Nucleic acid combination product, PCR (Polymerase Chain Reaction) premixed solution, kit and method for identifying respiratory pathogens

Abstract

The invention relates to the technical field of molecular biological detection, in particular to a nucleic acid combination product, a PCR premixed solution, a kit and a method for identifying respiratory pathogens. The nucleic acid combination product comprises a primer pair as shown in SEQ ID NO: 1 to SEQ ID NO: 21 and a probe. The primer pair and the probe combination can be mixed with other reagents required by PCR and then freeze-dried to prepare freeze-dried balls, the surfaces of the freeze-dried balls are smooth and round, nucleic acid sample liquid to be detected can be directly dropwise added into a PCR reaction tube filled with the freeze-dried balls and then can be detected on a machine, storage and transportation are facilitated, operation is convenient and fast, the combined detection effect of target pathogens is especially good, and the detection efficiency is high. 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 immunoparalysis 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 the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum and maximum values) of the numerical range, and every numerical value between the two numerical endpoints. When a numerical interval refers to only integers within the numerical interval, unless otherwise specified, the two endpoints and every integer between the two endpoints are included, and in this context, it is equivalent to directly listing every integer, for example, t is an integer selected from 1-10, which means t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein.

[0069] In the present application, unless otherwise specified, the temperature parameter allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5℃, ±4℃, ±3℃, ±2℃, ±1℃.

[0070] In the present 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 documents mentioned in the present application are incorporated by reference in the present application, as if each document is individually incorporated by reference. Unless and to the extent that the application purpose and / or technical solution of the present application is conflicted, the cited documents are incorporated by reference in their entirety. When referring to the cited documents in the present application, the definitions of the relevant technical features, terms, names, phrases, etc. in the cited documents are also incorporated by reference. When referring to the cited documents in the present application, the examples and preferred modes of the relevant technical features are also incorporated by reference, subject to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified according to the description in the present application.

[0072] Based on the previous research, the applicant developed 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 based on this, the patent application CN119242864A was filed. However, the previous primer and probe composition is mainly used for the development of liquid reagent products. The present application aims to promote product optimization and upgrading, and to provide primers and probes suitable for the development of freeze-dried reagents. Based on this, the present application is proposed.

[0073] In a first aspect, the present application provides a nucleic acid combination product for identifying a respiratory pathogen, which comprises a primer pair and a probe as shown in SEQ ID NO: 1 to SEQ ID NO: 21.

[0074] The nucleic acid combination product of the present application further comprises a primer pair and a probe for detecting an internal standard gene, which are not particularly limited in the present application, and can be, but are not limited to, a primer pair and a probe as shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0075] In some embodiments of the present application, the nucleic acid combination product comprises probes labeled with different fluorescent reporter groups. The fluorescent reporter groups and quencher groups of the probes are not particularly limited in the present application. The fluorescent reporter groups can be, but are not limited to, FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7 and AF 405. The quencher groups can be, but are not limited to, BHQ1, SQ2, SQ3 and SQ0.

[0076] In a second aspect, the present application provides a PCR premix for identifying a respiratory pathogen, which comprises the nucleic acid combination product and other PCR amplification reagents.

[0077] The other PCR amplification reagents are not particularly limited in the present application, and in some embodiments of the present application, the other PCR amplification reagents comprise one or more of a PCR buffer, Mg 2+ , dNTPs, Taq enzyme and RT enzyme. For example, in one embodiment of the present application, the other PCR amplification reagents comprise a PCR buffer, Mg 2+ , dNTPs, Taq enzyme and RT enzyme required for detection.

[0078] The product form of the PCR premix is not particularly limited in the present application, which can be in a liquid state or in a freeze-dried state. It can be understood that a freeze-drying protective agent can be added to the freeze-dried PCR premix to ensure good performance. The type and amount of the freeze-drying protective agent are not particularly limited in the present application. For example, trehalose is used as the freeze-drying protective agent, and the working concentration is 7 wt%. It can be understood that those skilled in the art can also select other suitable freeze-drying protective agents and determine the working concentration capable of playing a freeze-drying protective role.

[0079] In a third aspect, the present application provides a kit for identifying a respiratory pathogen, which comprises the nucleic acid combination product or the PCR premix.

[0080] 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.

[0081] In a fourth aspect of the embodiments of the present application, a method for identifying a respiratory pathogen for non-diagnostic purposes is provided, which comprises detecting the nucleic acid of a sample to be tested using the nucleic acid combination product, the PCR premix, or the kit.

[0082] The method is not particularly limited in the present application. The nucleic acid obtained by pretreatment of the sample to be tested can be manually added to the PCR premix for detection, or the nucleic acid can be mixed with the PCR premix directly without manual transfer for detection. For example, the sample to be tested is pretreated with the nucleic acid release reagent, and the pretreated product is mixed with the freeze-dried PCR premix as defined above for reaction, thereby detecting the nucleic acid of the sample to be tested. For example, the detection device and method described in the patent application CN116555007A can be used, which can simplify the process and avoid contamination.

[0083] The sample to be tested is not particularly limited in the present application, including but not limited to swab samples (such as throat swabs and nasal swabs) and plasmid samples.

[0084] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specifically indicated, and the guidelines given in the present application are preferred. The experimental methods in the art or the conditions recommended by the manufacturer can also be used, or the known experimental methods in the art can be used.

[0085] In the following specific examples, the amount of raw material components is measured, and if not specifically stated, there may be slight deviations within the weighing accuracy range. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operational accuracy.

[0086] The application designs specific primer and probe combinations for the conserved sequences of seven respiratory tract pathogens. Due to the use of multiplex PCR detection system, multiple primer probes exist in the reaction system at the same time, and there is a risk of primer dimer formation and non-specific amplification, but the primer probe combination of the application can avoid this risk. In terms of fluorescence detection channel, non-mainstream fluorescence channels such as QUASAR 705, ATTO 425, CY7, AF 405 are selected. At the same time, the risk of crosstalk is higher in 8-color fluorescence channel than in 4-color fluorescence channel, and false positive situations are more likely to occur due to crosstalk, but the application can also avoid this situation. The respiratory multi-pathogen joint detection reagent is prone to degradation and interaction in the liquid system during long-term storage, which restricts the performance stability of the reagent. The application upgrades the freeze-dried reagent, which effectively improves the long-term stability and detection sensitivity of the reagent at room temperature.

[0087] Example 1, the primer and probe designed by the application

[0088] Table 1

[0089]

[0090] Among them, the fluorescence reporter group of the probe represented by SEQ ID NO: 3 is FAM, and the quenching group is BHQ1; the fluorescence reporter group of the probe represented by SEQ ID NO: 6 is HEX, and the quenching group is BHQ1; the fluorescence reporter group of the probe represented by SEQ ID NO: 9 is ROX, and the quenching group is SQ2; the fluorescence reporter group of the probe represented by SEQ ID NO: 12 is CY5, and the quenching group is SQ2; the fluorescence reporter group of the probe represented by SEQ ID NO: 15 is QUASAR 705, and the quenching group is SQ3; the fluorescence reporter group of the probe represented by SEQ ID NO: 18 is ATTO 425, and the quenching group is SQ1; the fluorescence reporter group of the probe represented by SEQ ID NO: 21 is CY7, and the quenching group is SQ3; the fluorescence reporter group of the probe represented by SEQ ID NO: 24 is AF 405, and the quenching group is SQ0.

[0091] Example 2, the method for detecting pathogens

[0092] 1. Reagent preparation

[0093] The application corresponds to the PCR amplification kit, which is called respiratory seven-pathogen nucleic acid detection kit (fluorescence PCR method),

[0094] 1.1 Take out the reagents in the kit and place them at room temperature for standby.

[0095] Direct extension method nucleic acid release reagent: sample release agent (Shengxiang Biotechnology Co., Ltd.).

[0096] 1.2 Take 1000 μL of reconstitution solvent and add to the positive control (lyophilized), shake well, centrifuge at 2000 rpm for 10 seconds, and reserve.

[0097] 1.3 According to the quantity of the sample to be tested, negative control (normal saline), and positive control (pathogen mixed standard of Table 1), take the corresponding amount of reagent, and reserve.

[0098] 1.4 Transfer the prepared reagent above to the sample processing area, and wait for use.

[0099] 2. Sample processing and sample addition

[0100] The sample to be tested in the present application is a throat swab or a nose swab. The virus nucleic acid is lysed by the direct expansion method, and the following operations are performed in the sample processing chamber:

[0101] 2.1 Dilute the sample with the sample release agent 3:1 (v / v), mix well, and stand by.

[0102] 2.2 Add 25 μL of the prepared sample, negative control, and positive control to the PCR reaction tube (reaction system as shown in Table 2) containing the lyophilized reagent (prepared by freeze-drying the PCR reaction solution shown in Table 2), cover the tube cap (if there is a bubble, use a finger to hit and remove the bubble), and centrifuge at 2000 rpm for 10 seconds until there are no bubbles and obvious liquid beads on the tube wall.

[0103] Table 2

[0104]

[0105] 3. PCR amplification

[0106] On the SLAN-48S full-automatic medical PCR analysis system PCR instrument, PCR amplification is performed according to a certain temperature and time setting program. The PCR amplification program of the present application is shown in Table 3.

[0107] Table 3

[0108]

[0109] 4. Interpretation of test results

[0110] Specifically as shown in Table 4 and Table 5.

[0111] For the sample in which a typical S-shaped amplification curve is detected in the FAM channel, and Ct≤40, the report is human coronavirus positive; for the sample in which a typical S-shaped amplification curve is not detected in the FAM channel, or Ct>40, and an amplification curve is detected in the AF 405 channel, and Ct≤40, the report is human coronavirus negative.

[0112] Samples with a typical S-shaped amplification curve detected in the HEX channel and a Ct < 40 are reported as Haemophilus influenzae positive; samples with no typical S-shaped amplification curve detected in the HEX channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Haemophilus influenzae negative.

[0113] Samples with a typical S-shaped amplification curve detected in the ROX channel and a Ct < 40 are reported as Streptococcus pneumoniae positive; samples with no typical S-shaped amplification curve detected in the ROX channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Streptococcus pneumoniae negative.

[0114] Samples with a typical S-shaped amplification curve detected in the CY5 channel and a Ct < 40 are reported as Parainfluenza virus positive; samples with no typical S-shaped amplification curve detected in the CY5 channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Parainfluenza virus negative.

[0115] Samples with a typical S-shaped amplification curve detected in the QUASAR 705 channel and a Ct < 40 are reported as Group A Streptococcus positive; samples with no typical S-shaped amplification curve detected in the QUASAR 705 channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Group A Streptococcus negative.

[0116] Samples with a typical S-shaped amplification curve detected in the ATTO 425 channel and a Ct < 40 are reported as Bordetella pertussis positive; samples with no typical S-shaped amplification curve detected in the ATTO 425 channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Bordetella pertussis negative.

[0117] Samples with a typical S-shaped amplification curve detected in the CY7 channel and a Ct < 40 are reported as Chlamydia pneumoniae positive; samples with no typical S-shaped amplification curve detected in the CY7 channel or a Ct > 40 and an amplification curve in the AF 405 channel with a Ct < 40 are reported as Chlamydia pneumoniae negative.

[0118] Table 4

[0119]

[0120] Table 5

[0121]

[0122] The application integrates TaqMan fluorescence quantitative core and innovative multiplex detection design, and eight targets (including internal standard) can be accurately detected in a single tube. The optimized process significantly improves the throughput, reduces the burden and mistakes of manual operation, and the result interpretation is intuitive. The freeze-drying technology gives the reagent excellent stability, room temperature storage and transportation characteristics, effectively reduces the dependence on cold chain and the overall reagent cost, and greatly simplifies the operation steps.

[0123] Example 3, detection results of test samples of the application

[0124] Using the method of Example 2, 1 case of each of human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, chlamydia pneumoniae samples (all are clinical positive samples) were detected by PCR on the Macrostone fluorescence quantitative PCR instrument, and the results are shown in Figures 1 to 8 Figure 1 The human coronavirus is positive. Figure 2 Haemophilus influenzae is positive. Figure 3 The Streptococcus pneumoniae is positive. Figure 4 The parainfluenza virus is positive. Figure 5 The group A streptococcus is positive. Figure 6 The Bordetella pertussis is positive. Figure 7 The chlamydia pneumoniae is positive.

[0125] Similarly, using the method of Example 2, human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, chlamydia pneumoniae samples (all are clinical positive samples) were mixed in equal proportions to make a mixed sample, and PCR detection was performed, Figure 8 The joint target detection result is positive.

[0126] Example 4, sensitivity of the application

[0127] Gradient dilution of each pathogen standard was taken, and the concentrations were 2000, 1000, 500, 200, and 50 copies / mL, respectively. 25 μL was taken as a template for detection. The detection was performed according to the method described in Example 2.

[0128] The results show that the method of the application has high sensitivity, and the detection concentration can reach 100 copies / mL. As shown in Figures 9 to 15 The 100 copies / mL test results of coronavirus, Bordetella pertussis, group A streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and chlamydia pneumoniae targets are shown in Figure 9 The human coronavirus sensitivity detection result is shown in Figure 10 The Haemophilus influenzae sensitivity detection result is shown in Figure 11 The Streptococcus pneumoniae sensitivity detection result is shown in Figure 12 ​The results of the parainfluenza virus sensitivity test are shown. Figure 13 The results of the group A streptococcus sensitivity test are shown. Figure 14 The results of the Bordetella pertussis sensitivity test are shown. Figure 15 The results of the Chlamydia pneumoniae sensitivity test are shown.

[0129] Example 5, specificity of the present application

[0130] The sample processing and sample addition, detection steps for other pathogenic bacteria standards were exactly the same as in Example 2, and the non-specific amplification of each target was statistically analyzed. The results showed that there was no non-specific amplification in the present system among the following: new coronavirus, 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, and the specificity was high. For example, Figure 16 as shown. Figure 16 The results of the specificity test are shown. The results shown in the figure indicate that there is no cross-reaction with other pathogens.

[0131] Example 6, anti-interference property of the present application

[0132] The detection samples were artificial simulation samples prepared from each pathogen standard. The interference components were drugs, and the interference component concentrations were the actual use concentrations of the drugs. The artificial simulation samples were diluted with the drug solutions and TE buffer at the concentrations, respectively, for standby, and then the subsequent processing and sample addition, detection steps were the same as in Example 2.

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

[0134] The present system has strong anti-interference ability in 50 μg / mL dexamethasone, 50 μg / mL cefmenox hydrochloride, 100 μg / mL ribavirin, 100 μg / mL azithromycin, 320 μg / mL budesonide, 125 μg / mL phenylpropanolamine, 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 acetonide, 100 μg / mL arbidol, and 10 μg / mL hematin. For example, Figures 17 to 23 as shown. Figure 17 The results of the human coronavirus anti-interference test are shown. Figure 18 The results of the anti-interference test of Haemophilus influenzae are shown. Figure 19 The results of the anti-interference test of Streptococcus pneumoniae are shown. Figure 20The results of the anti-interference detection of parainfluenza virus are shown. Figure 21 The results of the anti-interference detection of group A streptococcus are shown. Figure 22 The results of the anti-interference detection of Bordetella pertussis are shown. Figure 23 The results of the anti-interference detection of Chlamydia pneumoniae are shown.

[0135] Comparative Example 1, other primers and probes are used

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

[0137] Table 6

[0138]

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

[0140] A control lyophilized reagent prepared using the primers and probes shown in Table 6 is provided, which is only different from the lyophilized reagent under item 2.2 of Example 2 in that the sequences of the primers and probes are different. 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 of which are clinically positive samples) are mixed in equal proportions to form a mixed sample, and PCR detection is performed, Figure 24 The results of the combined target detection are shown - positive. However, human coronavirus is missed, and the amplification curve has no amplification. Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae have obvious lagging Ct values.

[0141] The detection sensitivity of the primers and probes shown in Table 6 is verified using the method of Example 4, and the results show that the detection sensitivity of the primers and probes shown in Table 6 is significantly lower, with a detection concentration of 500 copies / mL. For example, Figures 25 to 31Shown are the test results for coronavirus, Bordetella pertussis, Streptococcus group A, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae targets at 500 copies / mL, respectively. Figure 25 Shown are the sensitivity test results for human coronavirus. Figure 26 Shown are the sensitivity test results for Haemophilus influenzae. Figure 27 Shown are the sensitivity test results for Streptococcus pneumoniae. Figure 28 Shown are the sensitivity test results for parainfluenza virus. Figure 29 Shown are the sensitivity test results for Streptococcus group A. Figure 30 Shown are the sensitivity test results for Bordetella pertussis. Figure 31 Shown are the sensitivity test results for Chlamydia pneumoniae.

[0142] Comparative Example 2, using other major raw material types, usage ratios, and freeze-drying auxiliary reagents

[0143] The preparation of nucleic acid detection freeze-dried reagents is a technology for converting a liquid reaction system into a stable solid state. The core process begins with precisely mixing and dispensing a liquid mixture containing primers, probes, DNA polymerase, dNTPs, and other necessary components with specific freeze-drying protectants (such as trehalose, sucrose, etc.). Subsequently, the sample is rapidly frozen at deep low temperature, causing water to solidify into ice crystals; then it is transferred to a vacuum environment, and through sublimation and desorption processes, most of the water is gradually removed, finally forming a loose structure, low water content solid-state microspheres or cake. This process can maximize the stability of enzyme activity and nucleic acid reagents, allowing the finished product to be stored and transported at room temperature, and ensuring that the reconstituted reaction system has higher sensitivity, specificity, and amplification efficiency than liquid reagents.

[0144] Improper selection of freeze-dried reagent raw materials will trigger a series of negative chain consequences. If the enzyme raw material activity is insufficient or the purity is poor, it will directly lead to low amplification efficiency or even complete failure of the reaction after reconstitution of the freeze-dried product; if the primer / probe sequence has deviations or impurities, it will seriously weaken the specificity of the detection, causing non-specific amplification or false negative results; if the freeze-drying protectant formula is incompatible with the core reaction components, not only can it not effectively maintain the stability of enzymes and nucleic acids during dehydration, but it may also inhibit subsequent PCR reactions, leading to collapse of the freeze-dried structure, slow or incomplete reconstitution. These raw material defects will collectively cause large batch-to-batch differences, significant stability decline, and shortened shelf life, ultimately severely affecting the accuracy and reliability of clinical detection.

[0145] The glycerol content of enzymes in liquid PCR reagents can seriously affect the freeze-drying effect, and often a single addition of a specific protective agent in the liquid reagent cannot successfully obtain freeze-dried reagents with excellent morphology and performance. Because glycerol itself as a cryoprotective agent can inhibit the formation of ice crystals, and hinder the effective sublimation of water during freeze-drying, resulting in the final product unable to form a stable porous structure, thereby affecting the long-term stability and reconstitution performance of the reagent.

[0146] The inventors also used different detection systems for the simultaneous detection of coronavirus, Streptococcus pneumoniae, Haemophilus influenzae, parainfluenza virus, group A streptococcus, Bordetella pertussis, and Chlamydia pneumoniae target points after freeze-drying, and compared the morphology and performance. Specifically:

[0147] (1) Freeze-dried reagent morphology

[0148] The inventors provided A-freeze-dried reagents, B-freeze-dried reagents, and C-freeze-dried reagents that differ only in the formulation of the reaction solution, as follows:

[0149] Referring to the scheme described in the conventional rapid molecular diagnostic scheme example CN119242864A, the inventors prepared an A-PCR reaction solution (see Table 7), and prepared the A-PCR reaction solution into an A-freeze-dried reagent; referring to the scheme described in the conventional rapid molecular diagnostic scheme example CN119242864A, the inventors prepared a B-PCR reaction solution by additionally adding a freeze-drying protective agent, and prepared the B-PCR reaction solution into a B-freeze-dried reagent; wherein the A-PCR reaction solution and the B-PCR reaction solution are both the scheme described in CN119242864A, and the difference between the B-PCR reaction solution and the A-PCR reaction solution is only that the freeze-drying protective agent-trehalose solution is added (the addition volume is 7.4 μL, and the working concentration is 7 wt%).

[0150] Table 7, A-PCR reaction solution (primers and probes are derived from Example 1 described above)

[0151]

[0152] At the same time, the inventors prepared a C-PCR reaction solution according to Table 2 in Example 2 of the present application, and prepared the C-PCR reaction solution into a C-freeze-dried reagent;

[0153] Figure 32 The different freeze-dried reagent morphologies corresponding to different PCR reaction solutions are shown in the figure. The results show that, compared with the C-freeze-dried reagent (C of Figure 32 ), the morphology of the A-freeze-dried reagent (A of Figure 32 ), and the B-freeze-dried reagent (B of Figure 32 ) shrinks, the surface is rough and not smooth, and therefore, the overall appearance effect is poor.

[0154] (2) Detection performance of the freeze-dried reagent

[0155] Human coronavirus, Haemophilus influenzae, Streptococcus pneumoniae, parainfluenza virus, group A streptococcus, Bordetella pertussis, Chlamydia pneumoniae mixed samples (clinical positive samples were mixed at the same proportion to prepare) were detected by using A-freeze-dried reagent, B-freeze-dried reagent and C-freeze-dried reagent, and the operation steps were the same as those in Example 2. The results are shown in Table 2. Figures 33 to 35 As shown in Table 2, the A-freeze-dried reagent had no amplification curve Figure 33 ), the B-freeze-dried reagent had low curve or missed detection Figure 34 ) of each target point, and the highest point of the curve was lower than the threshold line, indicating that the curve missed detection of the corresponding target point and had poor detection performance. The C-freeze-dried reagent had upright curve and no missed detection of each target point, and had excellent detection performance Figure 35 ).

[0156] Each technical feature of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered within the scope of the present disclosure.

[0157] The above-mentioned examples only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but should not be understood as limiting the scope of the patent protection. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms also fall within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided in the present application through logical analysis, reasoning or limited experiments, which are within the scope of the claims of the present application. Therefore, the scope of the patent protection of the present application should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents 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.

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 of any one of claims 1 to 4 and other PCR amplification reagents.

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.

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.

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 in the nucleic acid release reagent, quantitatively mixing the resulting pretreatment product with the lyophilized PCR premix as defined in claim 6, to achieve detection of nucleic acid in the sample to be tested; and, (2) The sample to be tested is a swab sample or a plasmid sample; the swab sample is a pharyngeal swab or a nasal swab.

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