Primer pairs, primer probe compositions, PCR master mixes, kits, and methods for detecting multiple respiratory pathogens

Abstract

This application relates to the field of biotechnology, and particularly to primer pairs, primer-probe compositions, PCR premixes, kits, and methods for detecting multiple respiratory pathogens. Nucleic acid sequences are shown in primer pairs as indicated by SEQ ID NO: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38, and 40-41. For target respiratory pathogens, this application designs specific amplification primer pairs, paired with suitable probes, enabling the simultaneous detection of eight targets. Furthermore, the detection process avoids interference from heme, trimethoprim, sulfamethoxazole, amphotericin B, itraconazole, fluconazole, azithromycin, adrenaline, and lidocaine hydrochloride in the test sample. It exhibits good anti-interference properties and shows no cross-reactivity with *Rhodococcus equi*, *Candida tropicalis*, and *Candida krusei*.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to primer pairs, primer-probe compositions, PCR premixes, kits, and methods for detecting multiple respiratory pathogens. Background Technology

[0002] Human coronavirus (HCoV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV), human enterovirus (EVs), human rhinovirus (HRV), Bordetella pertussis (BP), and human bocavirus (HBoV) are eight common respiratory pathogens. Accurate and rapid detection of these pathogens is crucial for clinical treatment.

[0003] Current technical solutions for detecting the above-mentioned common respiratory pathogens include:

[0004] CN120350178A discloses a primer set and kit for the simultaneous detection of multiple respiratory pathogens using 26-fold PCR, which describes primers and probes for detecting human coronavirus (HCoV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), human enterovirus (EVs), human rhinovirus (HRV), Bordetella pertussis (BP), and human bocavirus (HBoV), as well as detection methods. While this patent document relates to the combined detection of respiratory pathogens using multiplex PCR, it does not cover the combined detection of the aforementioned eight common respiratory infectious pathogens. Furthermore, this patent document only verifies specificity and does not verify the interference resistance of the protocol to common respiratory medications.

[0005] Similarly, although CN117144063A provides a fluorescent PCR melting curve kit for 12 respiratory pathogens, which describes primers, probes, and detection methods for detecting human coronavirus (HCoV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV), human rhinovirus (HRV), Bordetella pertussis (BP), and human bocavirus (HBoV), it does not cover the combined detection of the aforementioned eight common respiratory pathogens. Furthermore, this patent document only verifies specificity and does not verify the interference resistance of the protocol to common respiratory medications.

[0006] For the eight common respiratory pathogens mentioned above, how to achieve joint detection, and how to ensure that the joint detection scheme can avoid the influence of heme, trimethoprim, sulfamethoxazole, amphotericin B, itraconazole, fluconazole, azithromycin, adrenaline, and lidocaine hydrochloride in the test samples, and also avoid cross-reaction with Rhodococcus equi, Candida tropicalis, and Candida krusei during the detection process, are the technical problems that urgently need to be solved. Summary of the Invention

[0007] Based on this, one or more embodiments of this application provide primer pairs, primer-probe compositions, PCR premixes, kits, and methods for detecting multiple respiratory pathogens. These include the following technical solutions:

[0008] One or more embodiments of this application provide primer pairs for detecting multiple respiratory pathogens, comprising: primer pairs with nucleic acid sequences as shown in SEQ ID NO: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38 and 40-41.

[0009] In some embodiments of this application, the primer pair further includes a primer pair with nucleic acid sequences as shown in SEQ ID NO: 43-44.

[0010] One or more embodiments of this application provide a primer-probe composition for detecting multiple respiratory pathogens, comprising the primer pair described herein and a probe used in combination with the primer pair.

[0011] In some embodiments of this application, it includes probes with nucleic acid sequences such as SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42 and 45.

[0012] One or more embodiments of this application provide a PCR premix for detecting multiple respiratory pathogens, the PCR premix comprising:

[0013] (1) the primer pair or the primer-probe composition described herein; and,

[0014] (2) Other PCR amplification reagents;

[0015] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes;

[0016] Optionally, the PCR premix further includes a lyophilization protectant;

[0017] Optionally, the PCR premix is ​​in a lyophilized state.

[0018] One or more embodiments of this application provide a kit for detecting multiple respiratory pathogens, the kit comprising:

[0019] The primer pair mentioned above; or,

[0020] The primer-probe composition described above; or

[0021] The PCR premix mentioned above.

[0022] 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.

[0023] One or more embodiments of this application provide a method for detecting multiple respiratory pathogens, wherein the method uses the primer pair, the primer-probe composition, the PCR premix, or the kit to detect the nucleic acid of the sample to be tested.

[0024] In some embodiments of this application, the method includes performing the following operations in a closed reaction tube: pretreating the sample to be tested with a nucleic acid release reagent, and quantitatively mixing the resulting pretreatment product with a lyophilized PCR premix as defined above to achieve nucleic acid detection of the sample to be tested.

[0025] In some embodiments of this application, the sample to be tested is a swab sample or a plasmid sample.

[0026] In some embodiments of this application, the swab sample is a pharyngeal swab or a nasal swab.

[0027] Compared with traditional technologies, this application has the following advantages:

[0028] This application designs specific amplification primer pairs targeting target respiratory pathogens, namely human coronavirus (HCoV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV), human enterovirus (EVs), human rhinovirus (HRV), Bordetella pertussis (BP), and human bocavirus (HBoV). Based on these primer pairs and appropriate probes, multiple target respiratory pathogens can be detected using real-time fluorescence PCR in the same reaction system. Furthermore, the detection process avoids interference from heme, trimethoprim, sulfamethoxazole, amphotericin B, itraconazole, fluconazole, azithromycin, adrenaline, and lidocaine hydrochloride in the test samples. It exhibits good anti-interference properties and shows no cross-reaction with Rhodococcus equi, Candida tropicalis, and Candida krusei during the detection process. Attached Figure Description

[0029] 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.

[0030] Figure 1 The respiratory syncytial virus test result in Example 3 was positive.

[0031] Figure 2 The result of the human coronavirus test in Example 3 was positive.

[0032] Figure 3 The result of the parainfluenza virus test in Example 3 was positive.

[0033] Figure 4 The result of the human metapneumovirus test in Example 3 was positive.

[0034] Figure 5 The result of the human rhinovirus or respiratory enterovirus test in Example 3 was positive.

[0035] Figure 6 The result of the Bordetella pertussis test in Example 3 was positive.

[0036] Figure 7 The result of the human bocavirus test in Example 3 was positive.

[0037] Figure 8 The result of the combined target detection in Example 3 was positive.

[0038] Figure 9 The results are for respiratory syncytial virus sensitivity testing in Example 4.

[0039] Figure 10 The results are for the sensitivity detection of human coronaviruses in Example 4.

[0040] Figure 11 This is the result of the parainfluenza virus sensitivity test in Example 4.

[0041] Figure 12 The results are from the partial lung sensitivity test in Example 4.

[0042] Figure 13 The results are the sensitivity test results for human rhinovirus or respiratory enterovirus in Example 4.

[0043] Figure 14 The results of sensitivity testing for Bordetella pertussis in Example 4 are shown.

[0044] Figure 15 The results are the sensitivity test results for human bocavirus in Example 4.

[0045] Figure 16 This is a graph showing the detection results of the target mixed plasmids in each channel at a concentration of 500 copies / mL in Example 4.

[0046] Figure 17 This is the specific detection result in Example 5.

[0047] Figure 18 The results are for the respiratory syncytial virus interference detection in Example 6.

[0048] Figure 19 The results of the anti-interference test for human coronavirus in Example 6 are shown.

[0049] Figure 20 The results are the anti-interference detection results of the parainfluenza virus in Example 6.

[0050] Figure 21 The results of the anti-interference detection of human metapneumovirus in Example 6 are shown.

[0051] Figure 22 The results of the anti-interference test for human rhinovirus or respiratory enterovirus in Example 6 are shown.

[0052] Figure 23 The results of the anti-interference test for Bordetella pertussis in Example 6 are shown.

[0053] Figure 24 The results of the anti-interference test for human bocavirus in Example 6 are shown.

[0054] Figure 25 The results are for the detection of respiratory syncytial virus in the clinical sample of Comparative Example 1.

[0055] Figure 26 The results are for the detection of human coronavirus clinical samples in Comparative Example 1.

[0056] Figure 27 The results are for the detection of parainfluenza virus in clinical samples from Comparative Example 1.

[0057] Figure 28 The results are for the detection of human metapneumovirus in clinical samples from Comparative Example 1.

[0058] Figure 29 The results are for the detection of human rhinovirus or enterovirus clinical samples in Comparative Example 1.

[0059] Figure 30 The results are for the detection of Bordetella pertussis clinical samples in Comparative Example 1.

[0060] Figure 31 The results are for the detection of human bocavirus clinical samples in Comparative Example 1.

[0061] Figure 32 The results of combined target detection are shown in the nucleic acid lysis buffer mixture of 7 samples in Comparative Example 1.

[0062] Figure 33 This is the specificity verification result in Comparative Example 1.

[0063] Figure 34 The results of respiratory syncytial virus interference detection in Comparative Example 1 are shown.

[0064] Figure 35 The results of the coronavirus resistance test in Comparative Example 1 are shown.

[0065] Figure 36 The results of the anti-interference test for parainfluenza virus in Comparative Example 1 are shown.

[0066] Figure 37 The results of the anti-interference test for human metapneumovirus in Comparative Example 1 are shown.

[0067] Figure 38 The results of the anti-interference test for human rhinovirus or respiratory enterovirus in Comparative Example 1 are shown.

[0068] Figure 39 The results of anti-interference detection of Bordetella pertussis in Comparative Example 1 are shown.

[0069] Figure 40 The results of the anti-interference test for human bocavirus in Comparative Example 1 are shown. Detailed Implementation

[0070] 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.

[0071] 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.

[0072] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0073] 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").

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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℃.

[0084] 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.

[0085] 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.

[0086] One or more embodiments of this application provide primer pairs for detecting multiple respiratory pathogens, comprising: primer pairs with nucleic acid sequences as shown in SEQ ID NO: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38 and 40-41.

[0087] In some embodiments of this application, the primer pair further includes a primer pair with nucleic acid sequences as shown in SEQ ID NO: 43-44.

[0088] One or more embodiments of this application provide a primer-probe composition for detecting multiple respiratory pathogens, comprising the primer pair described herein and a probe used in combination with the primer pair.

[0089] In some embodiments of this application, it includes probes with nucleic acid sequences such as SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 and 42.

[0090] In this application, each probe is a Taqman fluorescent probe, with a fluorescent reporter group labeled at one end and a quencher group labeled at the other. The fluorescent reporter group for each probe is independently VIC, FAM, ROX, Cy5, QUASAR 705, ATTO 425, CY7, or AF 405. The quencher group for each probe is independently Super Quencher 0, Super Quencher 1, Super Quencher 2, Super Quencher-X, or Super Quencher 3.

[0091] One or more embodiments of this application provide a PCR premix for detecting multiple respiratory pathogens, the PCR premix comprising:

[0092] (1) the primer pair or the primer-probe composition described herein; and,

[0093] (2) Other PCR amplification reagents;

[0094] Optionally, the other PCR amplification reagents include PCR buffer, Mg... 2+ One or more of dNTPs, Taq enzymes, and RT enzymes;

[0095] Optionally, the PCR premix further includes a lyophilization protectant;

[0096] Optionally, the PCR premix is ​​in a lyophilized state.

[0097] One or more embodiments of this application provide a kit for detecting multiple respiratory pathogens, the kit comprising:

[0098] The primer pair mentioned above; or,

[0099] The primer-probe composition described above; or

[0100] The PCR premix mentioned above.

[0101] 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.

[0102] One or more embodiments of this application provide a method for detecting multiple respiratory pathogens, wherein the method uses the primer pair, the primer-probe composition, the PCR premix, or the kit to detect the nucleic acid of the sample to be tested.

[0103] 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 a 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.

[0104] In some embodiments of this application, the sample to be tested is a swab sample or a plasmid sample.

[0105] In some embodiments of this application, the swab sample is a pharyngeal swab or a nasal swab.

[0106] Quantitative Real-time PCR (qPCR) is a technique that involves adding a fluorescent dye to the PCR reaction system, using the accumulation of fluorescence signals to monitor the entire PCR process in real time, and finally quantifying unknown templates using a standard curve. It includes the non-specific SYBR Green I dye method and the specific TaqMan hydrolysis probe method. TaqMan qPCR is based on TaqMan fluorescent probes, which are oligonucleotides labeled with a fluorescent emitting group and a fluorescent quenching group at each end. When the probe is intact, the fluorescence signal emitted by the emitting group is absorbed by the quenching group. During PCR amplification, the 5'-3' exonuclease activity of Taq polymerase cleaves and degrades the probe, separating the fluorescent emitting and quenching groups. This allows the fluorescence monitoring system to receive the fluorescence signal; that is, for each DNA strand amplified, one fluorescent molecule is formed, achieving complete synchronization between the accumulation of fluorescence signal and the formation of PCR products, thus enabling quantification.

[0107] 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.

[0108] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0109] Example 1: Primers and probes used in this application

[0110] Table 1

[0111]

[0112] The fluorescent reporter group of probes SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12 is VIC; the fluorescent reporter group of probe SEQ ID NO: 15 is FAM; the fluorescent reporter group of probes SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 30 is ROX; the fluorescent reporter group of probes SEQ ID NO: 18 and SEQ ID NO: 21 is Cy5; the fluorescent reporter group of probes SEQ ID NO: 33 and SEQ ID NO: 36 is QUASAR 705; the fluorescent reporter group of probe SEQ ID NO: 39 is ATTO425; the fluorescent reporter group of probe SEQ ID NO: 42 is CY7; and the fluorescent reporter group of probe SEQ ID NO: 45 is AF 405.

[0113] Example 2: Method for detecting pathogens

[0114] 1. Reagent preparation

[0115] 1.1 Take the lyophilized reagents, positive control, negative control, reconstitution solvent, etc. and store them at room temperature for later use.

[0116] Table 2

[0117]

[0118] 1.2 Add 1000 μL of the reconstitution solvent to the positive control (lyophilized), shake thoroughly to mix, centrifuge at 2000 rpm for 10 seconds, and set aside.

[0119] 1.3 Based on the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of reagents for later use.

[0120] 1.4 Transfer the prepared reagents to the sample processing area for later use.

[0121] 2. Sample processing and loading

[0122] The samples tested in this application were pharyngeal swabs and nasal swabs. Viral nucleic acid was lysed using direct amplification, and the following procedures were performed in the sample processing room:

[0123] 2.1 Dilute the sample with the sample release agent at a ratio of 3:1 (v / v), mix thoroughly, and let stand for later use;

[0124] 2.2 Add 25 μL of the pre-treated test sample, negative control, and positive control to the PCR reaction tube containing the lyophilized reagent, respectively, cap the tube (if there are air bubbles, tap them with your finger to remove them), and centrifuge at 2000 rpm for 10 seconds until there are no air bubbles or obvious liquid droplets on the tube wall.

[0125] Table 3

[0126]

[0127] 3. PCR amplification

[0128] PCR amplification was performed on the SLAN-48S fully automated medical PCR analysis system PCR instrument according to a specific temperature and time setting program. The PCR amplification program of this application is shown in Table 4.

[0129] Table 4

[0130]

[0131] 4. Interpretation of test results

[0132] The details are shown in Table 5 below.

[0133] Samples with a typical S-type amplification curve detected in the FAM channel and Ct≤40 are reported as positive for respiratory syncytial virus (RSV); samples without a typical S-type 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 RSV.

[0134] Samples with a typical S-type amplification curve detected in the HEX channel and Ct≤40 are reported as positive for human coronavirus; 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 human coronavirus.

[0135] Samples with a typical S-type amplification curve detected in the ROX channel and Ct≤40 are reported as positive for parainfluenza virus; 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 parainfluenza virus.

[0136] Samples with a typical S-type amplification curve detected in the CY5 channel and Ct≤40 are reported as positive for human metapneumovirus (HMV); 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 HMV.

[0137] For samples where a typical S-type amplification curve is detected in the QUASAR 705 channel and Ct≤40, the report is positive for respiratory enterovirus or human rhinovirus; for samples where a typical S-type amplification curve is not detected in the QUASAR 705 channel, or Ct>40, and an amplification curve is present in the AF 405 channel and Ct≤40, the report is negative for respiratory enterovirus or human rhinovirus.

[0138] For samples where a typical S-type amplification curve is detected in the ATTO 425 channel and Ct≤40, the report is positive for Bordetella pertussis; for samples where a typical S-type amplification curve is not detected in the ATTO 425 channel, or Ct>40, and an amplification curve is detected in the AF 405 channel and Ct≤40, the report is negative for Bordetella pertussis.

[0139] Samples with a typical S-type amplification curve detected in the CY7 channel and Ct≤40 are reported as positive for human bocavirus; samples without a typical S-type amplification curve detected in the CY7 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for human bocavirus.

[0140] Table 5

[0141]

[0142] Example 3: Detection results of the test sample in this application

[0143] Using the method described in Example 2, one clinical sample each of respiratory syncytial virus, human coronavirus, parainfluenza virus, human metapneumovirus, human rhinovirus or enterovirus, Bordetella pertussis, and human bocavirus were detected by PCR on a Hongshi real-time PCR instrument. The results are as follows: Figures 1 to 8 As shown: Amplification curves (where...) Figures 1 to 7 This is a detection image for a single sample. Figure 8 (Image showing the combined target detection of a mixed sample of nucleic acid lysis buffers from 7 samples).

[0144] Example 4: Sensitivity of this application

[0145] Serially diluted target single plasmids of respiratory syncytial virus, human coronavirus, parainfluenza virus, human metapneumovirus, human rhinovirus, enterovirus, Bordetella pertussis, and human bocavirus were taken at concentrations of 50,000, 5,000, 500, and 50 copies / mL, respectively, and the four concentrations were detected. The results show that the method of this application has high sensitivity and can detect concentrations up to 500 copies / mL. Figures 9 to 15 The results shown are the detection results of single plasmids for each channel at concentrations of 50,000, 5,000, and 500 copies / mL. The above eight target plasmids were mixed and diluted to a concentration of 500 copies / mL for detection. 25 μL was used as a template. The results indicate that the method of this application exhibits high stability and repeatability at a detection concentration of 500 copies / mL for the mixed target plasmids. Figure 16 The image shows the detection results of target mixed plasmids in each channel at a concentration of 500 copies / mL.

[0146] Example 5, Specificity of this application

[0147] The method described in Example 2 was used to detect confirmed positive clinical samples of Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, influenza A virus, influenza B virus, Escherichia coli, Streptococcus pyogenes, Acinetobacter baumannii, Pneumocystis jirovecii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. No nonspecific amplification was observed, indicating high specificity. Figure 17 As shown, this indicates no cross-reactivity with other pathogens.

[0148] Example 6: Anti-interference capability of this application

[0149] Using the method of Example 2, one clinical sample each of the confirmed positive respiratory syncytial virus, human coronavirus, parainfluenza virus, human metapneumovirus, human rhinovirus or enterovirus, Bordetella pertussis, and human bocavirus were tested. During the PCR reaction system, the following interfering components were added at initial concentrations: 10 μg / mL mucin, 20 μg / mL heme, 20 μg / mL trimethoprim, 0.44 mg / mL sulfamethoxazole, 0.8 mg / mL amphotericin B, 0.1 g / mL itraconazole, 0.5 mg / mL fluconazole, 0.1 g / mL azithromycin, 1 mg / mL adrenaline, and 4 mg / mL lidocaine hydrochloride. The results showed that this application has strong anti-interference ability. Figures 18 to 24 As shown.

[0150] Comparative Example 1: Using other primers and probes

[0151] This comparative example provides another set of primers and probes to form a joint detection protocol for respiratory syncytial virus, human coronavirus, parainfluenza virus, human metapneumovirus, human rhinovirus, respiratory enterovirus, Bordetella pertussis, and human bocavirus.

[0152] The only difference between this comparative example and Examples 1 and 2 above is the use of the following primers and probes; all other aspects are the same as in Examples 1 and 2.

[0153] Table 6

[0154]

[0155] The test samples were examined according to the method described in Example 3. One clinical sample each of respiratory syncytial virus, human coronavirus, parainfluenza virus, human metapneumovirus, human rhinovirus or enterovirus, Bordetella pertussis, and human bocavirus were tested. The results are as follows: Figures 25 to 32 As shown: Amplification curves (where...) Figures 25 to 31 This is a detection image for a single sample. Figure 32 (This is a diagram showing the combined target detection of nucleic acid lysis buffers from 7 samples). The results indicate that the primers and probes shown in Table 6 can effectively detect the target pathogen.

[0156] Specificity verification was performed according to the method in Example 5, and the results are as follows: Figure 33 As shown in Table 6, the primers and probes used for target pathogen detection exhibit cross-reactivity with *Rhodococcus equi*, *Candida tropicalis*, and *Candida krusei*, indicating poor specificity.

[0157] The anti-interference capability was verified according to the method in Example 6, and the results are as follows: Figures 34 to 40 As shown in Table 6, the detection of the target antigen using the primers and probes is poorly protected against interference from heme, trimethoprim, sulfamethoxazole, amphotericin B, itraconazole, fluconazole, azithromycin, adrenaline, and lidocaine hydrochloride.

[0158] 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.

[0159] 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. Primer pairs for detecting multiple respiratory pathogens, characterized in that, It includes: Nucleic acid sequences are shown in primer pairs as indicated by SEQ ID NO: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-38 and 40-41; The primer pairs are used to detect the following respiratory pathogens: human coronavirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, human enterovirus, human rhinovirus, Bordetella pertussis, and human bocavirus.

2. The primer pair for detecting multiple respiratory pathogens according to claim 1, characterized in that, It also includes primer pairs with nucleic acid sequences as shown in SEQ ID NO: 43-44.

3. A primer and probe composition for detecting multiple respiratory pathogens, characterized in that, It includes the primer pair according to any one of claims 1 to 2 and the probe used in combination with the primer pair; The primer-probe composition detects the following respiratory pathogens: human coronavirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, human enterovirus, human rhinovirus, Bordetella pertussis, and human bocavirus.

4. The primer and probe composition for detecting multiple respiratory pathogens according to claim 3, characterized in that, It includes probes with nucleic acid sequences such as SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42 and 45.

5. A PCR premix for detecting multiple respiratory pathogens, characterized in that, The PCR premix includes: (1) The primer pair according to any one of claims 1 to 2 or the primer-probe composition according to any one of claims 3 to 4; and, (2) Other PCR amplification reagents; The PCR premix was used to detect the following respiratory pathogens: human coronavirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, human enterovirus, human rhinovirus, Bordetella pertussis, and human bocavirus.

6. The PCR premix for detecting multiple respiratory pathogens according to claim 5, characterized in that, The PCR premix 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 further includes a lyophilization protectant; and, (3) The PCR premix is ​​in a lyophilized state.

7. A kit for detecting multiple respiratory pathogens, characterized in that, The kit includes: The primer pair according to any one of claims 1 to 2; or, The primer and probe composition according to any one of claims 3 to 4; or The PCR premix according to any one of claims 5 to 6; The kit detects the following respiratory pathogens: human coronavirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, human enterovirus, human rhinovirus, Bordetella pertussis, and human bocavirus.

8. The kit for detecting multiple 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 detecting multiple respiratory pathogens for non-diagnostic purposes, characterized in that, The method uses the primer pair of any one of claims 1 to 2, the primer-probe composition of any one of claims 3 to 4, the PCR premix of any one of claims 5 to 6, or the kit of any one of claims 7 to 8 to detect the nucleic acid of the sample to be tested; The method detects the following respiratory pathogens: human coronavirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, human enterovirus, human rhinovirus, Bordetella pertussis, and human bocavirus.

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

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