Primer and probe combination products for respiratory infection detection

Abstract

This invention relates to the field of biotechnology, and in particular to a primer-probe combination product for respiratory infection detection. By integrating multiple specific primers and self-quenching probes into a single reaction system, simultaneous, rapid, and accurate detection of 15 respiratory pathogens (including influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci) is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a primer-probe combination product for the detection of respiratory infections. Background Technology

[0002] Respiratory tract infection (RTI) is the most common infectious disease worldwide, with extremely high morbidity and widespread socioeconomic impact. It includes acute upper respiratory tract infection, bronchitis, and pneumonia. These diseases are mainly caused by a range of viruses, bacteria, and atypical pathogens. Common pathogens include: influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), human parainfluenza virus (hPIV), mycoplasma pneumoniae (MP), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human respiratory adenovirus (hAdV), human rhinovirus / enterovirus (HRV / HEV), Bordetella pertussis (BP), human coronavirus (HCoV), human bocavirus (HBoV), Chlamydia pneumoniae (CP), human metapneumovirus (hMPV), and Chlamydia psittaci. All of the above-mentioned pathogens can cause respiratory infections. In addition, influenza virus, severe acute respiratory syndrome coronavirus 2, RSV, adenovirus mycoplasma pneumoniae and psittacosis chlamydia can even cause severe pneumonia, leading to more serious symptoms or complications.

[0003] Although most respiratory infections are self-limiting, their clinical symptoms (such as fever, runny nose, sore throat, cough, hoarseness, etc.) are highly similar, making etiological differentiation difficult based on clinical presentation alone. Lower respiratory tract infections, especially pneumonia, require early etiological identification to enable targeted treatment and reduce unnecessary antibiotic exposure. Accurate etiological identification is crucial for clinical decision-making: particularly in distinguishing between viruses and non-influenza viruses to avoid unnecessary antibiotic use; timely detection and management of highly contagious diseases such as pertussis to control outbreaks; and for high-risk patients, early pathogen identification helps initiate specific antiviral therapy or prevent secondary bacterial infections. Therefore, developing detection technologies capable of rapidly, accurately, and simultaneously identifying multiple respiratory pathogens is a key link in achieving precision medicine and effective public health control.

[0004] Existing methods for detecting respiratory pathogens mainly fall into three categories. Traditional virus isolation and culture methods, while considered the "gold standard," are time-consuming and have low sensitivity, failing to meet the needs of rapid diagnosis. Furthermore, they present significant challenges in culturing most viruses and fastidious bacteria, which are difficult to culture. Immunological rapid detection technologies (such as colloidal gold test strips) are simple to operate, but lack sufficient sensitivity and specificity, resulting in a high false-negative rate, especially in the early stages of infection or in asymptomatic or mildly symptomatic patients, where reliability is poor.

[0005] Molecular biology techniques such as real-time quantitative polymerase chain reaction (qPCR) have become authoritative methods for detecting respiratory pathogens due to their high sensitivity and specificity. However, qPCR technology has inherent limitations when dealing with the diversity of respiratory pathogens: its multiplexing capability is limited by the number of fluorescence optical channels in the instrument. Conventional qPCR instruments can typically only distinguish 4-5 fluorescence signals simultaneously, meaning that a single reaction can only perform a maximum of 4-5 detections. Given the wide variety of pathogens causing respiratory infections, clinical practice often requires screening for more than ten targets. Using qPCR requires splitting a sample into multiple reaction tubes, which leads to high testing costs, cumbersome procedures, low throughput, and large sample consumption, greatly limiting its widespread clinical application and large-scale screening.

[0006] While multi-channel detection platforms such as microfluidic chips exist, their core often still relies on multi-channel qPCR, failing to fundamentally address the limitation on detection throughput caused by the number of fluorescence channels. Furthermore, system development and instrument costs are high. Therefore, there is an urgent need in this field for an innovative molecular detection solution that can overcome the bottleneck of fluorescence channel count in a single, closed reaction system, enabling simultaneous, high-precision, and low-cost detection of more than ten major respiratory pathogens. This solution should also be suitable for integration with automated platforms to meet the pressing needs of rapid clinical and field testing. Summary of the Invention

[0007] This invention covers the following technical solutions:

[0008] One aspect of the present invention relates to a primer-probe combination product comprising any 5, 10, or more combinations listed in a)-p):

[0009]

[0010] Another aspect of the invention relates to a kit containing the primer-probe combination product as described above.

[0011] Another aspect of the present invention relates to a diagnostic device for respiratory pathogens, the diagnostic device comprising one or more independent modules, which, when in operation, are capable of performing PCR amplification detection steps, and at least one independent module contains a kit or components thereof as described above.

[0012] This invention provides a multiple primer-probe combination, a reagent kit, and a diagnostic device. By integrating multiple specific primers and self-quenching probes into a single reaction system, it enables the simultaneous, rapid, and accurate detection of 15 common and important respiratory pathogens (including influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci). Detailed Implementation

[0013] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0014] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0015] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the nucleic acid chemistry, molecular biology, and microbiology terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0016] As used herein, the terms “and / or,” “or / and,” and “and / or” encompass any one of two or more of the relevant listed items, as well as any and all combinations of the relevant listed items, including any two of the relevant listed items, any more of the relevant listed items, or a combination of all the relevant listed items.

[0017] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0018] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0019] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0020] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0021] The concentration values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%.

[0022] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0023] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0024] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0025] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0026] In this invention, the term "Respiratory Tract Infection (RTI)" refers to infectious diseases caused by viruses, bacteria, mycoplasma, chlamydia, or other pathogenic microorganisms invading the human respiratory system, including both upper and lower respiratory tract infections. The scope of this term also encompasses what is clinically known as Acute Respiratory Infection (ARI), which is an infectious disease with rapid onset, short course, and main symptoms such as cough, sore throat, runny nose, fever, or difficulty breathing. The respiratory tract infection mentioned above can involve the nasal cavity, pharynx, trachea, bronchi, or lung tissue, and the causative pathogens include, but are not limited to, influenza A / B viruses, respiratory syncytial virus (RSV), coronaviruses (including SARS-CoV-2), adenoviruses, parainfluenza viruses, human metapneumovirus (HMPV), rhinoviruses, enteroviruses, bocaviruses (HBoV), and other viruses, as well as atypical bacteria such as Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci, and other respiratory pathogens. This term does not limit the site of infection or the course of the disease; as long as the pathogen can cause respiratory symptoms or pathological changes, it falls under the category of "respiratory tract infection."

[0027] The first aspect of the present invention relates to a primer-probe combination product comprising any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 combinations or all of them listed in a)-p):

[0028]

[0029] The primer-probe combination described in this invention can simultaneously detect up to 15 common and important pathogens causing respiratory infections in a single reaction system, including common viral, bacterial, and atypical pathogens, achieving "multi-target, one-time detection." Compared with existing multiplex PCR reagents that typically detect only 4-8 pathogens, this invention significantly expands the detection spectrum and improves detection efficiency by more than 100%, obtaining a complete pathogen infection spectrum in a single amplification reaction, providing a high-throughput solution for rapid clinical screening and typing diagnosis.

[0030] In terms of technical implementation, constructing ultramultiplex PCR systems presents significant design challenges. As the number of targets increases, the number of primers and probes within the system grows exponentially, easily leading to problems such as sequence complementarity, dimer formation, and Tm (melting temperature) overlap, resulting in nonspecific amplification or signal interference. This invention, through precise design of each primer and probe sequence, controlling its GC content, length, and secondary structure, ensures the Tm differences between different targets, allowing the 15-fold detection system to maintain sensitivity and specificity comparable to single-target systems.

[0031] This invention, through systematic optimization of primers and probes, successfully realizes a highly complex and stable ultra-multiplex PCR system. This approach achieves levels of reaction compatibility, signal resolution, and clinical operability that are difficult to attain with existing technologies, providing a stable and industrially viable solution for simultaneous detection of multiple pathogens.

[0032] In some embodiments, the primer-probe combination product further includes primers and probes for detecting human internal reference nucleic acids.

[0033] The purpose of setting up human internal control nucleic acid is to conduct quality control throughout the entire process of sample collection, nucleic acid extraction and amplification, thereby determining whether there are sufficient human cell components in the sample and verifying the integrity of the reaction system.

[0034] Furthermore, the human internal reference nucleic acid can be derived from any gene that is stably expressed in various types of human cells, such as, but not limited to, the following categories:

[0035] (1) Ribosomes or housekeeping genes: such as 18S rRNA, β-Actin (ACTB), GAPDH, β2-Microglobulin (B2M), Hypoxanthine Phosphoribosyltransferase 1 (HPRT1), PeptidylprolylIsomerase A (PPIA, Cyclophilin A);

[0036] (2) Cell structure or metabolism-related genes: such as Tubulin (TUBB), Elongation Factor 1α (EEF1A1), Ubiquitin C (UBC), Ribosomal Protein L13A (RPL13A);

[0037] (3) Nucleic acid processing and RNA splicing genes: such as RNAse P (RPP30), RNA Polymerase II (POLR2A), TATA-binding Protein (TBP), etc.

[0038] These genes have constant copy numbers and stable transcription levels in different tissues or sample types, which can reflect the presence of human cells in the sample and the effectiveness of the reaction system.

[0039] In some embodiments, the human internal reference nucleic acid is derived from the RNase P gene.

[0040] In some embodiments, the human internal reference nucleic acid is derived from the RPP30 target site of the RNase P gene.

[0041] This target site, due to its conserved sequence, single copy number, and detectability in all human samples, has been widely adopted by various clinical nucleic acid detection systems (such as the CDC-recommended SARS-CoV-2 RT-PCR protocol), demonstrating good representativeness and reproducibility. The primers and probes used for detecting human internal control nucleic acid can employ molecular beacon structures or other self-quenching probe structures to ensure compatibility with pathogen detection systems and consistency in signal interpretation.

[0042] In some embodiments, the primers and probes for detecting human internal reference nucleic acid include a combination of primers with nucleotide sequences shown in SEQ ID NO: 49-50 and a probe shown in SEQ ID NO: 51.

[0043] Additionally, it should be noted that, in one respect, the concept of useful primers and probes should include nucleotide sequences having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one or more of the primers or probes shown in SEQ ID NO: 1-51. Modifications of such primers and probes and their ability to be prepared according to standard techniques are also considered.

[0044] The term "%identity" in the context of two or more nucleotide or amino acid sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured by one of the following sequence comparison algorithms or by visual inspection. For example, %identity is relative to the entire length of the coding region of the sequences to be compared.

[0045] For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to this reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates and algorithm parameters are specified if necessary. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified parameters. Percentage identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic AcidsRes25:17, 3389-402).

[0046] Primer and probe modifications can be performed using well-known methods. Modified versions of these primer and / or probe sequences may include, by non-limiting examples, adding one or more nucleotides to the 5' end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to both the 5' and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, shifting the sequence upstream or downstream by several bases, or any combination thereof.

[0047] Base modifications, such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5-propynyl-deoxycytidine, C-5-propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), reversed-dT, reversed-dideoxy-T, hydroxymethyl-dC, iso-dC, 5-methyl-dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNAs), including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, can achieve, for example, increased nucleic acid interaction at the 3' end of mutant-specific primers to increase Tm. The addition of stable double-stranded base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. Modified probes should retain the ability to distinguish between the mutant and wild-type sites to be detected.

[0048] In some embodiments, the probe is labeled with a detectable signal substance. In some embodiments, the signal substance is a fluorophore, colorimetric label, colloidal gold, quantum dot, biotin, and other tag molecules that can be used for detection (such as alkyne groups for Raman diffraction imaging, cycloalkenes for click reactions, and initiating groups for polymer labeling), or it may be selected from peptide / protein molecules, LNA / PNA, non-natural amino acids and their analogs (e.g., peptides), non-natural nucleic acids and their analogs (nucleotides), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation / assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalates, etc.).

[0049] In some implementations, the probes are self-quenching probes.

[0050] In this invention, the term "self-quenched probe" refers to a class of single-stranded oligonucleotide molecules with a fluorescent group and a quenching group attached to their 5′ and 3′ ends, respectively. When the probe is in a free or unbound state, the spatial distance between the fluorescent group and the quenching group is close or they are energy-coupled, causing the fluorescence energy to be partially or completely quenched. When the probe binds to a complementary target nucleic acid, is cleaved by a nuclease, or undergoes a conformational change, the energy transfer between the fluorescent group and the quenching group is interrupted or the distance increases, thereby releasing a detectable fluorescent signal. Thus, the probe itself can generate and eliminate the signal without the need for an external colorimetric or second reporter system.

[0051] Self-quenching probes known in the art can include various conformations or mechanisms of action. As long as they possess the characteristic of achieving fluorescence quenching and de-quenching through intramolecular or intermolecular energy transfer, they can be considered as "self-quenching probes" of this invention. These probes can be used in real-time fluorescence PCR, melting curve analysis, isothermal amplification, or multiplex amplification systems to achieve qualitative or quantitative detection of nucleic acid targets. The specific form of self-quenching probes is not limited to any particular structure, but preferably includes, but is not limited to, the following subtypes: stem-loop molecular beacons (MB), linear cleavage probes (TaqMan probes), built-in probes (Scorpion, LUX, or Amplifluor, etc.), and FRET-based dual-probe systems.

[0052] In some embodiments, the fluorescent emitting groups of each probe are independently selected from any one of AMCA, Pacific Blue, Atto425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor593, Texas Red, Atto 590, Cy5, Quasar 670, Cy5.5, and Cy5.5.

[0053] In some embodiments, the quenching groups of each probe are independently selected from any one of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, and MGB.

[0054] A second aspect of the invention also relates to a kit containing the primer-probe combination product as described above.

[0055] In this disclosure, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. The kit may further include instructions for use, supplementary reagents, and / or components or parts used in the methods or steps described in this disclosure.

[0056] In some embodiments, the kit also includes nucleic acid extraction reagents and / or PCR amplification reagents.

[0057] This design allows the entire detection process, from sample processing and nucleic acid extraction to amplification and detection, to be completed continuously in the same system, thereby improving operational convenience and detection consistency.

[0058] The nucleic acid extraction reagents and PCR amplification reagents can be flexibly configured according to the detection purpose and device structure. The components in the kit can be packaged in the form of solutions, solids, or test strips. Premixed or lyophilized forms are preferred to achieve rapid reconstitution and stable preservation. In some preferred embodiments, at least one component of the reagents or kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, or spots formed on the surface of a solid carrier. Therefore, components required for nucleic acid amplification (and preferably nucleic acid detection) can be provided in lyophilized form, particularly various enzymes, nucleic acid components, and reaction buffer components. In this way, the nucleic acid amplification (and preferably nucleic acid detection) process can be started directly in a user-friendly manner by adding the sample to be quantified and optionally other required components.

[0059] Furthermore, the nucleic acid extraction reagent may include any one or more of lysis buffer, washing buffer, and elution buffer. The lysis buffer may contain surfactants (such as Triton X-100, Tween-20, SDS), protein denaturants (such as guanidine salts, urea), chelating agents (such as EDTA), and buffer components; the washing buffer may contain appropriate amounts of alcohols or salts to remove impurities; the elution buffer is typically a low-salt buffer or nuclease-free water. The extraction method is not limited to any specific technical route and may include silica-based membrane adsorption, magnetic bead methods, or direct lysis amplification methods, etc. Those skilled in the art can select a suitable extraction scheme based on the detection equipment.

[0060] The PCR amplification reagents may include nucleic acid polymerase, dNTPs, buffer, and Mg. 2+ Salt, stabilizers, and auxiliary components required for fluorescence detection. The polymerase can be Taq, HotStart Taq, inhibition-resistant mutant Taq, or a mixture of reverse transcriptase and DNA polymerase; the reaction buffer can contain conventional components such as Tris-HCl, KCl, and (NH4)2SO4; stabilizers can be BSA, trehalose, glycerol, or polyvinylpyrrolidone (PVP). To improve transport and storage stability, the PCR amplification reagents are preferably encapsulated together with primers and probes in the reaction tube or microfluidic chip reaction chamber in the form of lyophilized powder or lyophilized microspheres, which can be reconstituted by adding samples before use.

[0061] By integrating nucleic acid extraction and PCR amplification functions in the same packaging unit, testing personnel can achieve closed-loop testing from sample to result without transferring reagents in an open environment. This is particularly suitable for rapid on-site testing, primary healthcare institutions, and fully enclosed automated nucleic acid testing devices.

[0062] A third aspect of the present invention also relates to a diagnostic device for respiratory pathogens, the diagnostic device comprising one or more independent modules, which, when in operation, are capable of performing PCR amplification detection steps, and at least one independent module comprises a kit or its components as described above.

[0063] The modules and reagent kits in the diagnostic device can be packaged independently or integrated as a whole. Preferably, at least some components of the reagent kit (including primers, probes, reaction buffers, polymerases, and internal control materials, etc.) are pre-filled or immobilized in the reaction module of the device so that the amplification reaction can be automatically executed by adding or loading samples during detection.

[0064] In a preferred embodiment, the diagnostic device employs a disposable, closed microfluidic chip as the core reaction carrier. This microfluidic chip contains multiple independent reaction chambers, each containing pre-lyophilized microspheres containing primers, probes, and amplification system components. During detection, samples are automatically dispensed into the corresponding reaction chambers for reconstitution, amplification, and signal acquisition, thereby achieving simultaneous detection of multiple targets. This chip structure helps prevent aerosol contamination during the detection process, improves detection speed and result stability, and facilitates parallel detection of multiple samples and long-term reagent preservation.

[0065] Those skilled in the art will understand that such diagnostic devices can employ any conventional or modified nucleic acid detection instrument structure, as long as they can complete sample loading, temperature control cycling, and fluorescence signal acquisition. Exemplary diagnostic devices include any one or more structures of the microfluidic or closed nucleic acid detection systems disclosed in Chinese patent applications CN202010571470.9, CN202021162806.8, CN202010769972.2, CN202021585862.2, and CN202210062076.1. The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following examples that do not specify specific conditions should preferably refer to the guidelines given in this invention, or be performed according to experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0066] The diagnostic device designed as described above can significantly reduce sample processing steps and contamination risks while maintaining the sensitivity and specificity of the amplification system.

[0067] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0068] Example 1

[0069] (1) Design of amplification primer and probe combination

[0070] Specific primer and probe sets were designed against the specific target sequences of influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci. The primer and probe set list is shown in Table 1.

[0071]

[0072]

[0073] Each probe has a fluorescent group at its 5' end and a quenching group at its 3' end;

[0074] The fluorescent group is FAM, Texas Red / ROX, Cy5, Cy5.5, CY7, or HEX / VIC, etc.

[0075] The quenching group is BHQ.

[0076] (2) Combined detection kit for influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci.

[0077] Using the aforementioned primer and probe combinations, detection kits were prepared for influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci. The kits also include nucleic acid extraction reagents and qPCR amplification reagents. RP (Reactive Probe) was used as an internal control to perform quality control throughout the entire sample collection and testing process.

[0078] (3) Nucleic acid amplification detection

[0079] Nucleic acid testing includes the following steps:

[0080] S1, Nucleic acid extraction from the sample to be tested: Lysis buffer and proteinase K are added to the sample to be tested, and the nucleic acid is extracted and purified by magnetic beads. Then, elution buffer is added to obtain the nucleic acid of the sample to be tested.

[0081] S2, Preparation of multiplex real-time PCR reaction system: 20uL template, 3.5uL Primer Mix (the above primer and probe combination), 25uL 2×PCR Mix, and water to make up to 50uL.

[0082] S3, the PCR amplification program was as follows: 50℃ reverse transcription for 10 min; 95℃ pre-denaturation for 5 min; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, 45 cycles, with fluorescence signals collected during the annealing and extension phase of each cycle; 95℃ denaturation for 1 min, 50℃ annealing for 1 min, and fluorescence signals collected during the 50→95℃ melting curve.

[0083] S4. Based on the melting curve results, the following interpretation rules are applied: Identify the following viruses in the sample: influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci.

[0084] (4) The interpretation of the dissolution curve is as follows:

[0085]

[0086] Example 2

[0087] Sensitivity testing for influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci.

[0088] Add 500 copies / mL of influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci samples to negative pharyngeal swabs, respectively. Add lysis buffer and proteinase K, extract and purify nucleic acids using magnetic beads, and then add elution buffer to obtain the nucleic acid of the sample to be tested.

[0089] The extracted nucleic acids of 15 pathogens were used as templates for amplification. After the amplification system was prepared, the mixture was stirred and then loaded onto the instrument.

[0090] The amplification system is shown in the table below:

[0091]

[0092] Place the PCR eight-pack in the sample chamber of the real-time PCR instrument, and after setting the amplification program, start the amplification.

[0093]

[0094] Experimental results

[0095]

[0096] The results showed that the detection sensitivity for influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci could all reach 500 copies / mL.

[0097] Example 3

[0098] The specific testing steps for detecting influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci are as follows:

[0099] 1. Using a negative throat swab as a negative matrix, add cross-reactant to the throat swab to achieve a viral cross-reactant concentration of 10. 5 PFU / mL, bacterial cross-reactive agent concentration was 10. 6 CFU / mL, which was used as the sample to be tested.

[0100] 2. Nucleic acid extraction

[0101] Add lysis buffer and proteinase K, extract and purify nucleic acid using magnetic beads, and then add elution buffer to obtain the nucleic acid of the sample to be tested.

[0102] 3. Preparation of qPCR amplification system:

[0103] After preparing the amplification system, mix thoroughly and then run the reaction mixture. The Primer Mix is ​​the amplification primer and probe combination from Example 1, and the reaction system is as follows:

[0104]

[0105] 4. Place the PCR eight-pack in the sample chamber of the real-time PCR instrument, and start amplification after setting the amplification program.

[0106]

[0107] 5. Test Results

[0108]

[0109]

[0110]

[0111]

[0112] The results showed that the combined detection kit for influenza A virus, influenza B virus, respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, mycoplasma pneumoniae, parainfluenza virus, rhinovirus, enterovirus, coronavirus, adenovirus, metapneumovirus, Bordetella pertussis, bocavirus, Chlamydia pneumoniae, and Chlamydia psittaci showed no cross-reactivity with the above 18 cross-reactants, demonstrating good specificity.

[0113] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A primer-probe combo product, characterized in that, Including any 5, 10 or more combinations listed in a)-p):

2. The primer-probe combination product according to claim 1, characterized in that, It also includes primers and probes for detecting human internal reference nucleic acids.

3. The primer-probe combination product according to claim 2, characterized in that, The human internal reference nucleic acid was derived from the RNase P gene.

4. The combined product according to claim 3, characterized in that, The human internal reference nucleic acid is derived from the RPP30 target site of the RNase P gene.

5. The combined product according to claim 4, characterized in that, The primers and probes used to detect human internal reference nucleic acid include a combination of primers with nucleotide sequences shown in SEQ ID NO: 49-50 and a probe shown in SEQ ID NO:

51.

6. The combined product according to any one of claims 1-5, characterized in that, All probes are self-quenching probes.

7. A reagent kit, characterized in that, A product containing the primer-probe combination according to any one of claims 1-6.

8. The reagent kit according to claim 7, characterized in that, It also includes nucleic acid extraction reagents and / or PCR amplification reagents.

9. The reagent kit according to claim 7 or 8, characterized in that, At least one component of the reagent or kit is a solid, and the solid includes at least one of lyophilized microspheres, lyophilized cakes, lyophilized powder, or spots formed on the surface of a solid carrier.

10. A diagnostic device for respiratory tract infection pathogens, characterized in that, The diagnostic device includes one or more independent modules that, when in operation, are capable of performing PCR amplification detection steps, and at least one independent module contains the kit or a component thereof as described in any one of claims 7-9.

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