and the various components contained therein (e.g., the mediator probe, the detection probe, the upstream oligonucleotide sequence, the downstream oligonucleotide sequence, the universal primer, the enzyme having 5' nuclease activity, the nucleic acid polymerase, the reagents for performing nucleic acid hybridization, the reagents for performing mediator probe cleavage, the reagents for performing nucleic acid extension, the reagents for performing nucleic acid amplification, the reagents for performing reverse transcription, or any combination thereof) are equally applicable thereto.

Those skilled in the art may make modifications, substitutions or combinations of various features of the invention based on the principles described in detail herein without departing from the spirit and scope of the invention. All such modifications and variations are intended to be included herein within the scope of the following claims and their equivalents.

Advantageous effects of the invention

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

(1) The methods, probe sets and kits of the invention enable the simultaneous detection (multiplex detection) of multiple target nucleic acid sequences/respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia and fungi that can infect the respiratory tract) using only one labeled probe (i.e., detection probe).

(2) The methods of the invention enable the simultaneous detection (multiplex detection) of multiple target nucleic acid sequences/respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract), and the maximum number of target nucleic acid sequences/targets that can be detected simultaneously far exceeds the number of labeled probes (i.e., detection probes) used.

Thus, the present invention provides a simple, efficient, low-cost multiplex assay that is capable of simultaneously detecting multiple respiratory pathogens (e.g., bacteria, chlamydia, mycoplasma, rickettsia, and fungi that can infect the respiratory tract). The maximum number of target nucleic acid sequences/targets (respiratory pathogens) that can be detected by the methods of the invention is not limited by the number of label probes (i.e., detection probes) used. That is, the present methods enable the simultaneous detection of a significantly greater number of target nucleic acid sequences/targets (respiratory pathogens) based on a relatively limited number of labeled probes (i.e., detection probes) (multiplex detection), 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 exemplary embodiment of the method of the invention to illustrate the basic principle of the method of the invention.

FIG. 1A schematically depicts the detection of 5 target nucleic acid molecules (each e.g., specific for one target, each target) using 1 detection probe and 5 mediator probesEach independently selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract). In this embodiment, a self-quenching detection probe (which carries a fluorescent group and a quenching group) is provided, and an upstream primer (upstream primer 1-5), a downstream primer (downstream primer 1-5), and a mediator probe (mediator probe 1-5) are designed and provided separately for each target nucleic acid molecule (T1-T5); wherein each mediator probe comprises a unique mediator sequence (mediator sequences 1-5) that is capable of hybridizing to the detection probe. The position at which each mediator sequence hybridises to the detection probe is unique, but there may be regions of overlap with each other. For example, as shown in FIG. 1A, the hybridization position of the mediator subsequence 1 on the detection probe overlaps with the mediator subsequence 2, the hybridization position of the mediator subsequence 4 on the detection probe overlaps with the mediator subsequence 5, and the hybridization position of the mediator subsequence 3 on the detection probe does not overlap with other mediator subsequences. In the detection process, 5 kinds of upstream primers, 5 kinds of downstream primers and 5 kinds of mediator probes are respectively hybridized (annealed) with the corresponding target nucleic acid molecules; subsequently, all the forward and reverse primers are extended by the action of nucleic acid polymerase, respectively, and the extension of each forward primer (forward primers 1-5) causes the corresponding mediator probe (mediator probes 1-5) to be cleaved by an enzyme having 5' nuclease activity, thereby releasing mediator fragments (mediator fragments 1-5); subsequently, the vector subsections 1-5 are hybridized to different positions of the detection probe, respectively, and extended by nucleic acid polymerase, thereby generating 5 extension products; the 5 extension products each have a different length and together with the detection probe form 5 extension products with different T_mValue of the duplex. From this, it can be confirmed that the melting curve has a specific T_mThe presence of a duplex of values, and thus the presence of a target nucleic acid molecule corresponding to the duplex, can be determined. Thus, in the methods of the invention, detection of 5 target nucleic acid molecules (and 5 targets corresponding thereto) can be achieved using 1 detection probe and 5 mediator probes.

FIG. 1B schematically depicts the use of 2Detection probes and 10 mediator probes to detect 10 target nucleic acid molecules (T1-T10, each specific for example to a target, each target independently selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract). In this embodiment, two self-quenching detection probes (first and second detection probes) are provided, each carrying a different fluorophore (fluorophore 1-2) and a different quencher (quencher 1-2); and for each target nucleic acid molecule (T1-T10), respectively designing and providing an upstream primer (upstream primer 1-10), a downstream primer (downstream primer 1-10) and a mediator probe (mediator probe 1-10); wherein each of the mediator probes comprises a unique mediator subsequence (mediator subsequences 1-10), and the mediator subsequences 1-5 are capable of hybridizing to the first detection probe and the mediator subsequences 6-10 are capable of hybridizing to the second detection probe. The position of hybridization of each mediator sequence to the detection probe is unique, but there may be overlapping regions with each other. For example, as shown in FIG. 1B, the hybridization position of the mediator subsequence 1 on the first detection probe overlaps with the mediator subsequence 2, the hybridization position of the mediator subsequence 4 on the first detection probe overlaps with the mediator subsequence 5, and the hybridization position of the mediator subsequence 3 on the first detection probe does not overlap with other mediator subsequences; the hybridization position of the vector subsequence 6 on the second detection probe is overlapped with the vector subsequence 7, the hybridization position of the vector subsequence 9 on the second detection probe is overlapped with the vector subsequence 10, and the hybridization position of the vector subsequence 8 on the second detection probe is not overlapped with other vector subsequences. In the detection process, 10 kinds of upstream primers, 10 kinds of downstream primers and 10 kinds of medium sub-probes are respectively hybridized (annealed) with the corresponding target nucleic acid molecules; subsequently, all the forward primers and the reverse primers are respectively extended under the action of nucleic acid polymerase, and the extension of each forward primer (the forward primers 1 to 10) causes the corresponding mediator probe (the mediator probes 1 to 10) to be cleaved by an enzyme having 5' nuclease activity, thereby releasing the mediator fragments (the mediator fragments 1 to 10); subsequently, the mediator fragments 1-5 hybridize to different positions of the first detection probe, respectivelyAnd extended by a nucleic acid polymerase, thereby producing 5 extension products; the 5 extension products each have a different length and together with the first detection probe form 5 extension products with different T_mValue duplexes. Similarly, the vector subsegments 6-10 hybridize to different positions on the second detection probe, respectively, and are extended by the nucleic acid polymerase, thereby generating 5 additional extension products; the 5 extension products each have a different length and together with the second detection probe form another 5 extension products having a different T_mValue duplexes. Subsequently, melting curve analysis is performed using fluorophores (fluorophores 1-2) on the first and second detection probes, respectively, and it is determined that the probe has a specific T_mThe presence of a duplex of values, and thus the presence of a target nucleic acid molecule corresponding to the duplex, can be determined. Thus, in the methods of the invention, detection of 10 target nucleic acid molecules (and corresponding 10 targets) can be achieved using 2 detection probes and 10 mediator probes.

FIG. 2 shows the results of testing a sample containing Mycoplasma pneumoniae using the reagents described in Table 1 and the testing protocol described in Table 2.

FIG. 3 shows the results of the detection of a sample containing Haemophilus influenzae and Moraxella catarrhalis using the reagents described in Table 1 and the detection protocol described in Table 2.

FIG. 4 shows the results of the detection of samples containing Acinetobacter baumannii, klebsiella pneumoniae and Pseudomonas aeruginosa using the reagents described in Table 1 and the detection protocol described in Table 2.

FIG. 5 shows the results of testing samples containing 19 targets using the reagents described in Table 1 and the testing protocol described in Table 2.

Sequence information

Information on the sequences to which this application refers (SEQ ID NOS: 1-66) is provided in Table 1.

Table 1: sequence information of SEQ ID NOS 1 to 66

Note: the base preceded by a "+" is a base modified with Locked Nucleic Acid (LNA); and, W, S, K, R, M, Y have meanings well known in the art (i.e., W = A or T; S = G or C; K = G or T; R = A or G; M = A or C; Y = C or T).

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.

Details of the detection probes, mediator probes, upstream oligonucleotides (upstream primers), downstream oligonucleotides (downstream primers), and universal primers used in the examples of the present application, as well as their working concentrations and detection targets, have been summarized in table 1. By using the reagents provided in table 1 (i.e., 7 detection probes, 20 mediator probes, 20 upstream oligonucleotides (upstream primers), 20 downstream oligonucleotides (downstream primers), and 1 universal primer), the method of the invention enables simultaneous detection of 19 respiratory pathogens and 1 control sequence (twenty-fold detection) using only 7 fluorescent probes (detection probes). The fluorescence detection channels used and melting points of the melting peaks detected are summarized in table 2.

Among the various detection probes and mediator probes described in Table 1, detection probes 1 and 2 are labeled with ROX and BHQ2, and the fluorescent signals thereof are detected through the ROX channel; the detection probes 3 and 4 are marked with FAM and BHQ1, and fluorescent signals of the detection probes are detected through a FAM channel; the detection probes 5 and 6 are marked with Cy5 and BHQ2, and fluorescent signals of the detection probes are detected through a Cy5 channel; the detection probe 7 is labeled with HEX and BHQ1, and the fluorescence signals of the detection probe are detected through a HEX channel. The medium subsequences of the 3 medium sub-probes shown in

SEQ ID NO

5, 8 and 11 can be respectively combined with the detection probe 1, and the 3 medium sub-probes are respectively used for detecting rickettsia, streptococcus pneumoniae and haemophilus influenzae. The medium subsequences of the 3 medium sub-probes shown in

SEQ ID NO

15, 18 and 21 can be respectively combined with the detection probe 2, and the 3 medium sub-probes are respectively used for detecting Moraxella catarrhalis, acinetobacter baumannii and Klebsiella pneumoniae. The medium subsequences of the 4 medium subsequences shown as SEQ ID NOS: 25, 28 and 31 can be combined with the detection probe 3 respectively, and the 3 medium subsequences are used for detecting staphylococcus epidermidis, chlamydia pneumoniae and staphylococcus aureus respectively. The vector subsequences of the 3 vector sub-probes shown in SEQ ID NOS 35, 38 and 41, respectively, are each capable of binding to the detection probe 4, and the 3 vector sub-probes are used for detecting Mycoplasma pneumoniae, candida albicans and Bordetella pertussis, respectively. The medium subsequences of the 3 medium sub-probes shown as SEQ ID NOS 45, 48 and 51 respectively can be combined with the detection probe 5, and the 3 medium sub-probes are used for detecting Escherichia coli, legionella pneumophila and cryptococcus respectively. The vector subsequences in the 3 vector sub-probes shown as SEQ ID NOS: 55, 58 and 61, respectively, are each capable of binding to the detection probe 6, and the 3 vector sub-probes are used for detecting Staphylococcus haemolyticus, pseudomonas aeruginosa and Streptococcus pyogenes, respectively. The mediator subsequences of the 2 mediator sub-probes shown in SEQ ID NO:65 and 68, respectively, are each capable of binding to the detection probe 7, and these 2 mediator sub-probes are used for detection of human ribonuclease P (used as a control) and/or Aspergillus fumigatus, respectively.

Table 2: detection scheme

Example 1 detection of Mycoplasma pneumoniae

In this example, a sample containing Mycoplasma pneumoniae was tested using the reagents described in Table 1 (7 detection probes, 20 mediator probes, 20 upstream primers, 20 downstream primers, and 1 universal primer) and the test protocol described in Table 2.

Briefly, in this example, real-time PCR was performed using 25 μ L of a PCR reaction system comprising: 1 XBuffer A (67 mM Tris-HCl,16.6mM (NH)₄)₂SO₄6.7 μ M EDTA and 0.085mg/mL BSA), 6.0mM MgCl₂0.2mM dNTPs,2.0U polymerase TaqHS (Takara), various reagents described in Table 1 (used at the indicated working concentrations), and 5. Mu.L of Mycoplasma pneumoniae DNA and control DNA (human RNase P gene) (the ratio of the amounts of the two is about 1. The reaction conditions of the real-time PCR are as follows: 95 ℃ for 5min; then 50 cycles (95 ℃,20s and 63 ℃,1 min). After completion of PCR, melting curve analysis was performed according to the following procedure: at 95 ℃ for 2min; at 40 ℃ for 2min; the temperature of the reaction system was then raised from 40 ℃ to 95 ℃ at a ramp rate of 0.4 ℃/step (the holding time per step was 5 s), and the fluorescence signals of the ROX, FAM, HEX and Cy5 channels were collected during this process. The laboratory instrument used was a Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, USA). The results of the detection are shown in FIG. 2.

The results in FIG. 2 show that in the fluorescence signal collected from the FAM channel, a characteristic melting peak corresponding to Mycoplasma pneumoniae was observed at 75.3 ℃; in the fluorescent signal collected in the HEX channel, a characteristic melting peak corresponding to the control DNA was observed at 64.7 ℃; also, in the fluorescence signals collected from the ROX and Cy5 channels, no melting peak was observed. The result shows that the designed detection system can be used for specifically detecting the mycoplasma pneumoniae and can accurately distinguish the mycoplasma pneumoniae from a control.

Example 2 detection of Haemophilus influenzae and Moraxella catarrhalis

In this example, samples containing Haemophilus influenzae and Moraxella catarrhalis were tested using the reagents described in Table 1 (7 detection probes, 20 mediator probes, 20 upstream primers, 20 downstream primers, and 1 universal primer) and the test protocol described in Table 2.

Briefly, in this example, real-time PCR was performed using a 25 μ L PCR reaction system comprising: 1 XBuffer A (67 mM Tris-HCl,16.6mM (NH)₄)₂SO₄6.7 μ M EDTA and 0.085mg/mL BSA), 6.0mM MgCl₂0.2mM dNTPs,2.0U of polymerase TaqHS (Takara), various reagents described in Table 1 (used at the indicated working concentrations), and 5. Mu.L of a mixture of Haemophilus influenzae DNA, moraxella catarrhalis DNA, and control DNA (human RNase P gene) (the ratio of the three used was about 3. The reaction conditions of the real-time PCR are as follows: 95 ℃ for 5min; then 50 cycles (95 ℃,20s and 63 ℃,1 min). After completion of PCR, melting curve analysis was performed according to the following procedure: at 95 ℃ for 2min; at 40 ℃ for 2min; the temperature of the reaction system was then raised from 40 ℃ to 95 ℃ at a ramp rate of 0.4 ℃/step (the holding time per step was 5 s), and the fluorescence signals of the ROX, FAM, HEX and Cy5 channels were collected during this process. The laboratory instrument used was a Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, USA). The results of the detection are shown in FIG. 3.

The results of FIG. 3 show that, in the fluorescence signal collected by the ROX channel, a characteristic melting peak corresponding to Haemophilus influenzae was observed at 66.8 ℃ and a characteristic melting peak corresponding to Moraxella catarrhalis was observed at 73.3 ℃; in the fluorescent signal collected in the HEX channel, a characteristic melting peak corresponding to the control DNA was observed at 64.7 ℃; also, in the fluorescence signals collected from FAM and Cy5 channels, no melting peak was observed. The result shows that the designed detection system can be used for specifically detecting haemophilus influenzae and moraxella catarrhalis, and can accurately distinguish haemophilus influenzae, moraxella catarrhalis and control. Further, from the peak heights of the characteristic melting peaks of Haemophilus influenzae and Moraxella catarrhalis, it was confirmed that the content of Haemophilus influenzae (DNA copy number) in the sample used in this example was higher than the content of Moraxella catarrhalis (DNA copy number).

Example 3 detection of Acinetobacter baumannii, klebsiella pneumoniae and Pseudomonas aeruginosa

In this example, samples containing Acinetobacter baumannii, klebsiella pneumoniae, and Pseudomonas aeruginosa were tested using the reagents described in Table 1 (7 detection probes, 20 mediator probes, 20 upstream primers, 20 downstream primers, and 1 universal primer) and the detection protocol described in Table 2.

Briefly, in this example, real-time PCR was performed using a 25 μ L PCR reaction system comprising: 1 XBuffer A (67 mM Tris-HCl,16.6mM (NH)₄)₂SO₄6.7 μ M EDTA and 0.085mg/mL BSA), 6.0mM MgCl₂0.2mm dntps,2.0u polymerase TaqHS (Takara), various reagents described in table 1 (used at the indicated working concentrations), and 5 μ L of a mixture of acinetobacter baumannii DNA, klebsiella pneumoniae DNA, pseudomonas aeruginosa DNA, and control DNA (human RNase P gene) (the ratio of the four was 4. The reaction conditions of the real-time PCR are as follows: 95 ℃ for 5min; then 50 cycles (95 ℃,20s and 63 ℃,1 min). After completion of PCR, melting curve analysis was performed according to the following procedure: at 95 ℃ for 2min; at 40 ℃ for 2min; the temperature of the reaction system was then raised from 40 ℃ to 95 ℃ at a ramp rate of 0.4 ℃/step (the holding time per step was 5 s), and the fluorescence signals of the ROX, FAM, HEX and Cy5 channels were collected during this process. The laboratory instrument used was a Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, USA). The results of the detection are shown in FIG. 4.

The results of fig. 4 show that, in the fluorescence signal collected by the ROX channel, a characteristic melting peak corresponding to acinetobacter baumannii was observed at 78.7 ℃ and a characteristic melting peak corresponding to klebsiella pneumoniae was observed at 85.5 ℃; in the fluorescence signal collected in the Cy5 channel, a melting peak characteristic to Pseudomonas aeruginosa was observed at 79.4 ℃ and in the fluorescence signal collected in the HEX channel, a melting peak characteristic to control DNA was observed at 64.7 ℃; also, in the fluorescence signal collected by the FAM channel, no melting peak was observed. The result shows that the designed detection system can be used for specifically detecting acinetobacter baumannii, klebsiella pneumoniae and pseudomonas aeruginosa, and can accurately distinguish the three bacteria and a control. In addition, it was confirmed that the sample used in this example had the highest acinetobacter baumannii content (cDNA copy number), the next highest pseudomonas aeruginosa content (DNA copy number), and the lowest klebsiella pneumoniae content (cDNA copy number), based on the peak heights of characteristic melting peaks of acinetobacter baumannii, klebsiella pneumoniae, and pseudomonas aeruginosa.

Example 4 twenty-fold detection

In this example, samples containing 19 targets (DNA from staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, and staphylococcus haemolyticus, respectively) and control DNA (human RNase P gene) were tested using the reagents described in table 1 (7 detection probes, 20 mediator probes, 20 upstream primers, 20 downstream primers, and 1 universal primer) and the detection protocol described in table 2.

Briefly, in this example, real-time PCR was performed using 25 μ L of a PCR reaction system comprising: 1 XBuffer A (67 mM Tris-HCl,16.6mM (NH)₄)₂SO₄6.7 μ M EDTA and 0.085mg/mL BSA), 6.0mM MgCl₂0.2mM dNTPs,2.0U of polymerase TaqHS (Takara), various reagents described in Table 1 (used at the indicated working concentrations), and 5. Mu.L of a nucleic acid mixture comprising the 19 target DNAs and a control DNA (human RNase P gene)). The reaction conditions of the real-time PCR are as follows: 95 ℃ for 5min; then 50 cycles (95 ℃,20s and 63 ℃,1 min). After completion of PCR, melting curve analysis was performed according to the following procedure: at 95 ℃ for 2min; at 40 ℃ for 2min; the temperature of the reaction system was then raised from 40 ℃ to 95 ℃ at a ramp rate of 0.4 ℃/step (the holding time per step was 5 s), and the fluorescence signals of the ROX, FAM, HEX and Cy5 channels were collected during this process. The laboratory instrument used was a Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, USA). The detection results are shown in FIG. 5As shown.

The results of FIG. 5 show that, in the fluorescence signal collected by the ROX channel, 6 characteristic melting peaks (peaks 1 to 6) were observed at 56.2 ℃, 60.8 ℃, 66.8 ℃, 73.3 ℃, 78.7 ℃ and 85.5 ℃ respectively corresponding to rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii and Klebsiella pneumoniae; in the fluorescence signal collected by the Cy5 channel, 6 characteristic melting peaks (peaks 7 to 12) corresponding to Escherichia coli, legionella pneumophila, cryptococcus, staphylococcus hemolyticus, pseudomonas aeruginosa and Streptococcus pyogenes were observed at 61.8 ℃, 66.2 ℃, 70.1 ℃, 73.8 ℃, 79.4 ℃ and 84.7 ℃ respectively; in the fluorescence signal collected by the FAM channel, 6 characteristic melting peaks (peaks 12 to 18) corresponding to Staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans and Bordetella pertussis, respectively, were observed at 61.8 ℃, 65.3 ℃, 69.8 ℃, 75.3 ℃, 81.6 ℃ and 85.0 ℃; also, in the fluorescence signal collected by the HEX channel, characteristic melting peaks (peaks 19 to 20) corresponding to the control DNA and Aspergillus fumigatus, respectively, were observed at 64.7 ℃ and 69.0 ℃. This result indicates that the designed detection system can be used to specifically detect the 19 targets and can accurately distinguish the 19 targets from the control.

These experimental results show that with the designed detection system and reagents (specifically, the designed 20 mediator sub-probes and 7 fluorescent probes), simultaneous detection and discrimination (i.e., twenty-fold detection) of 20 target sequences (19 targets and 1 control) can be achieved in a single assay. Thus, the methods and kits of the invention can be used for rapid, simple, sensitive, specific, stable, and reliable simultaneous detection of multiple targets (e.g., 19 or more targets).

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

<110> university of mansion

<120> a method for detecting respiratory pathogens

<130> IDC180030

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Universal primer

<400> 1

gcaagccctc acgtagcgaa 20

<210> 2

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 1

<400> 2

cccggcttgt cacctgtcct agagagcgta gagcccagaa cgatttgccg gg 52

<210> 3

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> rickettsia upstream primer

<400> 3

gcaagccctc acgtagcgaa cggatgaaac gggtgt 36

<210> 4

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> rickettsia downstream primer

<400> 4

gcaagccctc acgtagcgaa gccaccgctt ttaattcct 39

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> rickettsia mediator probe

<400> 5

ctcacacttc ttcaccaatc gcttcgtccc ggttca 36

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pneumoniae upstream primer

<400> 6

gcaagccctc acgtagcgaa acgcaatcta gcagatgaag 40

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pneumoniae downstream primer

<400> 7

gcaagccctc acgtagcgaa tcgtgcgttt taattccagc 40

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pneumoniae mediator Probe

<400> 8

cctctcacac ttgccgaaaa cgcttgatac agggag 36

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Haemophilus influenzae upstream primer

<400> 9

gcaagccctc acgtagcgaa tcactcaacg cttaactrg 39

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Haemophilus influenzae downstream primer

<400> 10

gcaagccctc acgtagcgaa cgcataggag ggaaatggt 39

<210> 11

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Haemophilus influenzae vector probe

<400> 11

tcacacctct caatgatgac aaaccttact agcggtaatt gggatcca 48

<210> 12

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 2

<400> 12

cggcggagtg ggcacggaga gcgctggaca gtgtggaccc acgtctcgca gcaggccgcc 60

g 61

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Moraxella catarrhalis upstream primer

<400> 13

gcaagccctc acgtagcgaa agtttcaatt attaccgctg ac 42

<210> 14

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Moraxella catarrhalis downstream primer

<400> 14

gcaagccctc acgtagcgaa ttctcgtaag gtaacaacat t 41

<210> 15

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Moraxella catarrhalis medium probe

<400> 15

gcgctctccg tgcttttgca gctgttagcc arcctaa 37

<210> 16

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> acinetobacter baumannii upstream primer

<400> 16

gcaagccctc acgtagcgaa ctcgtcgtat tggacttga 39

<210> 17

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Acinetobacter baumannii downstream primer

<400> 17

gcaagccctc acgtagcgaa cctcttgctg aggagtaatt tt 42

<210> 18

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Acinetobacter baumannii medium probe

<400> 18

ctgtccagcg ctctaaggaa gtgaagcgtg ttggttatgg c 41

<210> 19

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Klebsiella pneumoniae upstream primer

<400> 19

gcaagccctc acgtagcgaa cgtgaagcag ggatggatta t 41

<210> 20

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Klebsiella pneumoniae downstream primer

<400> 20

gcaagccctc acgtagcgaa taaaatgagt ttgccgcca 39

<210> 21

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Klebsiella pneumoniae-mediated mediator probe

<400> 21

gtccacactg tcatattggt ccccagacgc aggtcattg 39

<210> 22

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 3

<400> 22

aagcccaaaa aagagaacag tatcagtcac acggggctt 39

<210> 23

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus epidermidis upstream primer

<400> 23

gcaagccctc acgtagcgaa ggggcacaac atctaca 37

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus epidermidis downstream primer

<400> 24

gcaagccctc acgtagcgaa ggtgccttag cagtgtt 37

<210> 25

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus epidermidis mediator probe

<400> 25

gatactgttc tctgtgtctg ctaatcgtgg tgttgctcaa a 41

<210> 26

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Chlamydia pneumoniae upstream primer

<400> 26

gcaagccctc acgtagcgaa aacaaagtct gcgaccat 38

<210> 27

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Chlamydia pneumoniae downstream primer

<400> 27

gcaagccctc acgtagcgaa tccaatgtat ggcactaaag a 41

<210> 28

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Chlamydia pneumoniae mediator probe

<400> 28

gactgatact gttcatgaat ggcaagtagg agcctctcta tcttacag 48

<210> 29

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus aureus upstream primer

<400> 29

gcaagccctc acgtagcgaa atcgatccat atttaccata tcaatacttg 50

<210> 30

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus aureus downstream primer

<400> 30

gcaagccctc acgtagcgaa atccagtatg ttcaaatcct aagtt 45

<210> 31

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus aureus mediator probe

<400> 31

cgtgtgactg atcatgatgg cgagattaca ggtaatgctg gtaat 45

<210> 32

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 4

<400> 32

gcgcgccagc ggacgaggct gtgcaccggt cggaggtggg ggcgcgc 47

<210> 33

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Mycoplasma pneumoniae upstream primer

<400> 33

gcaagccctc acgtagcgaa caaaccttgt ttagtccgtt t 41

<210> 34

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Mycoplasma pneumoniae downstream primer

<400> 34

gcaagccctc acgtagcgaa aggtaacact gagcacaat 39

<210> 35

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Mycoplasma pneumoniae-mediated molecular probe

<400> 35

cacagcctcg tcgctaacgg caacacgtaa tcaggtca 38

<210> 36

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Candida albicans upstream primer

<400> 36

gcaagccctc acgtagcgaa tgaagaacgc agcgaaat 38

<210> 37

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Candida albicans downstream primer

<400> 37

gcaagccctc acgtagcgaa caacaccaaa cccagcg 37

<210> 38

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Candida albicans vector probe

<400> 38

gaccggtgca cagatattcg tgaatcatcg aatytttgaa cgcacatt 48

<210> 39

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Bordetella pertussis upstream primer

<400> 39

gcaagccctc acgtagcgaa gcgtgcagat tcgtcg 36

<210> 40

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Bordetella pertussis downstream primer

<400> 40

gcaagccctc acgtagcgaa atggcagggg ggtca 35

<210> 41

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Bordetella pertussis vector probe

<400> 41

cccacctccg accctcgatt cttccgtaca tcccgctac 39

<210> 42

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 5

<400> 42

gctgcaaaaa actcaacgat gtggaagtca gcagc 35

<210> 43

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> E.coli upstream primer

<400> 43

gcaagccctc acgtagcgaa cattattatt cgggcggt 38

<210> 44

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> E.coli downstream primer

<400> 44

gcaagccctc acgtagcgaa cggtccatta tccagttc 38

<210> 45

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> E.coli mediator Probe

<400> 45

catcgttgag tttatgtttg ccggtatccg tttttgccac ca 42

<210> 46

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> legionella pneumophila upstream primer

<400> 46

gcaagccctc acgtagcgaa gatgttaatc cggaagcaat 40

<210> 47

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Legionella pneumophila downstream primer

<400> 47

gcaagccctc acgtagcgaa ttttcatccg ctttcttatt g 41

<210> 48

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Legionella pneumophila mediator probe

<400> 48

tccacatcgt tgccactcat agcgtcttgc atgcctttag 40

<210> 49

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Cryptococcus upstream primer

<400> 49

gcaagccctc acgtagcgaa cctgttggac ttggatttgg 40

<210> 50

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Cryptococcus downstream primer

<400> 50

gcaagccctc acgtagcgaa agcaagccga agactacc 38

<210> 51

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Cryptococcus mediator probes

<400> 51

tgacttccac acgtcggctc gccttaaatg tgttagt 37

<210> 52

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 6

<400> 52

ccggcgggga gggaccgtcg tggcaggagg agcagctcac caggccgccg g 51

<210> 53

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> staphylococcus haemolyticus upstream primer

<400> 53

tgaatctggt ggcgacatta c 21

<210> 54

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> staphylococcus hemolyticus downstream primer

<400> 54

aggagctact gaatcccaag ta 22

<210> 55

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Staphylococcus hemolyticus mediator probe

<400> 55

ccacgacggt caaacccaag ttcaggtgca gcaggtaaat atc 43

<210> 56

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Pseudomonas aeruginosa upstream primer

<400> 56

gcaagccctc acgtagcgaa ggtctgggaa caggtcta 38

<210> 57

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Pseudomonas aeruginosa downstream primer

<400> 57

gcaagccctc acgtagcgaa cggagttgag gaaagaca 38

<210> 58

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Pseudomonas aeruginosa mediator probe

<400> 58

cctcctgcca cagcaacatc yacttcagtt gggacatcc 39

<210> 59

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pyogenes upstream primer

<400> 59

gcaagccctc acgtagcgaa caccttacta actgaacaat ctt 43

<210> 60

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pyogenes downstream primer

<400> 60

gcaagccctc acgtagcgaa gtgtagtgga ggtagaaggt t 41

<210> 61

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> Streptococcus pyogenes mediator Probe

<400> 61

cctggtgagc tcttttgaaa ctcttactgt ctctgacctt actaaaaagg ct 52

<210> 62

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> detection Probe 7

<400> 62

atcgccataa aagatagacc agagagagtc agagcggcga t 41

<210> 63

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> ribonuclease P upstream primer

<400> 63

gcaagccctc acgtagcgaa ggcggtgttt gcagatttg 39

<210> 64

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> ribonuclease P downstream primer

<400> 64

gcaagccctc acgtagcgaa gagcggctgt ctccacaagt 40

<210> 65

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> ribonuclease P-mediated daughter probe

<400> 65

ctctctctgg ttctgacctg aaggctctgc gcg 33

<210> 66

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> Aspergillus fumigatus upstream primer

<400> 66

gcaagccctc acgtagcgaa ctcggaatgt atcacctctt gcag 44

<210> 67

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Aspergillus fumigatus downstream primer

<400> 67

gcaagccctc acgtagcgaa tcctcggtcc aggca 35

<210> 68

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Aspergillus fumigatus vector probe

<400> 68

tctgactctc tgtcttatag ccgagggtgc aatgcg 36

Claims

1. A probe set comprising a detection probe and at least two mediator probes, wherein,

each of said mediator probes independently comprises in the 5 'to 3' direction a mediator subsequence comprising a sequence complementary to a target nucleic acid sequence specific to a target or a control sequence and a target-specific sequence comprising a sequence not complementary to said target nucleic acid sequence or control sequence, and each target is independently selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia and fungi capable of infecting the respiratory tract, and the mediator subsequences comprised by all mediator probes are different from each other; and

the detection probe comprises, in the 3 'to 5' direction, a capture sequence complementary to each mediator sequence or a portion thereof, and a template sequence; and the detection probe is labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and wherein the detection probe emits a signal when hybridized to its complement that is different from the signal when not hybridized to its complement.

2. The panel of claim 1, wherein the panel has one or more characteristics selected from the group consisting of:

(1) The probe set comprises 2,3,4,5, 6,7,8,9, 10 or more mediator probes;

(2) All the medium sub-probes comprise different medium sub-sequences; and, all mediator probes contain target-specific sequences that are different from each other;

(3) All mediator probes each target a different target nucleic acid sequence;

(4) At least one mediator probe or a target-specific sequence targeting control sequence comprised by it;

(5) The set of probes further comprises an upstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is upstream of the target-specific sequence of the mediator probe;

(6) The set of probes further comprises a downstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the downstream oligonucleotide sequence is located downstream of the target-specific sequence of the mediator probe.

3. The panel of claim 2, wherein said mediator probe or target-specific sequences comprised by it target specific nucleic acid sequences of 2,3,4,5, 6,7,8,9, 10 or more targets.

4. The panel of claim 2, wherein the different target nucleic acid sequences are each specific for the same or different targets.

5. The panel of claim 2, wherein the control sequence is a host-specific sequence.

6. The panel of claim 5, wherein said host-specific sequence is a human-specific sequence.

7. The panel of claim 1, wherein each target is independently selected from the group consisting of staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus haemolyticus, and any combination thereof.

8. The panel of claim 1, wherein at least one target is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia, moraxella catarrhalis, cryptococcus, legionella pneumophila, aspergillus flavus, candida tropicalis, candida glabrata, bordetella parapertussis, and chlamydia psittaci.

9. The panel of claim 1, wherein at least one target is selected from the group consisting of chlamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis, rickettsia catarrhalis, cryptococcus and legionella pneumophila.

10. The probe set of claim 2, wherein one upstream oligonucleotide sequence is provided for each target nucleic acid sequence and mediator probe.

11. The probe set of claim 2, wherein one downstream oligonucleotide sequence is provided for each target nucleic acid sequence and mediator probe.

12. The probe set of claim 2, wherein said probe set comprises said upstream and downstream oligonucleotide sequences and wherein the 5' ends of said upstream and downstream oligonucleotide sequences comprise an identical oligonucleotide sequence.

13. The probe set of claim 12, wherein said probe set further comprises a universal primer having a sequence complementary to said identical oligonucleotide sequence.

14. The set of probes of any of claims 1-13, wherein the media probe has one or more characteristics selected from the group consisting of:

(1) Each of said mediator probes independently comprises or consists of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(2) The lengths of the medium probes are respectively and independently 15-150nt;

(3) The lengths of the target specific sequences in the mediator probes are respectively and independently 10-140nt;

(4) The lengths of the medium subsequences in the medium subsebes are respectively and independently 5-140nt; and

(5) Each of said mediator probes independently has a 3'-OH terminus, or its 3' -terminus is blocked;

and/or the presence of a gas in the gas,

the detection probe has one or more characteristics selected from the group consisting of:

(1) The detection probe comprises or alternatively consists of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(2) The length of the detection probe is 15-1000nt;

(3) Each capture sequence in the detection probe is independently 10-500nt in length;

(4) The length of the template sequence in the detection probe is 1-900nt;

(5) The detection probe has a 3'-OH terminus, or its 3' -terminus is blocked;

(6) The detection probe is a self-quenching probe;

(7) The reporter group in the detection probe is a fluorescent group; and, the quenching group is a molecule or group capable of absorbing/quenching the fluorescence;

(8) The detection probe is resistant to nuclease activity;

(9) The detection probe is linear or has a hairpin structure; and

(10) The detection probe comprises a plurality of capture sequences; and, the plurality of capture sequences are arranged in an adjacent manner, in a spaced-apart manner with a linker sequence, or in an overlapping manner.

15. The probe set of claim 14, wherein the length of the mediator probe is 15 to 2nt, 20 to 30nt,30 to 40nt,40 to 50nt,50 to 60nt,60 to 70nt,70 to 80nt,80 to 90nt,90 to 100nt,100 to 110nt,110 to 120nt,120 to 130nt,130 to 140nt, or 140 to 150nt.

16. The probe set of claim 14, wherein the length of the target-specific sequence in the mediator probe is 10-20nt,20-30nt,30-40nt,40-50nt,50-60nt,60-70nt,70-80nt,80-90nt,90-100nt,100-110nt,110-120nt,120-130nt, or 130-140nt.

17. The probe set of claim 14, wherein the length of the mediator sequence in said mediator probe is 5 to 10nt,10 to 2nt, 20 to 30nt,30 to 40nt,40 to 50nt,50 to 60nt,60 to 70nt,70 to 80nt,80 to 90nt,90 to 100nt,100 to 110nt,110 to 120nt,120 to 130nt, or 130 to 140nt.

18. The probe set of claim 14, wherein the length of the detection probe is 15 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 100nt,100 to 200nt,200 to 300nt,300 to 400nt,400 to 500nt,500 to 600nt,600 to 700nt,700 to 800nt,800 to 900nt, or 900 to 1000nt.

19. The probe set according to claim 14, wherein the length of the capture sequence in the detection probe is 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 100nt,100 to 150nt,150 to 200nt,200 to 250nt,250 to 300nt,300 to 350nt,350 to 400nt,400 to 450nt, or 450 to 500nt.

20. The probe set according to claim 14, wherein the length of the template sequence in the detection probe is 1 to 5nt,5 to 10nt,10 to 2nt, 20 to 30nt,30 to 40nt,40 to 50nt,50 to 60nt,60 to 70nt,70 to 80nt,80 to 90nt,90 to 100nt,100 to 200nt,200 to 300nt,300 to 400nt,400 to 500nt,500 to 600nt,600 to 700nt,700 to 800nt, or 800 to 900nt.

21. The probe set of claim 14, wherein the detection probe is labeled with a reporter at its 5 'end or upstream and a quencher at its 3' end or downstream, or is labeled with a reporter at its 3 'end or downstream and a quencher at its 5' end or upstream.

22. The panel of claim 21, wherein the reporter and quencher are separated by a distance of 10-80nt or more.

23. The panel of claim 14, wherein said fluorophore is selected from the group consisting of 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, or any combination thereof.

24. The panel of claim 14, wherein said quencher group is selected from the group consisting of DABCYL, BHQ, ECLIPSE, and TAMRA.

25. The probe set of claim 24, wherein the BHQ is BHQ-1 or BHQ-2.

26. The panel of claim 14, wherein said detection probe is resistant to 5' nuclease activity.

27. The panel of claim 14, wherein said detection probe is resistant to 5 'to 3' exonuclease activity.

28. The panel of claim 14, wherein the backbone of the detection probe comprises a modification that is resistant to nuclease activity.

29. The probe set of claim 28, wherein said modification is selected from the group consisting of a phosphorothioate linkage, an alkylphosphotriester linkage, an arylphosphotriester linkage, an alkylphosphonate linkage, an arylphosphonate linkage, a hydrogenphosphate linkage, an alkylaminophosphate linkage, an arylaminophosphate linkage, a 2' -O-aminopropyl modification, a 2' -O-alkyl modification, a 2' -O-allyl modification, a 2' -O-butyl modification, and a 1- (4 ' -thio-PD-ribofuranosyl) modification.

30. A set according to any one of claims 2 to 13, wherein said upstream oligonucleotide sequence has one or more characteristics selected from:

(1) The upstream oligonucleotide sequences each independently comprise or consist of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof;

(2) The length of the upstream oligonucleotide sequences is 15-150nt independently;

(3) The upstream oligonucleotide sequences are each independently located at the upstream distal end of the mediator probe, or located adjacent to the upstream of the mediator probe, or have partially overlapping sequences with the target-specific sequence of the mediator probe after hybridization with the target nucleic acid sequence; and

(4) The upstream oligonucleotide sequences are each independently a primer specific for the target nucleic acid sequence or a probe specific for the target nucleic acid sequence;

and/or the presence of a gas in the gas,

the downstream oligonucleotide sequence has one or more characteristics selected from the group consisting of:

(1) The downstream oligonucleotide sequences each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof; and

(2) The downstream oligonucleotide sequences are each independently 15-150nt in length;

and/or the presence of a gas in the gas,

the universal primer has one or more characteristics selected from the group consisting of:

(1) The universal primer comprises or alternatively consists of a naturally occurring nucleotide, a modified nucleotide, a non-natural nucleotide, or any combination thereof; and

(2) The length of the universal primer is 8-50nt.

31. The probe set of claim 30, wherein the universal primer is 8-15nt,15-20nt,20-30nt,30-40nt, or 40-50nt in length.

32. The probe set of claim 30, wherein the upstream oligonucleotide sequence has a length of 15 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 100nt,100 to 110nt,110 to 120nt,120 to 130nt,130 to 140nt, or 140 to 150nt.

33. The probe set of claim 30, wherein the downstream oligonucleotide sequence has a length of 15-20nt,20-30nt,30-40nt,40-50nt,50-60nt,60-70nt,70-80nt,80-90nt,90-100nt,100-110nt,110-120nt,120-130nt,130-140nt, or 140-150nt.

34. The probe set of any one of claims 1 to 13, wherein the probe set further comprises a universal primer set as set forth in SEQ ID No. 1.

35. The panel of any of claims 1-13, wherein said panel is a panel selected from the group consisting of:

(1) A first probe group comprising a detection probe shown as SEQ ID NO. 2 and 3 mediator probes shown as SEQ ID NO. 5, 8 and 11, respectively;

(2) A second probe set comprising a detection probe shown as SEQ ID NO. 12 and 3 mediator sub-probes shown as SEQ ID NO. 15, 18 and 21, respectively;

(3) A third probe group, which comprises a detection probe shown as SEQ ID NO. 22 and 3 medium sub-probes shown as SEQ ID NO. 25, 28 and 31 respectively;

(4) A fourth probe group, which comprises a detection probe shown as SEQ ID NO. 32 and 3 medium sub-probes shown as SEQ ID NO. 35, 38 and 41 respectively;

(5) A fifth probe set comprising a detection probe shown as SEQ ID NO. 42 and 3 mediator sub-probes shown as SEQ ID NO. 45, 48 and 51, respectively;

(6) A sixth probe set comprising the detection probe shown as SEQ ID NO. 52 and 3 mediator probes shown as SEQ ID NO. 55, 58 and 61, respectively;

(7) A seventh probe set comprising, the detection probe set shown in SEQ ID NO:62, and 2 mediator sub-probes shown in SEQ ID NO:65 and 68, respectively.

36. The set of probes of claim 35, wherein said first set of probes further comprises: 3 upstream oligonucleotides as shown in SEQ ID NO 3, 6 and 9, respectively, 3 downstream oligonucleotides as shown in SEQ ID NO 4, 7 and 10, respectively, a universal primer as shown in SEQ ID NO 1, or any combination thereof.

37. The set of probes of claim 35, wherein said second set of probes further comprises: 13, 16 and 19 as shown in SEQ ID NO, 14, 17 and 20 as shown in SEQ ID NO, 1 as shown in the universal primer, or any combination thereof.

38. The set of probes of claim 35, wherein said third set of probes further comprises: 3 upstream oligonucleotides as shown in SEQ ID NO 23, 26 and 29, respectively, 3 downstream oligonucleotides as shown in SEQ ID NO 24, 27 and 30, respectively, a universal primer as shown in SEQ ID NO 1, or any combination thereof.

39. The set of probes of claim 35, wherein said fourth set of probes further comprises: 3 upstream oligonucleotides as shown in SEQ ID NO 33, 36 and 39, respectively, 3 downstream oligonucleotides as shown in SEQ ID NO 34, 37 and 40, respectively, a universal primer as shown in SEQ ID NO 1, or any combination thereof.

40. The set of probes of claim 35, wherein said fifth set of probes further comprises: 43, 46 and 49 as shown in SEQ ID NO, 44, 47 and 50 as shown in SEQ ID NO, 1 as shown in the universal primer, or any combination thereof.

41. The set of probes of claim 35, wherein said sixth set of probes further comprises: 3 upstream oligonucleotides as shown in SEQ ID NO 53, 56 and 59, respectively, 3 downstream oligonucleotides as shown in SEQ ID NO 54, 57 and 60, respectively, universal primers as shown in SEQ ID NO 1, or any combination thereof.

42. The set of probes of claim 35, wherein said seventh set of probes further comprises: 2 upstream oligonucleotides shown as SEQ ID NO 63 and 66, respectively, 2 downstream oligonucleotides shown as SEQ ID NO 64 and 67, respectively, a universal primer shown as SEQ ID NO 1, or any combination thereof.

43. A kit comprising one or more sets of probes as defined in any one of claims 1 to 42.

44. The kit of claim 43, wherein the kit further comprises: an enzyme having 5' nuclease activity, a nucleic acid polymerase, or any combination thereof.

45. The kit of claim 43, wherein the kit comprises 1, 2,3,4,5, 6,7,8,9, 10 or more probe sets.

46. The kit of claim 43, wherein the kit comprises one or more of the first to seventh set of probes as defined in any one of claims 36-42.

47. A kit, comprising: m detection probes and n mediator sub-probes, wherein n is an integer of 2 or more, m is an integer of 0 or more less than n,

each mediator sub-probe independently comprises in the 5 'to 3' direction a mediator sub-sequence and a target specific sequence, the target specific sequence comprising a sequence complementary to a target nucleic acid sequence or a control sequence specific to a target, the mediator sub-sequence comprising a sequence that is not complementary to the target nucleic acid sequence or the control sequence, and each target is independently selected from the group consisting of bacteria, chlamydia, mycoplasma, rickettsia, and fungi that are capable of infecting the respiratory tract, and the mediator sub-sequences comprised by all mediator sub-probes are different from each other; and

each detection probe independently comprises, in the 3 'to 5' direction, one or more capture sequences complementary to one or more mediator sequences or portions thereof, and a template sequence (mapping sequence); and, the m detection probes comprise a plurality of capture sequences that are complementary to the mediator sequences of each mediator probe, or a portion thereof, respectively; and the number of the first and second electrodes,

each detection probe is respectively and independently marked with a reporter group and a quenching group, wherein the reporter group can emit signals, and the quenching group can absorb or quench the signals emitted by the reporter group; and, each detection probe emits a signal when hybridized to its complement that is different from a signal when not hybridized to its complement.

48. The kit of claim 47, wherein n is an integer of 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.

49. The kit of claim 47, wherein said m detection probes comprise at least n capture sequences.

50. The kit of claim 47, wherein the kit has one or more characteristics selected from the group consisting of:

(1) The kit comprises 1, 2,3,4,5, 6, 8, 10 or more detection probes;

(2) The kit comprises 2,3,4,5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45 or more mediator probes;

(3) The kit further comprises an upstream oligonucleotide sequence comprising a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the upstream oligonucleotide sequence is upstream of the target-specific sequence of the mediator probe;

(4) The kit further comprises a downstream oligonucleotide comprising a sequence complementary to the target nucleic acid sequence; and, when hybridized to the target nucleic acid sequence, the downstream oligonucleotide sequence is located downstream of the target-specific sequence of the mediator probe;

(5) The kit further comprises a universal primer; and

(6) The kit further comprises: an enzyme having 5' nuclease activity, a nucleic acid polymerase, or any combination thereof.

51. The kit of claim 50, wherein the upstream oligonucleotide is as defined in any one of claims 2 to 13, 15 to 29, 31 to 33, 36 to 41.

52. The kit of claim 50, wherein the downstream oligonucleotide is as defined in any one of claims 2 to 13, 15 to 29, 31 to 33, 36 to 41.

53. The kit of claim 50, wherein the universal primers are as defined in any one of claims 2-13, 15-29, 31.

54. The kit of any one of claims 43-45, 47-50, having one or more characteristics selected from the group consisting of:

(1) The medium subsequences of all medium subsebes in the kit target different target nucleic acid sequences respectively;

(2) All the mediator probes in the kit comprise different mediator sequences;

(3) All mediator probes in the kit comprise target-specific sequences that are different from each other;

(4) All detection probes in the kit are respectively and independently labeled with the same or different reporter groups;

(5) The kit further comprises: a reagent for performing nucleic acid hybridization, a reagent for performing mediator probe cleavage, a reagent for performing nucleic acid extension, a reagent for performing nucleic acid amplification, a reagent for performing reverse transcription, or any combination thereof; and

(6) The enzyme having 5 'nuclease activity is a nucleic acid polymerase having 5' nuclease activity.

55. The kit of claim 54, wherein the enzyme having 5 'nuclease activity is a nucleic acid polymerase having 5' exonuclease activity.

56. The kit of claim 54, wherein the nucleic acid polymerase is a DNA polymerase.

57. The kit of claim 54, wherein the nucleic acid polymerase is a thermostable DNA polymerase.

58. The kit of claim 56, wherein the DNA polymerase is obtained from a bacterium selected from the group consisting of: thermus aquaticus (Taq), thermus thermophiles (Tth), thermus filiformis, thermus flavus, thermococcus literalis, thermus antalidanii, thermus caldophlus, thermus chalaranthius, thermus flavus, thermus agniterae, thermus lactis, thermus osidamia, thermus ruber, thermus rubens, thermus scodottus, thermus silvannus, thermus thermophilus, thermotoga maritima, thermotoga neocolitica, thermosipho africana, thermococcus litoralis, thermococcus barossi, thermococcus gordonarius, thermotoga maritima, thermotoga neocolitica, thermosiphofuragallinaceus, pyrococcus woesei, pyrococcus horikoshii, pyrococcus abyssi, pyrodium occultum, aquifexpyrophilius, and Aquifex aeolieus.

59. The kit of claim 56, wherein the DNA polymerase is Taq polymerase.

60. Use of a set of probes according to any one of claims 1 to 42 for the preparation of a kit for detecting the presence or level of said target in a sample or for diagnosing whether a subject is infected with said target.

61. The use of claim 60, wherein the sample comprises DNA, or RNA, or a mixture of nucleic acids.

62. The use of claim 60, wherein the target nucleic acid sequence is DNA or RNA; and/or, the target nucleic acid sequence is single-stranded or double-stranded.

63. The use of claim 60, wherein the sample is a sample obtained from a subject.

64. The use of claim 63, wherein the subject's sample is nasal secretion, nasal or pharyngeal swab, alveolar lavage, or sputum.

65. The use of claim 63, wherein the subject is a mammal.

66. The use of claim 65, wherein the mammal is a human.

67. The use of claim 60, wherein the target is selected from the group consisting of Staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus haemolyticus, aspergillus flavus, candida tropicalis, candida glabrata, enterobacter cloacae, enterococcus, proteus mirabilis, pseudomonas maltophilia, bordetella parapertussis, chlamydia psittaci, or any combination thereof.

68. The use of claim 60, wherein the target is selected from the group consisting of Staphylococcus epidermidis, chlamydia pneumoniae, staphylococcus aureus, mycoplasma pneumoniae, candida albicans, bordetella pertussis, rickettsia, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, acinetobacter baumannii, klebsiella pneumoniae, cryptococcus, escherichia coli, legionella pneumophila, pseudomonas aeruginosa, aspergillus fumigatus, streptococcus pyogenes, staphylococcus hemolyticus, or any combination thereof.

69. The kit of claim 47, wherein the detection probe is as defined in any one of claims 2 to 13, 15 to 29, 31 to 33, 36 to 42.

70. The kit of claim 47, wherein the mediator probe is as defined in any one of claims 2-13, 15-29, 31-33, 36-42.