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 a primer pair and a probe with sequences shown in SEQ ID NO: 1 to SEQ ID NO: 21. The nucleic acid combination product comprises a primer pair and a probe for detecting target pathogens, and in the process of detecting multiple targets based on the same reaction system, cross-reactions can be avoided, and the interference of drugs can also be avoided. In addition, the nucleic acid combination product is suitable for preparing a freeze-dried reagent, and through the selection of a freeze-drying protective agent component, primer probe damage can be avoided, and the compatibility of multiple components can be improved.

Description

Technical Field

[0001] This application relates to the field of molecular biology detection technology, and in particular to nucleic acid combination products, PCR premixes, kits and methods for identifying respiratory pathogens. Background Technology

[0002] Respiratory diseases refer to infectious or non-infectious conditions occurring in the nose, pharynx, larynx (upper respiratory tract) to the trachea and bronchi (lower respiratory tract), and are generally divided into upper respiratory tract infections and lower respiratory tract infections. Pathogens that cause respiratory diseases include bacteria, viruses, mycoplasma, chlamydia, Legionella, etc. Among them, the novel coronavirus (SARS-CoV-2), influenza A virus, human rhinovirus (HRV), respiratory syncytial virus (RSV), influenza B virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and adenovirus (ADV) are the core pathogens causing human respiratory diseases.

[0003] How to accurately detect the aforementioned core pathogens using multiplex PCR technology is an urgent technical problem to be solved. In this regard, the applicant discovered:

[0004] On the one hand, the aforementioned core pathogens often cross-infect with other common pathogens that cause similar symptoms. This requires that during the multiplex PCR detection of the aforementioned core pathogens, the test results be more reliable if there is no cross-reaction with the aforementioned core pathogens. Other common pathogens include Escherichia coli, Streptococcus pyogenes, Acinetobacter baumannii, Pneumocystis jirovecii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Epstein-Barr virus, human metapneumovirus, measles virus, mumps virus, norovirus, rotavirus, Candida albicans, and Mycobacterium tuberculosis.

[0005] On the other hand, current medications for treating respiratory diseases interfere with multiplex PCR detection of the aforementioned core pathogens. Detection results are more reliable when these drug interferences are avoided. These medications include dexamethasone, cefotaxime hydrochloride, ribavirin, azithromycin, budesonide, beclomethasone, mometasone, fluticasone, histamine hydrochloride, lopinavir, triamcinolone, and arbidol.

[0006] Based on the above findings, this application is filed. Summary of the Invention

[0007] Based on this, one or more embodiments of this application provide nucleic acid combination products, PCR premixes, kits and methods for identifying respiratory pathogens.

[0008] One or more embodiments of this application provide a nucleic acid combo product for identifying respiratory pathogens, the nucleic acid combo product comprising primer pairs and probes with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 21.

[0009] In some embodiments of this application, the nucleic acid combo product further includes primer pairs and probes for detecting internal standard genes;

[0010] Optionally, the nucleic acid combination product further includes primer pairs and probes with sequences as shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0011] In some embodiments of this application, the types of fluorescent reporter groups labeled on each probe in the nucleic acid combination product are different;

[0012] Optionally, the fluorescent reporter group labeled on each probe is selected from any one of FAM, HEX, ROX, CY5, QUASAR 705, ATTO425, CY7 and AF 405.

[0013] One or more embodiments of this application provide a PCR premix for identifying respiratory pathogens, the PCR premix comprising the aforementioned nucleic acid combination product and other PCR amplification reagents;

[0014] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes.

[0015] One or more embodiments of this application provide a lyophilized PCR premix for identifying respiratory pathogens, the lyophilized PCR premix comprising a lyophilization protectant, a lyophilization excipient, primer pairs and probes, and other PCR amplification reagents;

[0016] The freeze-drying protectant includes trehalose;

[0017] The freeze-drying excipient includes dextran;

[0018] Optionally, the working concentration of the trehalose is 5wt%-10wt%;

[0019] Optionally, the working concentration of the dextran is 2wt%-5wt%;

[0020] Optionally, the primer pair and probe are used to identify one or more of the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus;

[0021] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of the following: dNTPs, Taq enzymes, and RT enzymes;

[0022] In some embodiments of this application, the sequences of primer pairs and probes for detecting the novel coronavirus are shown in SEQ ID NO: 1 to SEQ ID NO: 3;

[0023] The sequences of the primer pairs and probes for detecting influenza A virus are shown in SEQ ID NO: 4 to SEQ ID NO: 6;

[0024] The sequences of the primer pairs and probes for detecting human rhinovirus are shown in SEQ ID NO: 7 to SEQ ID NO: 9;

[0025] The sequences of the primer pairs and probes for detecting respiratory syncytial virus are shown in SEQ ID NO: 10 to SEQ ID NO: 12;

[0026] The sequences of the primer pairs and probes for detecting influenza B virus are shown in SEQ ID NO: 13 to SEQ ID NO: 15;

[0027] The sequences of the primer pairs and probes for detecting Middle East Respiratory Syndrome Coronavirus are shown in SEQ ID NO: 16 to SEQ ID NO: 18;

[0028] The sequences of the primer pairs and probes for detecting adenovirus are shown in SEQ ID NO: 19 to SEQ ID NO: 21;

[0029] Optionally, the lyophilized PCR premix further includes primer pairs and probes for detecting internal standard genes; optionally, the lyophilized PCR premix includes primer pairs and probes with sequences as shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0030] Optionally, the lyophilized PCR premix contains different types of fluorescent reporter groups labeled with each probe; optionally, the fluorescent reporter group labeled with each probe is selected from any one of FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7 and AF405.

[0031] One or more embodiments of this application provide a kit for identifying respiratory pathogens, the kit comprising the nucleic acid combination product or the PCR premix.

[0032] 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.

[0033] One or more embodiments of this application provide a method for identifying respiratory pathogens, wherein the method uses the nucleic acid combination product, the PCR premix, or the kit described above to detect the nucleic acid of the sample to be tested;

[0034] Optionally, the method includes: enriching the nucleic acid with magnetic beads, collecting the nucleic acid adsorbed on the magnetic beads, adding the collected nucleic acid to the PCR premix, and performing detection; or, lysing the sample to be tested with a nucleic acid release reagent, adding the resulting lysate to the PCR premix, and performing detection.

[0035] In some embodiments of this application, the method includes performing the following operations in a closed reaction tube: placing the sample to be tested in the nucleic acid release reagent for pretreatment, and quantitatively mixing the resulting pretreatment product with the lyophilized PCR premix to achieve nucleic acid detection of the sample to be tested.

[0036] In some embodiments of this application, the sample to be tested is a swab sample or a plasmid sample; optionally, the swab sample is a nasopharyngeal swab, an oropharyngeal swab, or a nasal swab.

[0037] Compared to traditional technologies, the beneficial effects of this application include:

[0038] Based on a comprehensive study of targets including novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus, this application designs a nucleic acid combo product capable of identifying respiratory pathogens. This nucleic acid combo product includes primer pairs and probes for detecting the target pathogen. Using this nucleic acid combo product, multiple targets can be detected using the same reaction system, avoiding cross-reactions and drug interference. Furthermore, this nucleic acid combo product is suitable for preparing lyophilized reagents; the selection of the lyophilization protectant components can prevent primer and probe damage and improve multi-component compatibility. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The image shows a positive result for the novel coronavirus in Example 3.

[0041] Figure 2 The image shows a positive result for influenza A virus in Example 3.

[0042] Figure 3 The image shows a positive result for human rhinovirus in Example 3.

[0043] Figure 4 The image shows a positive result for respiratory syncytial virus in Example 3.

[0044] Figure 5 The image shows a positive result for influenza B virus in Example 3.

[0045] Figure 6 The image shows a positive result for Middle East Respiratory Syndrome Coronavirus in Example 3.

[0046] Figure 7 The image shows adenovirus positive in Example 3.

[0047] Figure 8 The result shown is a positive result for the combined target detection in Example 3.

[0048] Figure 9 The results of the novel coronavirus sensitivity test are shown in Example 4.

[0049] Figure 10 The results of the sensitivity test for influenza A virus in Example 4 are shown.

[0050] Figure 11 The results of human rhinovirus sensitivity testing are shown in Example 4.

[0051] Figure 12 The results of respiratory syncytial virus sensitivity testing in Example 4 are shown.

[0052] Figure 13 The results of the influenza B virus sensitivity test in Example 4 are shown.

[0053] Figure 14 The results of sensitivity testing for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Example 4 are shown.

[0054] Figure 15 The results of adenovirus sensitivity testing are shown in Example 4.

[0055] Figure 16 The results of specific detection in Example 5 are shown.

[0056] Figure 17 The results of the novel coronavirus anti-interference detection in Example 6 are shown.

[0057] Figure 18 The results of the anti-interference test for influenza A virus in Example 6 are shown.

[0058] Figure 19 The results of the anti-interference test for human rhinovirus in Example 6 are shown.

[0059] Figure 20 The results of the respiratory syncytial virus anti-interference test in Example 6 are shown.

[0060] Figure 21 The results of the anti-interference test for influenza B virus in Example 6 are shown.

[0061] Figure 22 The results of the anti-interference test for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Example 6 are shown.

[0062] Figure 23 The results of the adenovirus anti-interference test in Example 6 are shown.

[0063] Figure 24 The results shown are the sensitivity detection results of using other primers and probes at 1000 copies / mL in Comparative Example 1.

[0064] Figure 25 The figures show the morphology of the lyophilized reagents in Comparative Example 2; Figure A shows the morphology of the S-lyophilized reagent, and Figure B shows the morphology of the CK-lyophilized reagent.

[0065] Figure 26 The results of the anti-interference test for multiple pathogens in Example 6 are shown.

[0066] Figure 27 The results show the sensitivity detection results of the CK-lyophilized reagent in Comparative Example 2.

[0067] Figure 28 The results of the stability test of the S-lyophilized reagent in Comparative Example 2 are shown.

[0068] Figure 29 The results of the stability test of the CK-lyophilized reagent in Comparative Example 2 are shown.

[0069] Figure 30 The results shown are the specificity detection results using other primers and probes in Comparative Example 1.

[0070] Figure 31 The results shown are the sensitivity detection results of using other primers and probes at 200 copies / mL in Comparative Example 1.

[0071] Figure 32 The results of multi-pathogen detection with other primers and probes in Comparative Example 1 are shown. Detailed Implementation

[0072] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0073] 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 herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0074] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0075] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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 "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0076] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0077] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0078] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0079] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0080] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0081] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0082] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0083] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0084] 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.

[0085] 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℃.

[0086] 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.

[0087] 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.

[0088] 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 with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 21.

[0089] 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, primer pairs and probes with sequences as shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0090] 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 be, but are not limited to: FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7, and AF 405.

[0091] 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;

[0092] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes.

[0093] A third aspect of this application provides a lyophilized PCR premix for identifying respiratory pathogens, the lyophilized PCR premix comprising a lyophilization protectant, a lyophilization excipient, primer pairs and probes, and other PCR amplification reagents.

[0094] The freeze-drying protectant includes trehalose;

[0095] The freeze-drying excipient includes dextran;

[0096] Optionally, the working concentration of the trehalose is 5wt%-10wt% (e.g., 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%).

[0097] Optionally, the working concentration of the dextran is 2wt%-5wt% (e.g., 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%).

[0098] Optionally, the primer pair and probe are used to identify one or more of the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus;

[0099] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes;

[0100] In some embodiments of this application, the sequences of primer pairs and probes for detecting the novel coronavirus are shown in SEQ ID NO: 1 to SEQ ID NO: 3;

[0101] The sequences of the primer pairs and probes for detecting influenza A virus are shown in SEQ ID NO: 4 to SEQ ID NO: 6;

[0102] The sequences of the primer pairs and probes for detecting human rhinovirus are shown in SEQ ID NO: 7 to SEQ ID NO: 9;

[0103] The sequences of the primer pairs and probes for detecting respiratory syncytial virus are shown in SEQ ID NO: 10 to SEQ ID NO: 12;

[0104] The sequences of the primer pairs and probes for detecting influenza B virus are shown in SEQ ID NO: 13 to SEQ ID NO: 15;

[0105] The sequences of the primer pairs and probes for detecting Middle East Respiratory Syndrome Coronavirus are shown in SEQ ID NO: 16 to SEQ ID NO: 18;

[0106] The sequences of the primer pairs and probes for detecting adenovirus are shown in SEQ ID NO: 19 to SEQ ID NO: 21;

[0107] Optionally, the lyophilized PCR premix further includes primer pairs and probes for detecting internal standard genes; optionally, the lyophilized PCR premix includes primer pairs and probes with sequences as shown in SEQ ID NO: 22 to SEQ ID NO: 24.

[0108] Optionally, the lyophilized PCR premix contains different types of fluorescent reporter groups labeled with each probe; optionally, the fluorescent reporter group labeled with each probe is selected from any one of FAM, HEX, ROX, CY5, QUASAR 705, ATTO 425, CY7 and AF405.

[0109] A fourth 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.

[0110] 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.

[0111] A fifth aspect of this application provides a method for identifying respiratory pathogens, 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.

[0112] The method described in this application can be a method for non-diagnostic purposes or a method for diagnostic purposes.

[0113] 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 and PCR premix can be directly mixed and detected 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 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.

[0114] In some embodiments of this application, the method includes: enriching the nucleic acid with magnetic beads, collecting the nucleic acid adsorbed on the magnetic beads, and adding the collected nucleic acid to the PCR premix for detection; or, lysing the sample to be tested using a nucleic acid release reagent, adding the resulting lysis product to the PCR premix for detection. In some embodiments of this application, the sample to be tested is a swab sample or a plasmid sample; optionally, the swab sample is a nasopharyngeal swab, an oropharyngeal swab, or a nasal swab.

[0115] 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.

[0116] 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.

[0117] The diverse range of respiratory pathogens makes samples more complex, necessitating the design of primers / probes with broad coverage and high specificity to avoid cross-reactivity and reduce drug interference. Introducing lyophilization significantly improves reagent stability at room temperature. Optimizing the composition of the lyophilization protectant avoids primer and probe damage and enhances multi-component compatibility. The low pathogen load in early-stage infected patient samples increases the risk of missed detection; the fully reconstituted nature of lyophilized reagents significantly optimizes nucleic acid utilization, thereby improving the signal detection threshold.

[0118] Example 1: Primers and probes used in this application

[0119] Table 1

[0120]

[0121] Among them, the fluorescent reporter group of probe SEQ ID NO: 3 is FAM; the fluorescent reporter group of probe SEQ ID NO: 6 is HEX; the fluorescent reporter group of probe SEQ ID NO: 9 is ROX; the fluorescent reporter group of probe SEQ ID NO: 12 is CY5; the fluorescent reporter group of probe SEQ ID NO: 15 is QUASAR 705; the fluorescent reporter group of probe SEQ ID NO: 18 is ATTO425; the fluorescent reporter group of probe SEQ ID NO: 21 is CY7; the fluorescent reporter group of probe SEQ ID NO: 24 is AF405; all of the above probes have a double quenched group as their fluorescent reporter group.

[0122] Example 2: Method for detecting pathogens

[0123] 1. Reagent preparation

[0124] 1.1 Remove the reagents from the kit and place them at room temperature until they reach room temperature before use.

[0125] This application corresponds to the PCR amplification kit: Seven Respiratory Pathogen Nucleic Acid Detection Kit (Fluorescent PCR Method);

[0126] Magnetic bead-based nucleic acid extraction kit (Sansure Biotech Inc., S50016E / S10015): Reagent for nucleic acid extraction or purification;

[0127] Direct amplification method nucleic acid release kit (Sansure Biotech Inc., X1009E): Sample release agent.

[0128] 1.2 Add 1000 μL of the reconstitution solvent to the positive control (lyophilized), shake thoroughly to mix, centrifuge at 2000 rpm for 10 s, and set aside.

[0129] 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.

[0130] 1.4 Transfer the prepared reagents to the sample processing area for later use.

[0131] 2. Sample processing and loading

[0132] The sample used in this application is a nasopharyngeal swab.

[0133] 2.1 Viral nucleic acid was extracted using the magnetic bead method. The following procedures were performed in the sample processing room:

[0134] (1) Take a 1.5 mL cryovial and add glass beads. Add 300 μL of nasopharyngeal swab sample and 300 μL of TE buffer to the 1.5 mL cryovial containing the mixed glass beads and grind them using a grinder. After grinding, remove the cryovial and centrifuge briefly. Aspirate the supernatant liquid as the sample to be tested for later use.

[0135] (2) Take several 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube.

[0136] (3) Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cap the tube, shake to mix for 30 s, and heat at 60 °C for 10 min.

[0137] (4) Let stand at room temperature for 1 minute, then centrifuge at low speed for a short time. Place the centrifuge tube on a magnetic separator and slowly remove the waste liquid after 5 minutes (*be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall).

[0138] (5) Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake to mix for 30 s, centrifuge briefly at low speed, and then place the centrifuge tube in a magnetic separator. Magnetically aspirate for 3 min to completely remove and discard the liquid.

[0139] (6) Place the centrifuge tube in the centrifuge and centrifuge at low speed for a short time. Then place the centrifuge tube back into the magnetic separator. Magnetize for 3 minutes to completely remove the liquid from the bottom of the tube.

[0140] (7) Add 30-100 μL of elution buffer S (80 μL is recommended for elution), shake to mix for 30 seconds, wash the magnetic beads on the centrifuge tube wall to the bottom of the tube, let stand at room temperature for 3 minutes; centrifuge at low speed for a short time, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0141] In subsequent embodiments, unless otherwise specified, the magnetic bead method is used to extract viral nucleic acid.

[0142] 2.2 The nucleic acid of the sample was lysed using the direct amplification method, and the following operations were performed in the sample processing room:

[0143] Samples collected using the sample release agent were sonicated for 120 seconds on an ultrasound instrument with a frequency of 10kHz-80kHz. After sonication, the samples were allowed to stand for 3 minutes to cool down before use.

[0144] Add 25 μL of the test sample (prepared by freeze-drying the PCR reaction solution shown in Table 2) as described in items 2.1 and 2.2 above, 25 μL of the negative control and 25 μL of the positive control to the PCR reaction tubes containing the lyophilized reagent (prepared by freeze-drying the PCR reaction solution shown in Table 2). Cap the tubes (if there are air bubbles, tap them with your finger to remove them). Centrifuge at 2000 rpm for 10 seconds until there are no air bubbles or obvious liquid droplets on the tube walls.

[0145] Table 2

[0146]

[0147] 3. PCR amplification

[0148] PCR amplification was performed on the SLAN-48S fully automated medical PCR analysis system according to a specific temperature and time setting program. The amplification program of this application is shown in Table 3.

[0149] Table 3

[0150]

[0151] 4. Interpretation of test results

[0152] The details are shown in Table 4 below.

[0153] Samples with a typical S-shaped amplification curve detected in the FAM channel and Ct≤40 are reported as positive for novel 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 novel coronavirus.

[0154] Samples with a typical S-type amplification curve detected in the HEX channel and Ct≤40 are reported as positive for influenza A virus; 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 influenza A virus.

[0155] Samples with a typical S-type amplification curve detected in the ROX channel and Ct≤40 are reported as positive for human rhinovirus; samples without a typical S-type amplification curve detected in the ROX channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for human rhinovirus.

[0156] Samples with a typical S-type amplification curve detected in the CY5 channel and Ct≤40 are reported as positive for respiratory syncytial virus (RSV); 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 RSV.

[0157] Samples with a typical S-type amplification curve detected in the QUASAR 705 channel and Ct≤40 are reported as positive for influenza B virus; samples without a typical S-type amplification curve detected in the QUASAR 705 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for influenza B virus.

[0158] Samples with a typical S-shaped amplification curve detected in the ATTO 425 channel and Ct≤40 are reported as positive for Middle East Respiratory Syndrome Coronavirus (MERS-CoV); samples without a typical S-shaped amplification curve detected in the ATTO 425 channel, or Ct>40, but with an amplification curve in the AF405 channel and Ct≤40, are reported as negative for MERS-CoV.

[0159] Samples with a typical S-type amplification curve detected in the CY7 channel and Ct≤40 are reported as adenovirus positive; 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 adenovirus negative.

[0160] Table 4

[0161]

[0162] Table 5

[0163]

[0164] Example 3: Detection results of the test sample in this application

[0165] Using the method described in Example 2, one sample each of novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus (all clinical samples) were detected by PCR on the Hongshi Real-Time PCR instrument. The results are as follows: Figure 1 The result shown is a positive result for the novel coronavirus. Figure 2 The result shows a positive result for influenza A virus. Figure 3 The result showed a positive result for human rhinovirus. Figure 4 The result shows a positive result for respiratory syncytial virus (RSV). Figure 5 The result shows a positive result for influenza B virus. Figure 6 The result shown is positive for Middle East Respiratory Syndrome Coronavirus. Figure 7 The result shows adenovirus positive.

[0166] Using the same method as in Example 2, PCR testing was performed on artificially simulated samples (prepared from a mixture of novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus). The results are as follows: Figure 8 The result shown is a positive result from the combined target detection.

[0167] Example 4: Sensitivity of this application

[0168] Serially diluted plasmids were taken at concentrations of 5000, 2500, 1000, 500, and 50 copies / mL, and detection was performed at each of the five concentrations. 25 μL of each concentration was used as a template. The results show that the method of this application has high sensitivity, and the detection concentration can reach 200 copies / mL. Figures 9 to 15 Results of target testing for novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus. Figure 9 The results of the novel coronavirus sensitivity test are shown. Figure 10 The results of the influenza A virus sensitivity test are shown. Figure 11 The results of the human rhinovirus sensitivity test are shown. Figure 12 The results of respiratory syncytial virus (RSV) sensitivity testing are shown. Figure 13 The results of the influenza B virus sensitivity test are shown. Figure 14 The results of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) sensitivity test are shown. Figure 15 The results of adenovirus sensitivity testing are shown.

[0169] Example 5, Specificity of this application

[0170] Nucleic acid extraction, processing, sample addition, and detection steps for other pathogenic bacteria standards were performed 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 high specificity, with no non-specific amplification observed in Escherichia coli, Streptococcus pyogenes, Acinetobacter baumannii, Pneumocystis jirovecii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Epstein-Barr virus, human metapneumovirus, measles virus, mumps virus, norovirus, rotavirus, Candida albicans, and Mycobacterium tuberculosis. Figure 16 The results show the specificity test results, indicating no cross-reactivity with other pathogens.

[0171] Example 6: Anti-interference capability of this application

[0172] The test sample was a nasopharyngeal swab sample (the pathogen had been identified by the hospital). The interfering component was a drug, and the concentration of the interfering component was the actual concentration of the drug used. The nasopharyngeal swab sample was diluted with the drug solution of this concentration. Then, the resulting diluted solution and TE buffer were added to a cryovial containing mixed glass beads, as in Example 2. Subsequent processing, sample addition, and detection steps were the same as in Example 2.

[0173] If the results of the group with added interfering components are no different from those of the control group which uses other diluents (the only difference from the drug solution is that no drug is added), it indicates that the reagent has strong anti-interference ability.

[0174] The results showed that the system described in this application exhibited strong anti-interference ability 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 phenformin, 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 : Figure 17 The image shows the results of the novel coronavirus anti-interference test. Figure 18 The results of the anti-interference test for influenza A virus are shown. Figure 19 The results of the human rhinovirus anti-interference test are shown. Figure 20 The results of the respiratory syncytial virus (RSV) anti-interference test are shown. Figure 21 The results of the interference resistance test for influenza B virus are shown. Figure 22 The image shows the interference resistance test results for Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Figure 23 The results of the adenovirus anti-interference test are shown.

[0175] Following the above protocol, PCR testing was performed on artificially simulated samples (prepared from a mixture of novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus). The results are as follows: Figure 26 The results show the interference resistance of 50 μg / mL dexamethasone for multi-pathogen detection. The results indicate that the method described in this application is effective against interference from multiple drugs during multi-pathogen detection.

[0176] Comparative Example 1: Using other primers and probes

[0177] The inventors also designed other control group primers and probes to form different detection systems, which are also used for the joint detection of targets of novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus.

[0178] The sequences of the primers and probes for the control group are shown in Table 6.

[0179] Table 6

[0180]

[0181] The fluorescent reporter group of the probe shown in SEQ ID NO: 27 is FAM; the fluorescent reporter group of the probe shown in SEQ ID NO: 30 is HEX; the fluorescent reporter group of the probe shown in SEQ ID NO: 33 is ROX; the fluorescent reporter group of the probe shown in SEQ ID NO: 36 is CY5; the fluorescent reporter group of the probe shown in SEQ ID NO: 39 is QUASAR 705; the fluorescent reporter group of the probe shown in SEQ ID NO: 42 is ATTO 425; the fluorescent reporter group of the probe shown in SEQ ID NO: 45 is CY7; and the fluorescent reporter group of the probe shown in SEQ ID NO: 48 is AF 405. All of the fluorescent reporter groups of the above probes are double-quenched. W represents an A / T degenerate base.

[0182] Referring to Example 3, the primers and probes listed in Table 6 were used for detection, and the results are as follows: Figure 24 As shown, serially diluted plasmids at concentrations of 5000, 2500, 1000, 500, and 50 copies / mL were used for detection at each of the five concentrations, with 25 μL used as template. The results indicate that the detection sensitivity using the primers and probes listed in Table 6 can reach a concentration of 1000 copies / mL. Figure 31 Results of target testing for novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus. The results show that the primers and probes shown in Table 6 can achieve normal detection of these seven pathogens. However, when testing with a template concentration of 200 copies / mL, the primers and probes shown in Table 6 exhibited false negatives, as shown in the following results. Figure 31 As shown in Table 6, the detection sensitivity of the primers and probes is weaker than that of the primers and probes used in this application.

[0183] Referring to Example 5, the specificity of the primers and probes in Table 6 was verified. High-concentration clinical real samples were tested using the primers and probes shown in Table 6, and the results are as follows: Figure 30As shown, some amplification curves exhibited secondary amplification, which does not conform to the standard of conventional PCR amplification curves. This situation is caused by interactions with primers and probes from other channels. The results indicate that non-specific amplification occurred between primers and probes during the detection of the seven pathogens using the primers and probes shown in Table 6.

[0184] Referring to Example 6, the anti-interference ability of the primers and probes in Table 6 was verified, and the results are as follows: Figure 32 As shown. PCR testing was performed on artificially simulated samples (prepared from a mixture of novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus). Figure 32 The results show the anti-interference detection results of 50 μg / mL cefotaxime hydrochloride against multiple pathogens. According to the results, the interference from cefotaxime hydrochloride, a commonly used drug in the diagnosis and treatment of respiratory diseases, cannot be avoided when using the primers and probes shown in Table 6 to detect seven pathogens.

[0185] Comparative Example 2: Using other lyophilization auxiliary reagents

[0186] In the preparation of lyophilized reagents for nucleic acid detection, lyophilization auxiliary reagents (protectants and excipients) are usually added. Protectants protect active ingredients such as nucleases, primers, and probes from damage during lyophilization and ensure reagent stability during long-term storage. Excipients maintain the physical structure of the reagent, prevent disintegration after lyophilization, and rapidly restore activity upon reconstitution, thereby improving batch consistency and detection sensitivity. However, improper use of auxiliary reagents can cause the lyophilized reagent to collapse, or even lead to a decline in detection performance. To ensure optimal stability, reconstitution efficiency, and detection performance after lyophilization, it is necessary to repeatedly optimize the ratio and amount of auxiliary reagents, gradually adjust the concentration of each component, and compare the protective effects of different formulations to select the optimal one.

[0187] The inventors also added other lyophilized auxiliary reagents to form different detection systems, which are also used for the joint detection of targets such as novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus. Specifically:

[0188] (1) Lyophilized reagent form

[0189] Referring to Table 2 in Example 2, the inventors prepared an S-PCR reaction solution and then prepared the S-PCR reaction solution into an S-lyophilized reagent.

[0190] Meanwhile, the inventors also prepared a CK-PCR reaction solution and prepared the CK-PCR reaction solution into a CK-lyophilized reagent; the only difference between the CK-PCR reaction solution and the S-PCR reaction solution is that mannitol is used instead of trehalose and PEG8000 is used instead of dextran.

[0191] The results showed that, compared to the S-lyophilized reagent ( Figure 25 Figure A), CK-lyophilized reagent ( Figure 25 The shape of the morphology in Figure B is shrunken, and the surface is rough and not rounded, such as Figure 25 As shown, the overall appearance is poor. Figure 25 The image shows a comparison of different lyophilized reagent forms.

[0192] (2) Detection performance of lyophilized reagents

[0193] The artificially simulated low-concentration sample (same as control example 1) was tested using CK-lyophilized reagent, following the same procedure as in Example 2. The results are as follows: Figure 27 As shown, some curves are low, and the highest point of the curve is below the threshold line, indicating that the target point corresponding to this curve is missed and the detection performance is poor.

[0194] (3) Stability of lyophilized reagents

[0195] The S-lyophilized reagent and CK-lyophilized reagent prepared in step (1) were placed at 50°C for 7 days, and then used to simulate the detection of low-concentration samples (same as comparative example 1). The operation steps were the same as in Example 2.

[0196] The results are as follows Figure 28 and Figure 29 As shown. Compared to that obtained using S-lyophilization reagent. Figure 28 Obtained using CK-lyophilized reagent Figure 29 The overall curves are dispersed and do not aggregate, and the fluorescence intensity of some curves shows a significant decline, indicating that the thermally accelerated stability of the CK-lyophilized reagent is poor.

[0197] 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.

[0198] 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 with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 21; The nucleic acid combination product identifies the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus.

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

24.

3. A PCR premix for identifying respiratory pathogens, characterized in that, The PCR premix comprises the nucleic acid combination product according to any one of claims 1 to 2 and other PCR amplification reagents; the other PCR amplification reagents include PCR buffer, Mg 2+ One or more of dNTPs, Taq enzymes, and RT enzymes; The PCR premix identifies the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus.

4. A lyophilized PCR premix for identifying respiratory pathogens, characterized in that, The lyophilized PCR premix includes a lyophilization protectant, a lyophilization excipient, primer pairs and probes, and other PCR amplification reagents; the lyophilization protectant includes trehalose; the lyophilization excipient includes dextran; the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes; The primer pairs and probes are used to identify the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus; The sequences of primer pairs and probes for detecting the novel coronavirus are shown in SEQ ID NO: 1 to SEQ ID NO: 3; The sequences of the primer pairs and probes for detecting influenza A virus are shown in SEQ ID NO: 4 to SEQ ID NO: 6; The sequences of the primer pairs and probes for detecting human rhinovirus are shown in SEQ ID NO: 7 to SEQ ID NO: 9; The sequences of the primer pairs and probes for detecting respiratory syncytial virus are shown in SEQ ID NO: 10 to SEQ ID NO: 12; The sequences of the primer pairs and probes for detecting influenza B virus are shown in SEQ ID NO: 13 to SEQ ID NO: 15; The sequences of the primer pairs and probes for detecting Middle East Respiratory Syndrome Coronavirus are shown in SEQ ID NO: 16 to SEQ ID NO: 18; The sequences of the primer pairs and probes for detecting adenovirus are shown in SEQ ID NO: 19 to SEQ ID NO:

21.

5. The lyophilized PCR premix for identifying respiratory pathogens according to claim 4, characterized in that, The lyophilized PCR premix includes primer pairs and probes with sequences as shown in SEQ ID NO: 22 to SEQ ID NO:

24.

6. A kit for identifying respiratory pathogens, characterized in that, The kit comprises the nucleic acid combination product of any one of claims 1 to 2, or the PCR premix of any one of claims 3 to 5; The kit identifies the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus.

7. 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 2, the PCR premix of any one of claims 3 to 5, or the kit of claim 6 to detect the nucleic acid of the sample to be tested; The method identifies the following respiratory pathogens: novel coronavirus, influenza A virus, human rhinovirus, respiratory syncytial virus, influenza B virus, Middle East respiratory syndrome coronavirus, and adenovirus.

8. The method for identifying respiratory pathogens for non-diagnostic purposes according to claim 7, characterized in that, The method satisfies one or more of the following conditions: (1) The method includes: enriching the nucleic acid with magnetic beads, collecting the nucleic acid adsorbed on the magnetic beads, adding the collected nucleic acid to the PCR premix, and performing detection; or, lysing the sample to be tested with a nucleic acid release reagent, adding the resulting lysate to the PCR premix, and performing detection. (2) 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 of any one of claims 4 to 5 to achieve detection of nucleic acid in the sample to be tested; and, (3) The sample to be tested is a plasmid sample.

Images