Nucleic acid panel, pcr master mix, kit, and method for identifying respiratory pathogens

Abstract

The application relates to the technical field of molecular biology detection, in particular to a nucleic acid combination product for identifying respiratory tract pathogens, a PCR premix, a kit and a method. The nucleic acid combination product comprises primer pairs and probes shown in SEQ ID NO: 1 to SEQ ID NO: 21. The primer pairs and the probes can be mixed with other reagents required by PCR, and then freeze-dried to prepare a freeze-dried ball. The freeze-dried ball is smooth and round in surface, and after a nucleic acid sample liquid to be detected is directly dropped into a PCR reaction tube containing the freeze-dried ball, machine detection can be carried out. The freeze-dried ball is not only beneficial to storage and transportation, but also convenient to operate. In particular, the effect of joint detection of target pathogens is good, and the detection sensitivity is also improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology detection, in particular to a nucleic acid combination product for identifying respiratory pathogens, a PCR premix, a kit and a method. BACKGROUND

[0002] Currently, mixed infection of respiratory pathogens, especially the complex clinical scenario of secondary bacterial infection as the initial pathogen of virus, has constituted a significant challenge in the field of global public health. Respiratory viruses represented by coronavirus and parainfluenza virus (PIV) can create a niche for subsequent bacterial colonization and invasion by destroying the integrity of airway mucosa and inducing temporary immune paralysis in the early stage of infection. Under this background, Haemophilus influenzae (HI) and Streptococcus pneumoniae (SP) as typical secondary pathogenic bacteria can cause the clinical course from mild upper respiratory tract infection to severe pneumonia, sinusitis and otitis media and other complications. In addition, Group A Streptococcus (GAS) can cause severe pharyngitis or invasive infection on the basis of viral damage, while Chlamydia Pneumoniae (CP) shows the dual characteristics of independent pathogenicity and participation in multi-pathogen synergistic pathogenicity, often leading to prolonged illness. In the pediatric population, the prodromal symptoms of viral infection are easily confused with the paroxysmal spasmodic cough caused by Bordetella pertussis (BP), further exacerbating the complexity of diagnosis and treatment. Such multiple infections not only significantly aggravate the clinical condition of patients and prolong the hospitalization period, but also are the key driving factors for the occurrence and death risk of severe pneumonia.

[0003] In the face of diagnostic difficulties such as overlapping clinical manifestations and insufficient sensitivity of traditional pathogen detection technology, clinical practice highly relies on biomarkers such as procalcitonin (PCT) to guide empirical anti-infective therapy. In order to cope with the increasingly complex pathogen spectrum and the challenge of antimicrobial drug resistance, promoting early accurate pathogen identification based on molecular diagnostic technology has become the core direction to optimize treatment strategies and improve patient outcomes.

[0004] For the above common respiratory pathogens, the traditional rapid molecular diagnosis scheme, for example, CN119242864A, discloses a composition for detecting respiratory infection related pathogens, which includes upstream and downstream primers and probes for detecting coronavirus, Bordetella pertussis, Streptococcus group A, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae and Chlamydia pneumoniae, and discloses a kit comprising the primers and probes and a method for multi-target joint detection using the same. However, the PCR reaction liquid and enzyme mixed liquid in the kit are in liquid state, and when used, the corresponding amount of components is taken according to the proportion (PCR reaction liquid 26 μL / person + enzyme mixed liquid 4 μL / person), and the PCR mixed liquid is fully mixed and centrifuged for standby. However, liquid reagents have disadvantages such as poor storage and transportation. In addition, the PCR reaction liquid and enzyme mixed liquid packaged independently will also make the detection process cumbersome.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] Based on this, one or more embodiments of the present application provide a nucleic acid combination product, a PCR premix, a kit and a method for identifying respiratory pathogens. The technical solutions include the following:

[0007] One or more embodiments of the present application provide a nucleic acid combination product for identifying respiratory pathogens, which comprises primer pairs and probes shown in SEQ ID NO: 1 to SEQ ID NO: 21.

[0008] In some embodiments of the present application, the nucleic acid combination product further comprises primer pairs and probes shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0009] In some embodiments of the present application, the nucleic acid combination product, the types of fluorescent reporter groups labeled on each probe are different.

[0010] In some embodiments of the present application, the fluorescent reporter groups labeled on each probe are selected from any one of FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7 and AF 405.

[0011] One or more embodiments of the present application provide a PCR premix for identifying respiratory pathogens, which comprises the nucleic acid combination product and other PCR amplification reagents.

[0012] In some embodiments of the present application, the other PCR amplification reagents include one or more of PCR buffer, Mg 2+ , dNTPs, Taq enzyme and RT enzyme.

[0013] In some embodiments of the present application, the PCR premix further comprises a lyoprotectant.

[0014] In some embodiments of the present application, the PCR premix is freeze-dried into a lyophilized state.

[0015] One or more embodiments of the present application provide a kit for identifying respiratory pathogens, which comprises the nucleic acid combination product or the PCR premix.

[0016] In some embodiments of the present application, the kit further comprises one or more of a sampling tool, a sample preservation reagent, a nucleic acid release reagent, a nucleic acid extraction reagent, a negative control, a positive control, and a reconstitution reagent.

[0017] One or more embodiments of the present application provide a method for identifying respiratory pathogens for non-diagnostic purposes, which detects nucleic acid of a sample to be tested by using the nucleic acid combination product, the PCR premix, or the kit.

[0018] In some embodiments of the present application, the method comprises the following steps in a closed reaction tube: pre-treating the sample to be tested with the nucleic acid release reagent, and mixing the pre-treatment product with the lyophilized PCR premix defined above to detect the nucleic acid of the sample to be tested.

[0019] In some embodiments of the present application, the sample to be tested is a swab sample or a plasmid sample.

[0020] In some embodiments of the present application, the swab sample is a throat swab or a nasal swab.

[0021] Compared with the conventional technology, the present application designs specific primer pairs and probe combinations for seven respiratory pathogens, which can be mixed with other reagents required for PCR and freeze-dried into lyophilized balls. The lyophilized balls are smooth and round, and the nucleic acid sample solution to be tested can be directly added to the PCR reaction tube containing the lyophilized balls for machine detection. This not only facilitates storage and transportation, but also is convenient to operate, especially the target pathogen co-detection effect is good, and the detection sensitivity is also improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 Shown is a human coronavirus positive in Example 3.

[0024] Figure 2 Shown is a Haemophilus influenzae positive in Example 3.

[0025] Figure 3 Shown is a Streptococcus pneumoniae positive in Example 3.

[0026] Figure 4 Shown is a Parainfluenza virus positive in Example 3.

[0027] Figure 5 Shown is a Group A Streptococcus positive in Example 3.

[0028] Figure 6 Shown is a Bordetella pertussis positive in Example 3.

[0029] Figure 7 Shown is a Chlamydia pneumoniae positive in Example 3.

[0030] Figure 8 Shown is a combined target detection result - positive in Example 3.

[0031] Figure 9 Shown is a human coronavirus sensitivity detection result in Example 4.

[0032] Figure 10 Shown is a Haemophilus influenzae sensitivity detection result in Example 4.

[0033] Figure 11 Shown is a Streptococcus pneumoniae sensitivity detection result in Example 4.

[0034] Figure 12 Shown is a Parainfluenza virus sensitivity detection result in Example 4.

[0035] Figure 13 Shown is a Group A Streptococcus sensitivity detection result in Example 4.

[0036] Figure 14 Shown is a Bordetella pertussis sensitivity detection result in Example 4.

[0037] Figure 15 Shown is a Chlamydia pneumoniae sensitivity detection result in Example 4.

[0038] Figure 16 Shown is a specificity detection result in Example 5.

[0039] Figure 17 Shown is a human coronavirus anti-interference detection result in Example 6.

[0040] Figure 18 Shown are the results of anti-interference detection of Haemophilus influenzae in Example 6.

[0041] Figure 19 Shown are the results of anti-interference detection of Streptococcus pneumoniae in Example 6.

[0042] Figure 20 Shown are the results of anti-interference detection of Parainfluenza virus in Example 6.

[0043] Figure 21 Shown are the results of anti-interference detection of Group A Streptococcus in Example 6.

[0044] Figure 22 Shown are the results of anti-interference detection of Bordetella pertussis in Example 6.

[0045] Figure 23 Shown are the results of anti-interference detection of Chlamydia pneumoniae in Example 6.

[0046] Figure 24 Shown are the results of primer probe combination target point test in Comparative Example 1.

[0047] Figure 25 Shown are the results of human coronavirus sensitivity detection in Comparative Example 1.

[0048] Figure 26 Shown are the results of Haemophilus influenzae sensitivity detection in Comparative Example 1.

[0049] Figure 27 Shown are the results of Streptococcus pneumoniae sensitivity detection in Comparative Example 1.

[0050] Figure 28 Shown are the results of Parainfluenza virus sensitivity detection in Comparative Example 1.

[0051] Figure 29 Shown are the results of Group A Streptococcus sensitivity detection in Comparative Example 1.

[0052] Figure 30 Shown are the results of Bordetella pertussis sensitivity detection in Comparative Example 1.

[0053] Figure 31 Shown are the results of Chlamydia pneumoniae sensitivity detection in Comparative Example 1.

[0054] Figure 32 Shown are the comparison diagrams of different lyophilized reagents corresponding to different PCR reaction solutions in Comparative Example 2; wherein, A diagram shows the morphology of A-lyophilized reagent, B diagram shows the morphology of B-lyophilized reagent, and C diagram shows the morphology of C-lyophilized reagent.

[0055] Figures 33 to 35The detection results of different lyophilized reagents corresponding to different PCR reaction solutions in Comparative Example 2 are shown. DETAILED DESCRIPTION

[0056] The application will be further described below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to explain the application and not intended to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the disclosure of the application more thoroughly and comprehensively understood. It should also be understood that the application can be implemented in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the application, and the equivalent forms obtained thereby also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

[0058] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0059] The selection range of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0060] In the present application, “plurality”, “a plurality of”, “multiple times”, “multiple”, and the like, if not specifically limited, refer to greater than or equal to 2 in number. For example, “one or more” means one or greater than or equal to two.

[0061] As used herein, “combinations thereof”, “any combination thereof”, “any combination manner thereof”, and the like, include all suitable combination manners of any two or more of the listed items.

[0062] As used herein, “suitable”, “suitable manner”, “any suitable manner”, and the like, refer to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0063] As used herein, “preferably”, “better”, “more preferably”, and the like, are merely used to describe embodiments or examples with better effects, and should be understood as not constituting a limitation on the protection scope of the present application.

[0064] In the present application, “further”, “more further”, “in particular”, and the like, are used for the purpose of description, indicating differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0065] In the present application, “optionally”, “optional”, and the like, mean that it can or can not exist, i.e., it means selecting either of the two parallel schemes “yes” or “no”. If there are multiple “optionally” in a technical solution, and there is no specific description, and no contradictory relationship or mutual restriction, each “optionally” is independent.

[0066] In the present application, in the terms “first aspect”, “second aspect”, “third aspect”, “fourth aspect”, and the like, the terms “first”, “second”, “third”, “fourth”, and the like, are only used for the purpose of description, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, “first”, “second”, “third”, “fourth”, and the like, only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0067] In the present application, in the technical features described in an open manner, both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features are included.

[0068] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0069] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0070] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0071] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0072] Based on previous research, the applicant has developed a composition for detecting respiratory infection-related pathogens. This composition includes upstream and downstream primers and probes for detecting coronaviruses, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae, and has filed patent application CN119242864A based on this. However, previous primer and probe compositions were mainly used for developing liquid reagent products. This application aims to promote product optimization and upgrades, providing primers and probes suitable for developing lyophilized reagents. Therefore, this application is filed.

[0073] A first aspect of this application provides a nucleic acid combination product for identifying respiratory pathogens, the nucleic acid combination product comprising primer pairs and probes shown in SEQ ID NO: 1 to SEQ ID NO: 21.

[0074] The nucleic acid combination product of this application also includes primer pairs and probes for detecting internal standard genes. This application does not make any special limitations on these, and they can be, but are not limited to, the primer pairs and probes shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0075] In some embodiments of this application, the types of fluorescent reporter groups labeled on each probe in the nucleic acid combination product are different. This application does not specifically limit the fluorescent reporter groups and quencher groups labeled on each probe. Fluorescent reporter groups may include, but are not limited to: FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7, and AF 405. Quencher groups may include, but are not limited to: BHQ1, SQ2, SQ3, and SQ0.

[0076] A second aspect of this application provides a PCR premix for identifying respiratory pathogens, the PCR premix comprising the aforementioned nucleic acid combination product and other PCR amplification reagents.

[0077] This application does not specifically limit other PCR amplification reagents. In some embodiments of this application, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes. For example, in one embodiment of this application, other PCR amplification reagents include the PCR buffer required for detection, Mg... 2+ dNTPs, Taq enzymes, and RT enzymes.

[0078] This application does not impose any particular limitation on the product form of the PCR premix; it can be liquid or lyophilized. It is understood that a lyophilization protectant may be added to ensure good performance of the lyophilized PCR premix. This application does not impose any particular limitation on the type and amount of the lyophilization protectant; for example, trehalose can be used as a lyophilization protectant at a working concentration of 7 wt%. It is understood that those skilled in the art can also choose other suitable lyophilization protectants and determine the working concentration that can exert the lyophilization protection effect.

[0079] A third aspect of this application provides a kit for identifying respiratory pathogens, the kit comprising the aforementioned nucleic acid combination product or the aforementioned PCR premix.

[0080] In some embodiments of this application, the kit further includes one or more of the following: sampling tools, sample preservation reagents, nucleic acid release reagents, nucleic acid extraction reagents, negative controls, positive controls, and reconstitution solvents.

[0081] A fourth aspect of this application provides a method for identifying respiratory pathogens for non-diagnostic purposes, wherein the method uses the aforementioned nucleic acid combination product, the aforementioned PCR premix, or the aforementioned kit to detect the nucleic acid of the sample to be tested.

[0082] This application does not impose any particular limitation on the method. The sample to be tested can be pretreated first, and then the nucleic acid obtained from the pretreatment can be manually added to the PCR premix for detection. Alternatively, the nucleic acid can be directly mixed with the PCR premix for detection without relying on manual transfer of nucleic acid. For example, the following operation can be performed in a closed reaction tube: the sample to be tested is placed in the nucleic acid release reagent for pretreatment, and the resulting pretreatment product is quantitatively mixed with the lyophilized PCR premix defined above to achieve detection of the nucleic acid in the sample to be tested. For example, the detection device and detection method described in the invention patent application with publication number CN116555007A can be used, which simplifies the process and avoids contamination.

[0083] This application does not impose any special limitations on the samples to be tested, including but not limited to: swab samples (e.g., throat swabs, nasal swabs), plasmid samples.

[0084] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0085] 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. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0086] This application designs specific primer and probe combinations targeting the conserved sequences of seven respiratory pathogens. Since a multiplex PCR detection system is used, multiple sets of primers and probes exist simultaneously in the reaction system, posing a risk of primer dimer formation and non-specific amplification. The primer and probe combinations in this application avoid this risk. For the fluorescence detection channels, non-mainstream fluorescence channels such as QUASAR 705, ATTO 425, CY7, and AF 405 were selected. Furthermore, 8-color fluorescence channels have a higher risk of crosstalk than 4-color fluorescence channels, making false positives more likely. This application also avoids this situation. Respiratory multi-pathogen detection reagents are prone to degradation and interactions during long-term storage in liquid systems, limiting reagent performance stability. This application upgrades the reagent to a lyophilized reagent, effectively improving the long-term stability and detection sensitivity of the reagent under room temperature conditions.

[0087] Example 1: Primers and probes designed in this application

[0088] Table 1

[0089]

[0090] The fluorescent reporter group of the probe shown in SEQ ID NO: 3 is FAM and the quencher group is BHQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 6 is HEX and the quencher group is BHQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 9 is ROX and the quencher group is SQ2; the fluorescent reporter group of the probe shown in SEQ ID NO: 12 is CY5 and the quencher group is SQ2; the fluorescent reporter group of the probe shown in SEQ ID NO: 15 is QUASAR 705 and the quencher group is SQ3; the fluorescent reporter group of the probe shown in SEQ ID NO: 18 is ATTO 425 and the quencher group is SQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 21 is CY7 and the quencher group is SQ3; and the fluorescent reporter group of the probe shown in SEQ ID NO: 24 is AF 405 and the quencher group is SQ0.

[0091] Example 2: Method for detecting pathogens

[0092] 1. Reagent preparation

[0093] The corresponding PCR amplification kit in this application is designated as the Respiratory Seven Pathogen Nucleic Acid Detection Kit (Fluorescent PCR Method).

[0094] 1.1 Remove the reagents from the kit and store at room temperature for later use.

[0095] Direct amplification method nucleic acid release reagent: Sample release agent (Sansure Biotech Co., Ltd.).

[0096] 1.2 Add 1000 μL of the reconstitution solvent to the positive control (lyophilized), shake thoroughly to mix, centrifuge at 2000 rpm for 10 seconds, and set aside.

[0097] 1.3 Based on the quantity of the sample to be tested, negative control (physiological saline), and positive control (the mixed standard of pathogens in Table 1), take the corresponding amount of reagents and set them aside.

[0098] 1.4 Transfer the prepared reagents to the sample processing area for later use.

[0099] 2. Sample processing and loading

[0100] The samples tested in this application were pharyngeal swabs and nasal swabs. Viral nucleic acid was lysed using direct amplification, and the following procedures were performed in the sample processing room:

[0101] 2.1 Dilute the sample with the sample release agent at a ratio of 3:1 (v / v), mix thoroughly, and let stand for later use.

[0102] 2.2 Add the above-treated test sample, negative control and positive control 25 μL each to the PCR reaction tube containing the lyophilized reagent (prepared by freeze-drying the PCR reaction solution shown in Table 2) and the reaction system as shown in Table 2. Cover the tube (if there are air bubbles, you can tap them with your finger to remove them), and centrifuge at 2000 rpm for 10 seconds until there are no air bubbles and obvious liquid droplets on the tube wall.

[0103] Table 2

[0104]

[0105] 3. PCR amplification

[0106] PCR amplification was performed on the SLAN-48S fully automated medical PCR analysis system PCR instrument according to a specific temperature and time setting program. The PCR amplification program of this application is shown in Table 3.

[0107] Table 3

[0108]

[0109] 4. Interpretation of test results

[0110] The details are shown in Tables 4 and 5 below.

[0111] Samples with a typical S-shaped amplification curve detected in the FAM channel and Ct≤40 are reported as positive for human coronavirus; samples without a typical S-shaped amplification curve detected in the FAM channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for human coronavirus.

[0112] Samples with a typical S-type amplification curve detected in the HEX channel and Ct≤40 are reported as positive for Haemophilus influenzae; samples without a typical S-type amplification curve detected in the HEX channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for Haemophilus influenzae.

[0113] For samples where a typical S-type amplification curve is detected in the ROX channel and Ct≤40, the report is positive for Streptococcus pneumoniae; for samples where a typical S-type amplification curve is not detected in the ROX channel, or Ct>40, but an amplification curve is present in the AF 405 channel and Ct≤40, the report is negative for Streptococcus pneumoniae.

[0114] Samples with a typical S-type amplification curve detected in the CY5 channel and Ct≤40 are reported as positive for parainfluenza virus; samples without a typical S-type amplification curve detected in the CY5 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for parainfluenza virus.

[0115] For samples where a typical S-shaped amplification curve is detected in the QUASAR 705 channel and Ct≤40, the report is positive for Group A Streptococcus; for samples where a typical S-shaped amplification curve is not detected in the QUASAR 705 channel, or Ct>40, and an amplification curve is detected in the AF 405 channel and Ct≤40, the report is negative for Group A Streptococcus.

[0116] For samples where a typical S-type amplification curve is detected in the ATTO 425 channel and Ct≤40, the report is positive for Bordetella pertussis; for samples where a typical S-type amplification curve is not detected in the ATTO 425 channel, or Ct>40, and an amplification curve is detected in the AF 405 channel and Ct≤40, the report is negative for Bordetella pertussis.

[0117] Samples with a typical S-type amplification curve detected in the CY7 channel and Ct≤40 are reported as positive for Chlamydia pneumoniae; samples without a typical S-type amplification curve detected in the CY7 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for Chlamydia pneumoniae.

[0118] Table 4

[0119]

[0120] Table 5

[0121]

[0122] This application integrates the TaqMan Quantitative Real-Time (QRP) core with an innovative multiplex detection design, enabling precise detection of eight targets (including internal standard) in a single tube. Optimized workflow significantly increases throughput, reduces manual operation and errors, and provides intuitive result interpretation. Lyophilization technology endows reagents with excellent stability and room-temperature storage and transportation characteristics, effectively reducing reliance on cold chain and overall reagent costs, while greatly simplifying the operation steps.

[0123] Example 3: Detection results of the test sample in this application

[0124] Using the method described in Example 2, one sample each of human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae (all clinically positive samples) were detected by PCR on a Hongshi real-time quantitative PCR instrument. The results are as follows: Figures 1 to 8 As shown: Figure 1 The result indicates a positive result for human coronavirus. Figure 2 Haemophilus influenzae positive. Figure 3 The result shows a positive result for Streptococcus pneumoniae. Figure 4 The result shows a positive result for parainfluenza virus. Figure 5 The result shows a positive result for Group A Streptococcus. Figure 6 The result shows a positive result for Bordetella pertussis. Figure 7 The result shows a positive result for Chlamydia pneumoniae.

[0125] Using the same method as in Example 2, human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae samples (all clinically positive samples) were mixed in equal proportions to prepare a pooled sample for PCR detection. Figure 8 The result shown is a positive result from the combined target detection.

[0126] Example 4: Sensitivity of this application

[0127] Serially diluted pathogen standards were prepared at concentrations of 2000, 1000, 500, 200, and 50 copies / mL. Five concentrations were tested, and 25 μL of each was used as a template. The tests were performed according to the method described in Example 2.

[0128] The results show that the method of this application has high sensitivity, and the detection concentration can reach 100 copies / mL. Figures 9 to 15 The figures show the test results for 100 copies / mL of targets for coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae, respectively. Figure 9 The results shown are from a human coronavirus sensitivity test. Figure 10 The results of the sensitivity test for Haemophilus influenzae are shown. Figure 11 The results of the Streptococcus pneumoniae sensitivity test are shown. Figure 12The results of the parainfluenza virus sensitivity test are shown. Figure 13 The results of the sensitivity test for Group A Streptococcus are shown. Figure 14 The results of the sensitivity test for Bordetella pertussis are shown. Figure 15 The results of the Chlamydia pneumoniae sensitivity test are shown.

[0129] Example 5, Specificity of this application

[0130] The sample processing, addition, and detection steps for other pathogenic bacteria standards were exactly the same as in Example 2. Non-specific amplification of each target was statistically analyzed. The results showed that the system in this application exhibited no non-specific amplification in SARS-CoV-2, influenza virus, Staphylococcus epidermidis, human rhinovirus, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Epstein-Barr virus, human metapneumovirus, SARS virus, mumps virus, norovirus, rotavirus, Candida albicans, and Mycobacterium tuberculosis, demonstrating high specificity. Figure 16 As shown. Figure 16 The results show the specificity test results. The illustration indicates no cross-reactivity with other pathogens.

[0131] Example 6: Anti-interference capability of this application

[0132] The test samples were artificial simulants prepared from the standards of each pathogen. The interfering component was a drug, and the concentration of the interfering component was the actual concentration of the drug used. The artificial simulants were diluted with the drug solution and TE buffer at the same concentrations for later use. Then, the subsequent processing, sample addition, and detection steps were the same as in Example 2.

[0133] If there is no significant difference between the group with added interfering components and the group with samples diluted with TE buffer, it indicates that the reagent has strong anti-interference ability.

[0134] The system described in this application exhibits strong anti-interference capabilities in 50 μg / mL dexamethasone, 50 μg / mL cefotaxime hydrochloride, 100 μg / mL ribavirin, 100 μg / mL azithromycin, 320 μg / mL budesonide, 125 μg / mL beclomethasone, 50 μg / mL beclomethasone, 100 μg / mL mometasone, 200 μg / mL fluticasone, 200 μg / mL histamine hydrochloride, 100 μg / mL lopinavir, 100 μg / mL triamcinolone, 100 μg / mL arbidol, and 10 μg / mL heme. Figures 17 to 23 As shown: Figure 17 The image shows the results of the human coronavirus anti-interference test. Figure 18 The results of the anti-interference test for Haemophilus influenzae are shown. Figure 19 The results of the anti-interference test for Streptococcus pneumoniae are shown. Figure 20The results of the parainfluenza virus anti-interference test are shown. Figure 21 The results of the interference resistance test for Group A Streptococcus are shown. Figure 22 The results of the anti-interference test for Bordetella pertussis are shown. Figure 23 The results of the anti-interference test for Chlamydia pneumoniae are shown.

[0135] Comparative Example 1: Using other primers and probes

[0136] The sequences of the comparison primers and probes are shown in Table 6.

[0137] Table 6

[0138]

[0139] The fluorescent reporter group of the probe shown in SEQ ID NO: 27 is FAM and the quencher group is BHQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 30 is HEX and the quencher group is BHQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 33 is ROX and the quencher group is SQ2; the fluorescent reporter group of the probe shown in SEQ ID NO: 36 is CY5 and the quencher group is SQ2; the fluorescent reporter group of the probe shown in SEQ ID NO: 39 is QUASAR 705 and the quencher group is SQ3; the fluorescent reporter group of the probe shown in SEQ ID NO: 42 is ATTO 425 and the quencher group is SQ1; the fluorescent reporter group of the probe shown in SEQ ID NO: 45 is CY7 and the quencher group is SQ3; and the fluorescent reporter group of the probe shown in SEQ ID NO: 48 is AF 405 and the quencher group is SQ0.

[0140] A control lyophilized reagent prepared using the primers and probes shown in Table 6 is provided. This control lyophilized reagent differs from the lyophilized reagent under section 2.2 of Example 2 only in the sequence of the primers and probes. Using the control lyophilized reagent and the method of Example 2, human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A Streptococcus, Bordetella pertussis, and Chlamydia pneumoniae samples (all clinically positive samples) were mixed in equal proportions to prepare a mixed sample for PCR detection. Figure 24 The result shown is a positive result for the combined target detection. However, there was a false negative for human coronaviruses, with no amplification in the amplification curve. The Ct values ​​for Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A Streptococcus, Bordetella pertussis, and Chlamydia pneumoniae showed a significant lag.

[0141] The detection sensitivity of the primers and probes shown in Table 6 was verified using the method described in Example 4. The results showed that the detection sensitivity using the primers and probes shown in Table 6 was significantly low, with a detection concentration of 500 copies / mL. Figures 25 to 31The results shown are the 500 copies / mL test results for coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae, respectively. Figure 25 The results shown are from a human coronavirus sensitivity test. Figure 26 The results of the sensitivity test for Haemophilus influenzae are shown. Figure 27 The results of the Streptococcus pneumoniae sensitivity test are shown. Figure 28 The results of the parainfluenza virus sensitivity test are shown. Figure 29 The results of the sensitivity test for Group A Streptococcus are shown. Figure 30 The results of the sensitivity test for Bordetella pertussis are shown. Figure 31 The results of the Chlamydia pneumoniae sensitivity test are shown.

[0142] Comparative Example 2: Other main raw materials, dosage ratios, and lyophilization auxiliary reagents used.

[0143] The preparation of lyophilized reagents for nucleic acid detection is a technology that transforms a liquid reaction system into a stable solid form. Its core process begins with the precise mixing and dispensing of a liquid mixture containing primers, probes, DNA polymerase, dNTPs, and other necessary components with a specific lyophilization protectant (such as trehalose or sucrose). Subsequently, the sample is rapidly frozen at deep cryogenic temperatures, solidifying the water into ice crystals. It is then transferred to a vacuum environment, where most of the water is gradually removed through sublimation and desorption, ultimately forming a loosely structured, extremely low-water-content solid microsphere or cake-like substance. This process maximizes the preservation of enzyme activity and the stability of the nucleic acid reagents, allowing the finished product to be stored and transported at room temperature, and ensuring that the reconstituted reaction system possesses higher sensitivity, specificity, and amplification efficiency than the liquid reagent.

[0144] Improper selection of raw materials for lyophilization reagents can trigger a series of negative consequences. Insufficient enzyme activity or poor purity can directly lead to low amplification efficiency after rehydration of the lyophilized product, or even complete reaction failure. Deviated primer / probe sequences or impure modifications can severely weaken the specificity of the detection, leading to non-specific amplification or false negative results. Incompatibility between the lyophilization protectant formulation and the core reaction components can not only fail to effectively maintain the stability of the enzyme and nucleic acid during dehydration but may also inhibit subsequent PCR reactions, causing lyophilized structure collapse, slow or incomplete rehydration. These raw material defects collectively result in significant batch-to-batch variations, a marked decrease in stability, and a shortened shelf life, ultimately severely impacting the accuracy and reliability of clinical testing.

[0145] The glycerol content of enzymes in liquid PCR reagents significantly affects lyophilization results. Simply adding a specific cryoprotectant to liquid reagents often fails to yield lyophilized reagents with excellent morphology and performance. This is because glycerol, as a cryoprotectant, inhibits ice crystal formation and hinders effective water sublimation during lyophilization, preventing the final product from forming a stable porous structure and thus affecting the long-term stability and reconstitution properties of the reagent.

[0146] The inventors compared the morphology and performance of different detection systems after freeze-drying for the combined detection of coronaviruses, Streptococcus pneumoniae, Haemophilus influenzae, parainfluenza virus, Group A Streptococcus, Bordetella pertussis, and Chlamydia pneumoniae. Specifically:

[0147] (1) Lyophilized reagent form

[0148] The inventors have provided three lyophilized reagents, A-lyophilized reagent, B-lyophilized reagent, and C-lyophilized reagent, which differ only in the formulation of the reaction solution, as follows:

[0149] Referring to the protocol described in CN119242864A, a traditional rapid molecular diagnostic protocol, the inventors prepared an A-PCR reaction solution (see Table 7) and prepared the A-PCR reaction solution as an A-lyophilized reagent. Referring to the protocol described in CN119242864A, the inventors added an additional lyophilization protectant to prepare a B-PCR reaction solution and prepared the B-lyophilized reagent. Both the A-PCR reaction solution and the B-PCR reaction solution are based on the protocol described in CN119242864A. The only difference between the B-PCR reaction solution and the A-PCR reaction solution is the addition of a lyophilization protectant—trehalose solution (7.4 μL, working concentration 7 wt%).

[0150] Table 7. A-PCR reaction solution (primers and probes are derived from Example 1 of this application)

[0151]

[0152] Meanwhile, the inventors prepared a C-PCR reaction solution with reference to Table 2 in Example 2 of this application, and prepared the C-PCR reaction solution as a C-lyophilized reagent;

[0153] Figure 32 The image shows a comparison of different lyophilized reagent forms corresponding to different PCR reaction solutions. The results indicate that, compared to C-lyophilized reagents ( Figure 32 (Figure C), A-lyophilized reagent ( Figure 32 Figure A), B-lyophilized reagent ( Figure 32 The shape of the part (Figure B) is shrunken, the surface is rough and not rounded, so the overall appearance is poor.

[0154] (2) Detection performance of lyophilized reagents

[0155] The mixed sample (prepared by mixing equal proportions of clinically positive samples) of human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A Streptococcus, Bordetella pertussis, and Chlamydia pneumoniae was tested using lyophilized reagents A, B, and C. The procedure was the same as in Example 2. The results are as follows: Figures 33 to 35 As shown, lyophilized reagent A showed no amplification curve. Figure 33 ), B-lyophilized reagent, low curves for each target point or missed detection ( Figure 34 If the highest point of the curve is below the threshold line, it indicates that the target point corresponding to that curve has missed detection, resulting in poor detection performance. The C-lyophilized reagent shows strong curves for each target point with no missed detections, indicating excellent detection performance. Figure 35 ).

[0156] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0157] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A nucleic acid combination product for identifying respiratory pathogens, characterized in that, The nucleic acid combo product includes primer pairs and probes shown in SEQ ID NO: 1 to SEQ ID NO: 21; The nucleic acid combination product identifies the following respiratory pathogens: human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae.

2. The nucleic acid combination product for identifying respiratory pathogens according to claim 1, characterized in that, The nucleic acid combo product also includes the primer pairs and probes shown in SEQ ID NO: 22 to SEQ ID NO:

24.

3. The nucleic acid combination product for identifying respiratory pathogens according to any one of claims 1 to 2, characterized in that, In the nucleic acid combination product, each probe is labeled with a different type of fluorescent reporter group.

4. The nucleic acid combination product for identifying respiratory pathogens according to claim 3, characterized in that, The fluorescent reporter group labeled on each probe is selected from any one of FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7 and AF 405.

5. A PCR premix for identifying respiratory pathogens, characterized in that, The PCR premix includes the nucleic acid combination product according to any one of claims 1 to 4 and other PCR amplification reagents; The PCR premixed solution identifies the following respiratory pathogens: human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae.

6. The PCR premix for identifying respiratory pathogens according to claim 5, characterized in that, The PCR premix solution meets one or more of the following conditions: (1) The other PCR amplification reagents include PCR buffer, Mg 2+ One or more of dNTPs, Taq enzymes, and RT enzymes; (2) The PCR premix also includes a lyophilization protectant; and, (3) The PCR premixed solution is freeze-dried.

7. A kit for identifying respiratory pathogens, characterized in that, The kit comprises the nucleic acid combination product of any one of claims 1 to 4, or the PCR premix of any one of claims 5 to 6; The kit identifies the following respiratory pathogens: human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, Group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae.

8. The kit for identifying respiratory pathogens according to claim 7, characterized in that, The kit also includes one or more of the following: sampling tools, sample preservation reagents, nucleic acid release reagents, nucleic acid extraction reagents, negative controls, positive controls, and reconstitution solvents.

9. A method for identifying respiratory pathogens for non-diagnostic purposes, characterized in that, The method uses the nucleic acid combination product of any one of claims 1 to 4, the PCR premix of claim 6, or the kit of claim 8 to detect the nucleic acid of the sample to be tested; The method identifies the following respiratory pathogens: human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae.

10. The method for identifying respiratory pathogens for non-diagnostic purposes according to claim 9, characterized in that, The method satisfies one or more of the following conditions: (1) The method includes performing the following operations in a closed reaction tube: pretreating the sample to be tested with a nucleic acid release reagent, quantitatively mixing the resulting pretreatment product with the lyophilized PCR premix as defined in condition (3) of claim 6, to achieve detection of nucleic acid in the sample to be tested; and, (2) The sample to be tested is a plasmid sample.

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