Primer probe combination for broad-spectrum screening of common respiratory pathogens and application of primer probe combination

Abstract

The invention relates to the technical field of biomedicine, in particular to a primer probe combination for broad-spectrum screening of common respiratory pathogens and application of the primer probe combination. Through screening and optimization, the respiratory pathogen nucleic acid detection primer probe combination for broad-spectrum screening of common acute respiratory infection and community-acquired pneumonia is obtained; the primer probe combination covers 18 respiratory tract pathogens, has the advantages of high accuracy, good specificity, strong anti-interference capability, good inclusiveness, high sensitivity, hyperjoint detection and the like, can realize parallel detection of 20 targets in cooperation with totally-closed single-tube PCR amplification, greatly improves early diagnosis of respiratory tract diseases with complex pathogen structures, and has wide application prospects. The method has very important significance on prevention, control and treatment of infectious diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to a primer probe combination for screening common respiratory pathogens and application thereof. BACKGROUND

[0002] Respiratory tract infection is one of the most common diseases worldwide. Respiratory tract infection is divided into upper respiratory tract infection and lower respiratory tract infection. Upper respiratory tract infection is often accompanied by symptoms such as rhinitis, cough, pharyngitis, and fever. Lower respiratory tract infection includes tracheitis, bronchitis, and pneumonia. Acute respiratory tract infection (ARTI) can occur in any part of the respiratory tract and is one of the most common infectious diseases, especially lower respiratory tract infection, which is the main cause of all infectious diseases. There are many types of pathogens causing respiratory tract infection, including bacteria, viruses, mycoplasma, and chlamydia, etc. Studies have shown that Mycoplasma pneumoniae pneumonia accounts for a high proportion of community-acquired pneumonia (CAP). In domestic clinical diagnosis, due to the limitations of diagnosis and treatment conditions, many patients with respiratory tract infection cannot receive more accurate diagnosis, and often use broad-spectrum antibiotics for treatment. About 25% to 20% of the antibiotics used in clinical practice are not reasonable. This not only makes it difficult to achieve the best therapeutic effect, but also may delay the disease, or induce the generation of bacterial drug resistance and more complex superinfection, etc. Respiratory tract infection often involves mixed infection of multiple pathogens, and clinicians need to consider the role of a pathogen or multiple pathogens in causing pneumonia in hospitalized patients.

[0003] Due to the complex pathogen spectrum of respiratory diseases, high incidence, few multi-association nucleic acid detection products, and the need to improve the specificity and sensitivity of the detection reagent for early diagnosis of ARTI and CAP respiratory diseases, most ARTI and CAP are acute diseases and concentrated outbreaks, so the detection method requires simple operation, rapidity, and high-throughput detection capability. Therefore, it is particularly necessary to develop a multi-association product or method with good specificity, high sensitivity, simple operation, rapidity, and high-throughput detection. SUMMARY

[0004] Therefore, the present application aims to provide a primer probe combination for screening common respiratory pathogens and application thereof.

[0005] The present application provides a primer probe combination for detecting respiratory pathogens, wherein the target fragment of the primer probe combination comprises:

[0006] an influenza B virus (IFB) target fragment with a nucleotide sequence as shown in SEQ ID NO. 82; and / or

[0007] Mycoplasma pneumoniae (MP) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 83; and / or

[0008] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF1ab gene target fragment having a nucleotide sequence as set forth in SEQ ID NO. 84; and / or

[0009] Parainfluenza virus 1 (PIV-1) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 85; and / or

[0010] Parainfluenza virus 2 (PIV2) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 86; and / or

[0011] Parainfluenza virus 3 (PIV3) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 87; and / or

[0012] Parainfluenza virus 4 (PIV4) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 88; and / or

[0013] Coronavirus 229E (HCOV-229E) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 89; and / or

[0014] Coronavirus HKU (HCOV-HKU1) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 90; and / or

[0015] Coronavirus OC43 (HCOV-OC43) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 91; and / or

[0016] Coronavirus NL63 (HCOV-NL63) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 92; and / or

[0017] Bocavirus (HBOV) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 93; and / or

[0018] Chlamydia pneumoniae (CP) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 94; and / or

[0019] Rhinovirus (HRV) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 95; and / or

[0020] Human metapneumovirus (HMPV) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 96; and / or

[0021] an adenovirus (HADV) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 97; and / or

[0022] a respiratory syncytial virus (RSV) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 98; and / or

[0023] a novel coronavirus (SARS-CoV-2) N gene target fragment having a nucleotide sequence as set forth in SEQ ID NO. 99; and / or

[0024] an influenza A virus (IFA) target fragment having a nucleotide sequence as set forth in SEQ ID NO. 100.

[0025] Further, the target fragment also includes an internal standard target fragment having a nucleotide sequence as set forth in SEQ ID NO. 101.

[0026] The target fragment (or primer probe combination) for detecting respiratory pathogens according to the present application is a target fragment (or primer probe combination) for detecting 18 respiratory pathogens (19 target sequences), specifically, the 18 respiratory pathogens (19 target sequences) include: influenza B virus (IFB) toxin, Mycoplasma pneumoniae (MP), novel coronavirus (SARS-CoV-2, ORF1ab gene target fragment and N gene target fragment), parainfluenza virus type 1 (PIV-1), parainfluenza virus type 2 (PIV2), parainfluenza virus type 3 (PIV3), parainfluenza virus 4 (PIV4), coronavirus 229E (HCOV-229E), coronavirus HKU (HCOV-HKU1), coronavirus OC43 (HCOV-OC43), coronavirus NL63 (HCOV-NL63), bocavirus (HBOV), Chlamydia pneumoniae (CP), rhinovirus (HRV), metapneumovirus (HMPV), adenovirus (HADV) respiratory syncytial virus (RSV), and influenza A virus (IFA).

[0027] In the detection of the above-mentioned 18 respiratory pathogens, the pathogens can be detected in combination according to actual needs, and the combinations containing different target sequences of the pathogens (or combinations of primer probes) can be generated, and each combination needs an internal standard as a reference; specifically, the combination of target fragments includes at least one of the following combinations 1-3:

[0028] Combination 1: a combination containing any one of the target fragments of the 18 respiratory pathogens and an internal standard target fragment;

[0029] Combination 2: a combination containing any multiple of the target fragments of the 18 respiratory pathogens and an internal standard target fragment;

[0030] Combination 3: a combination containing all the target fragments of the 18 respiratory pathogens and the internal standard target fragment;

[0031] In specific embodiments of the present application, the target fragments of the above-mentioned pathogens can be randomly combined, and the detection results are not limited by the types of pathogens, and have high accuracy, precision, sensitivity and stability, etc. under any combination. Under the conditions allowed by the equipment, the effect of detecting 18 kinds of pathogens together is better, and the detection time can be saved.

[0032] Further, in the present application, the primer set and probe for detecting influenza B virus (IFB) include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2, and a probe with a nucleotide sequence as shown in SEQ ID NO. 41 and / or SEQ ID NO. 42; and / or

[0033] The primer set and probe for detecting Mycoplasma pneumoniae (MP) include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, and a probe with a nucleotide sequence as shown in SEQ ID NO. 43 and / or SEQ ID NO. 44; and / or

[0034] The primer set and probe for detecting SARS-CoV-2 include: primer set and probe combination 1 and primer set and probe combination 2; the primer set and probe combination 1 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, and a probe with a nucleotide sequence as shown in SEQ ID NO. 45 and / or SEQ ID NO. 46; the primer set and probe combination 2 includes: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 35, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 36, and a probe with a nucleotide sequence as shown in SEQ ID NO. 75 and / or SEQ ID NO. 76; and / or

[0035] A primer set and a probe for detecting parainfluenza virus 1 (PIV-1), the primer set and the probe for detecting parainfluenza virus 1 (PIV-1) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 7, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 8, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 47 and / or SEQ ID NO. 48; and / or

[0036] A primer set and a probe for detecting parainfluenza virus 2 (PIV2), the primer set and the probe for detecting parainfluenza virus 2 (PIV2) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 9, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 10, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 49 and / or SEQ ID NO. 50; and / or

[0037] A primer set and a probe for detecting parainfluenza virus 3 (PIV3), the primer set and the probe for detecting parainfluenza virus 3 (PIV3) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 11, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 12, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 51 and / or SEQ ID NO. 52; and / or

[0038] A primer set and a probe for detecting parainfluenza virus 4 (PIV4), the primer set and the probe for detecting parainfluenza virus 4 (PIV4) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 13, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 14, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 53 and / or SEQ ID NO. 54; and / or

[0039] A primer set and a probe for detecting coronavirus 229E (HCOV-229E), the primer set and the probe for detecting coronavirus 229E (HCOV-229E) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 15, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 16, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 55 and / or SEQ ID NO. 56; and / or

[0040] A primer set and a probe for detection of coronavirus HKU (HCOV-HKU1) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 17, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 18, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 57 and / or SEQ ID NO. 58; and / or

[0041] A primer set and a probe for detection of coronavirus OC43 (HCOV-OC43) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 19, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 20, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 59 and / or SEQ ID NO. 60; and / or

[0042] A primer set and a probe for detection of coronavirus NL63 (HCOV-NL63) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 21, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 22, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 61 and / or SEQ ID NO. 62; and / or

[0043] A primer set and a probe for detection of bocavirus (HBOV) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 23, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 24, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 63 and / or SEQ ID NO. 64; and / or

[0044] A primer set and a probe for detection of chlamydia pneumoniae (CP) comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 25, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 26, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 65 and / or SEQ ID NO. 66; and / or

[0045] A primer set and a probe for detection of rhinovirus (HRV) include an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 27, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 28, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 67 and / or SEQ ID NO. 68; and / or

[0046] A primer set and a probe for detection of metapneumovirus (HMPV) include an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 29, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 30, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 69 and / or SEQ ID NO. 70; and / or

[0047] A primer set and a probe for detection of adenovirus (HADV) include an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 31, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 32, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 71 and / or SEQ ID NO. 72; and / or

[0048] A primer set and a probe for detection of respiratory syncytial virus (RSV) include an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 33, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 34, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 73 and / or SEQ ID NO. 74; and / or

[0049] A primer set and a probe for detection of influenza A virus (IFA) include an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 37, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 38, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 77 and / or SEQ ID NO. 78.

[0050] In the present application, two probes are provided for each specific pathogen, and the nucleotide sequences of the two probes are partially reverse-complemented.

[0051] In the present application, two probes are used for each specific pathogen, either of which has a fluorescent reporter group and a fluorescent quencher group; the fluorescent reporter group includes FAM, HEX, JOE, TET, VIC, ROX, CY3, CY5, CY7 or NED, and more preferably FAM, VIC, ROX, CY3, CY5, TET or JOE; the quencher group includes BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, BBQ 650 or TAMRA, and more preferably BHQ1, BHQ2, BHQ3 or Dabcyl. The other of the probes has a blocking group, which includes phosphte, dNTP, NH2, O-ME, and more preferably phosphte or dNTP.

[0052] Further, the primer-probe combination of the present application further comprises a primer set and a probe for detecting an internal reference.

[0053] The primer set and the probe for detecting the internal reference include an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 39, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 40, and a probe with a nucleotide sequence as shown in SEQ ID NO. 79, SEQ ID NO. 80 and / or SEQ ID NO. 81.

[0054] Correspondingly, the primer-probe combination can also be combined in the form of the combinations of the target fragments as shown in Combination 1 to Combination 3 to obtain different groups of the preferred primer-probe combinations for simultaneously detecting different pathogens.

[0055] The present application provides a reagent comprising the primer-probe combination of the present application, and further comprising a solvent, a stabilizer, a preservative and / or an antioxidant, etc., which are not limited in the present application.

[0056] The present application provides a kit comprising at least one of the primer-probe combination of the present application or the reagent of the present application and a PCR reagent.

[0057] Further, the PCR detection reagent comprises a DNA polymerase, a reverse transcriptase, an RNAse inhibitor and / or dNTPs.

[0058] The DNA polymerase includes Taq enzyme.

[0059] Still further, the kit further comprises a negative control and a positive control; the negative control is a human internal reference gene; and the positive control contains the amplification sequence of the detected IFB, RSV, CP and human internal reference gene.

[0060] The application provides at least one of i) to iii) for use in the detection of a pathogenic respiratory sample for non-diagnostic purposes.

[0061] i) the primer probe combination according to the application;

[0062] ii) the reagent according to the application;

[0063] iii) the kit according to the application.

[0064] Further, the respiratory sample includes sputum, saliva and / or swab.

[0065] The application provides a method for detecting a respiratory pathogen for non-diagnostic purposes, which detects a sample by using at least one of i) to iii) as follows:

[0066] i) the primer probe combination according to the application;

[0067] ii) the reagent according to the application;

[0068] iii) the kit according to the application.

[0069] In the application, the method for detecting a respiratory pathogen for non-diagnostic purposes is a method for detecting multiple respiratory pathogens for non-diagnostic purposes; but in specific use, it can also be used for detecting a single pathogen, and the application does not limit this.

[0070] Further, the method includes mixing a sample, the reagent (primer probe mixture) according to the application and the PCR reagent (enzyme mixture) in the reagent according to the application, and then performing PCR detection.

[0071] In the application, the volume ratio of the sample, the reagent (primer probe mixture) according to the application and the PCR reagent (enzyme mixture) in the reagent according to the application is 5:3:4.

[0072] In the reaction, the concentration of the upstream primer in the primer probe combination is 100nM to 300nM, preferably 200nM; the concentration of the downstream primer is 100nM to 300nM, preferably 200nM; and the concentration of the probe is 50nM to 200nM, preferably 120nM.

[0073] In the PCR amplification reagent, the DNA polymerase is Taq enzyme; the concentration of the Taq enzyme is 1 U / 20 μL-3 U / 20 μL, preferably 2 U; the concentration of the reverse transcriptase is 1 U / 20 μL-4 U / 20 μL, preferably 2 U; the concentration of the RNase inhibitor is 5 U / 20 μL-20 U / 20 μL, preferably 10 U; and the concentration of the dNTPs is 100 μM-300 μM, preferably 200 μM.

[0074] In the present application, the concentration is a final concentration, more specifically, a use concentration, which is a concentration when reacting with a target substance.

[0075] The PCR detection procedure is as follows: 55°C for 5 min; 95°C for 2 min; 95°C for 5 s, 60°C for 30 s, 69°C for 10 s, 45 cycles; 95°C for 10 s; 25°C for 3 min; and continuously increasing the temperature from 25 to 68°C at a rate of 0.03°C / s.

[0076] In the detection method, the "60°C for 30 s" and "continuously increasing the temperature from 25 to 68°C" stages of the PCR detection further include a step of detecting a fluorescence signal; and the PCR detection further includes a step of interpreting the results.

[0077] The detection method further includes a step of independently detecting a positive quality control and a negative quality control, which are used for quality control; and the step of detecting the positive quality control and the negative quality control includes the following steps: mixing the positive quality control / negative quality control, the reagent (primer probe mixture) and the PCR reagent (enzyme mixture) in the reagent to perform PCR detection.

[0078] In specific embodiments of the present application, the sample, the positive quality control and the negative quality control can be nucleic acid solutions obtained by nucleic acid extraction from a pathogen-containing sample, or can be specific pathogen-containing samples without nucleic acid extraction; the present application does not limit this; and in the present application, nucleic acid solutions obtained by nucleic acid extraction from a pathogen-containing sample are preferred.

[0079] In the present application, the detection method includes a detection method for diagnostic purposes and a detection method for non-diagnostic purposes, and the detection method for non-diagnostic purposes includes a method for developing a detection product or a detection method, and determining the influence of a specific parameter change on the performance of the detection product or the advantages and disadvantages of the detection method.

[0080] In the detection method, a single-tube detection method is used, each reaction contains 2-6 fluorescence channels, preferably 4; but after design, 20-fold detection can be performed in one tube under the conditions allowed by the equipment, and 18 pathogens are detected.

[0081] The present application aims to break through the limitation of the existing PCR technology in completing multiplexed and differentiated detection on the fluorescent PCR device, and provides a primer probe combination for screening common respiratory pathogens of acute and community-acquired pneumonia, which has the advantages of high accuracy, good specificity, strong anti-interference ability, good inclusivity, high sensitivity, super combined detection and simple operation, which are the results of the joint action of various technical parameters, and specifically comprises the following steps: selecting a specific conservative region as a target sequence for the amplified primer probe combination, comparing, screening and evaluating the specificity of the conservative sequence and the primer, effectively avoiding non-specific amplification and non-specific cross reaction between multiple primers, and simultaneously having the advantages of strong specificity and wide coverage; meanwhile, the concentration of the primer probe combination is optimized, and the composition of the enzyme mixture is screened and the concentration is optimized, so as to further improve the sensitivity, anti-interference ability and specificity of the detection; further, the present application is designed by two probes, so that the two probes interact to provide a stable Tm value, thereby being more beneficial to the specificity and accuracy of the multiplexed detection result (each Tm value is determined by the corresponding two probes).

[0082] Further, the above optimization makes the present application have the following advantages:

[0083] (1) Strong specificity: the present application selects a specific conservative region as a target sequence for the amplified primer probe combination, compares, screens and evaluates the specificity of the conservative sequence and the primer, effectively avoids non-specific amplification and non-specific cross reaction between multiple primers, and has the advantages of strong specificity and wide coverage;

[0084] (2) Strong anti-interference ability: the present application uses clinical samples to add interference substances (the interference substances are selected in the largest potential concentration (worst condition)) to test the interference substance tolerance, and the results show that the interference substances do not interfere with the detection results;

[0085] (3) Good inclusivity: the present application can detect different types of pathogens, which indicates that the present application has good inclusivity;

[0086] (4) High sensitivity: the present application can detect 100 copies / ml of pathogen concentration by optimizing the amount of primer, the amount of probe and the amplification efficiency of the reaction system;

[0087] (5) Multi-index combined detection: the present application can simultaneously detect up to 18 kinds of ARTI and CAP respiratory pathogens in a single tube, and takes into account viruses and atypical pathogens and other types, has wide coverage and multiple types of pathogens, and can provide an accurate, multi-target parallel detection for medical institutions;

[0088] (6) Combined with a fully automated nucleic acid detection device, the present application realizes fully automated detection and reduces the possibility of contamination;

[0089] (7) Adopting internal standard system: capable of showing occurrence of false negative, avoiding false negative caused by inhibitor existing in sample or operation mistake;

[0090] (8) Simple operation, whole-process closed tube detection: no need of expensive and special instrument equipment, whole-process closed tube detection, no need of opening cover, effectively avoiding pollution.

[0091] The present application provides a nucleic acid detection primer probe combination for wide-spectrum detection of respiratory tract pathogens of acute and community-acquired pneumonia and an expected matching detection method by optimizing primer probe sequences, optimizing primer probes, screening and optimizing concentrations of each component in PCR amplification reagents, and optimizing PCR amplification procedures, so that 20 target detections can be met at one time, 18 common respiratory tract pathogens can be identified, and the nucleic acid detection primer probe combination has the advantages of high accuracy, good specificity, strong anti-interference ability, good inclusivity, high sensitivity, super joint detection and simple operation.

[0092] The present application provides a nucleic acid detection primer probe combination for wide-spectrum detection of respiratory tract pathogens of acute and community-acquired pneumonia and an expected matching detection method by optimizing primer probe sequences, optimizing primer probes, screening and optimizing concentrations of each component in PCR amplification reagents, and optimizing PCR amplification procedures, so that 20 target detections can be met at one time, 18 common respiratory tract pathogens can be identified, and the nucleic acid detection primer probe combination has the advantages of high accuracy, good specificity, strong anti-interference ability, good inclusivity, high sensitivity, super joint detection and simple operation. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 Cross-specificity detection results are shown;

[0094] Figure 2 Inclusivity detection results of adenovirus (HADV) are shown;

[0095] Figure 3 Inclusivity detection results of influenza A virus (IFA) are shown;

[0096] Figure 4 Inclusivity detection results of novel coronavirus (SARS-CoV-2) are shown;

[0097] Figure 5 Detection results of 18 heavy pathogens are all positive. DETAILED DESCRIPTION

[0098] The present application provides a primer probe combination for screening common respiratory pathogens and its application. Those skilled in the art can refer to the content herein and appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application to realize and apply the present application technology.

[0099] According to the nucleic acid information of the respiratory pathogens, homology comparison analysis is performed, specific primers and probes are designed for the conserved sequences of the pathogens, and the primers and probes are shown in Table 1.

[0100] Table 1. Primer sequences for detecting pathogens and internal standards

[0101]

[0102]

[0103] Table 2. Probe sequences for detecting pathogens and internal standards

[0104]

[0105]

[0106] In Table 2, the two ends (5' end and 3' end) of the fluorescent probe have a fluorescent reporter group (located at the 5' end or 3' end) and a fluorescent quencher group located at the 5' end or 3' end), in the examples, the fluorescent reporter group is one or more of FAM, VIC, ROX and CY5, and the fluorescent quencher group is one or more of BHQ1, BHQ2 and BHQ3; one end (5' end or 3' end) of the auxiliary probe has a blocking group, in the examples, the blocking group is phosphte. And in Table 1 and Table 2, in addition to the normal a, t, g, c four base symbols, other letters such as r, y, m, k and the like appear in the nucleotide chain. These other letters are degenerate bases, wherein r represents that the base is a or g, y represents that the base is c or t, m represents that the base is a or c, k represents that the base is g or t, s represents that the base is c or g, w represents that the base is a or t, h represents that the base is a or c or t, b represents that the base is c or g or t, v represents that the base is a or c or g, d represents that the base is a or g or t, and n represents that the base is a or c or g or t;

[0107] Table 3. Conserved sequences of pathogens and internal standard sequences for detection

[0108]

[0109]

[0110]

[0111] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The present application is further described below in combination with examples:

[0112] Example 1 Kit composition

[0113] The present example provides a primer probe set for broad-spectrum screening of common acute and community-acquired respiratory pathogens and application, including enzyme mixture, primer probe mixture, DEPC treated water, negative control and positive control. The enzyme mixture contains reverse transcriptase, RNase inhibitor, Taq enzyme, PCR buffer, dNTPs, salt ions. The negative control is a human internal standard gene; the positive control contains the detection of IFB, RSV, CP and human internal standard gene target amplification sequence.

[0114] The primer probe mixture includes amplification primer set and probe set. The final concentration of the primer is 100nM-300nM (200nM is more optimal). The final concentration of the probe is 50nM-200nM (120nM is more optimal).

[0115] Table 4. Final concentration of each main component in enzyme mixture

[0116] Main components Final concentration Reverse transcriptase 1 U ~ 4 U (2 U is more optimal) RNase inhibitor 5 U ~ 20 U (10 U is more optimal) Taq enzyme 1 U ~ 3 U (2 U is more optimal) dNTPs 100 μM ~ 300 μM (200 μM is more optimal)

[0117] PCR reaction system preparation: (enzyme mixture, primer probe mixture, DEPC treated water, nucleic acid template), formula as shown in Table 5:

[0118] Table 5. Reagent preparation

[0119] Components Amount added (μL / test) Enzyme mix 4 Primer / probe mix 3 DEPC-treated water 8 Nucleic acid template 5

[0120] PCR amplification procedure: stage 1, 55℃, 5min, 1 cycle; stage 2, 95℃, 2min, 1 cycle; stage 3, 95℃, 5s, 60℃ (collect fluorescence), 30s, 69℃, 10s, 45 cycles; stage 4, 95℃, 10s, 25℃, 3min, 25-68℃, collect fluorescence signal, 1 cycle.

[0121] Result determination: the detection channel and melting temperature range of each pathogen are shown in the following table 6. The positive sample should have peak height change with negative control (NC) melting curve within the corresponding pathogen detection melting temperature range, to determine the specific type of pathogen.

[0122] Table 6. Detection channel and melting temperature range of each pathogen

[0123]

[0124]

[0125] Detection of clinical samples in Example 2

[0126] Specifically, using the detection method described in Example 1 above, this example shows the detection data of clinical samples. The clinical samples are swab or sputum samples of ARTI and CAP patients known to be infected with pathogens.

[0127] The nucleic acid template was obtained by extracting the nucleic acid of the samples in Table 7, and the reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0128] According to the analysis of the detection results, there are 60 positive samples and 20 negative samples. The PCR amplification products of the above positive samples were sequenced to obtain product sequences, which were input into the NCBI database and analyzed by blast software to confirm whether the types of pathogens were consistent with the application detection results. According to the detection results, the sequencing feedback results are consistent with the application detection results, indicating that the detection accuracy of the application is high.

[0129] Table 7. Detection results of clinical samples

[0130]

[0131]

[0132] Sensitivity test in Example 3

[0133] The analysis sensitivity test procedure is as follows:

[0134] The clinical samples with a fixed value were diluted to 500, 100, and 50 copies / ml, and the nucleic acid was extracted to obtain the nucleic acid template. The specific sample information is shown in Table 8.

[0135] The reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0136] The detection was repeated 20 times, and the detection results are shown in Table 8.

[0137] Table 8. Analysis sensitivity sample information

[0138] Sample name Concentration (copies / ml) Sample name Concentration (copies / ml) IFB 500、100、50 OC43 500、100、50 MP 500、100、50 NL63 500、100、50 SARS-CoV-2 500、100、50 HBOV 500、100、50 PIV1 500、100、50 CP 500、100、50 PIV2 500、100、50 HRV 500、100、50 PIV3 500、100、50 HMPV 500、100、50 PIV4 500、100、50 HADV 500、100、50 229E 500、100、50 RSV 500、100、50 HKU1 500、100、50 IFA 500、100、50

[0139] According to the detection results in Table 9, the lowest detection limit of the application is 100 copies / ml, which has high sensitivity.

[0140] Table 9. Statistical results

[0141]

[0142] Repeatability test of Example 4

[0143] The repeatability test procedure is as follows:

[0144] Each sample in Table 10 is a mixed sample of multiple pathogens, which contains the types of pathogens as shown in "pathogen target combination"; and has been confirmed by clinical detection;

[0145] The nucleic acids of the samples in Table 10 are extracted, the reaction system is prepared according to Table 5 in Example 1, and PCR amplification is performed according to the procedure in Example 1.

[0146] According to the detection results in Table 10, the detection method of the present application has good repeatability.

[0147] Table 10. Detection results

[0148]

[0149]

[0150] Specificity test of Example 5

[0151] The specificity test procedure is as follows:

[0152] Other pathogens except the 18 pathogens described in the present application are used as templates for detection, and the types and concentrations of pathogens in the templates are as shown in Table 11.

[0153] The nucleic acids of the samples in Table 11 are extracted, the reaction system is prepared according to Table 5 in Example 1, and PCR amplification is performed according to the procedure in Example 1.

[0154] The detection results of other pathogens except the 12 pathogens described in the present application are shown in Table 11, and according to the detection results, the present application has high specificity and no cross-reaction (as shown in Table 11). Figure 1

[0155] Table 11. Cross-specificity detection results

[0156]

[0157]

[0158] Anti-interference ability of Example 6

[0159] The anti-interference ability test procedure is as follows:

[0160] ​The nucleic acid extraction was performed after adding the interfering substances to the sample containing the pathogen, and the interfering substances were selected at the potential maximum concentration ("worst condition"). The pathogen type, interfering substance type and concentration are shown in Table 12. The nucleic acid extracted from the sample containing the pathogen to which the interfering substance was added was used as the nucleic acid template.

[0161] The reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0162] The samples to which the interfering substances were added were detected according to the detection method in the above examples. According to the detection results in Table 12, it can be seen that the listed drugs do not interfere with the detection results, and the present application has good anti-interference ability.

[0163] Table 12. Detection results

[0164]

[0165]

[0166]

[0167] Inclusion test case 7

[0168] The procedure of the inclusion test is as follows:

[0169] Different types of pathogens were used as templates for independent detection. The specific pathogen typing types are shown in Table 13. All of them are samples confirmed by clinical detection, and are used as nucleic acid templates after nucleic acid extraction. Each test detects a specific type of pathogen. The pathogen is extracted to obtain a nucleic acid template;

[0170] The reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0171] The different type samples were detected according to the above steps. According to the detection results in Table 13, the detection method of the present application can normally detect the listed different type pathogens, which shows that the present application has good inclusivity. For example, the inclusion detection results of adenovirus (HADV) are shown in Figure 2 , the inclusion detection results of influenza A virus (IFA) are shown in Figure 3 , and the inclusion detection results of novel coronavirus (SARS-CoV-2) are shown in Figure 4 .

[0172] Table 13. Detection results

[0173]

[0174]

[0175]

[0176] Example 8 Multiplex test

[0177] The procedure of the multiplex test is as follows:

[0178] The nucleic acid templates obtained after nucleic acid extraction of the clinically determined mixed pathogen samples containing the mixed pathogens shown in Table 14 are detected;

[0179] The reaction system is prepared according to Table 5 in Example 1, and PCR amplification is performed according to the procedure in Example 1.

[0180] The composite infection samples are detected according to the above procedure.

[0181] According to the detection results in Table 13, the detection method of the present application can normally detect the multiplex composite infection samples (20x18). Figure 5 The joint detection results of 18 pathogens (20x18) are shown in the figure.

[0182] Table 14. Detection results

[0183]

[0184] As can be seen from the above examples, the present application provides a detection primer probe set for nucleic acids of common respiratory pathogens of acute and community-acquired pneumonia and its application, which has the advantages of good specificity, high sensitivity, multiple detection targets, simple and rapid operation, etc. The present application can simultaneously detect 18 kinds of ARTI and CAP respiratory pathogens in a single tube, has wide coverage and multiple pathogen types, can provide an accurate, multi-target parallel detection for medical institutions such as disease control centers and hospitals, and realize full automation detection in combination with full-automatic nucleic acid detection equipment. The setting of the internal standard system can control the quality of the false negative results caused by potential PCR inhibitors, instruments, reagents and operations during the detection process. The setting of the negative control and the positive control can control the quality of the sample nucleic acid extraction process.

[0185] Example 9 Concentration optimization

[0186] In the development process of the present application, the amounts of various components in the kit were compared and optimized to obtain the best one. Table 15 exemplarily lists a part of them.

[0187] Table 15. Final concentration of each component in the comparison test

[0188]

[0189] The test procedure is as follows:

[0190] The clinical samples positive for each pathogen are diluted into two concentration levels (concentration 1: 1.00x105 copies / mL, concentration 2: 5.00 x 10 3 copies / mL, 1.00 x 10

[0191] The primers and probes in Tables 1 and 2 above were prepared into corresponding three groups of primer probe mixtures 3-5 according to the requirements of the final concentrations in Table 15; and other components in Table 15 were prepared into corresponding three groups of enzyme mixtures 3-5 according to the requirements of the final concentrations.

[0192] The corresponding PCR reaction systems (enzyme mixtures 3-5, primer probe mixtures 3-5, DEPC treated water, nucleic acid templates) were prepared, and the formulations are shown in Table 5.

[0193] PCR amplification was performed according to the procedure in Example 1.

[0194] Each concentration level of sample was detected twice in three reaction systems, and the Ct value results are shown in Table 16.

[0195] Table 16. Ct value summary of comparative detection

[0196]

[0197]

[0198] Comparative case 1

[0199] During the development of the present application, a plurality of sets of primers and probes were designed for 18 pathogens, and the best one was obtained through comparative tests. For the purpose of reducing the length, a part of them are exemplarily listed in Table 17.

[0200] Table 17. Primer sequences for detecting pathogens and internal standards

[0201]

[0202]

[0203]

[0204] The screening test steps are as follows:

[0205] The clinical samples positive for each pathogen were diluted into high, medium and low concentration levels, and the nucleic acids were extracted synchronously with the negative control; wherein the high, medium and low concentration levels respectively represent the concentrations of 1.00 x 10 6 copies / mL, 1.00 x 10 5 copies / mL, 1.00 x 10 4copies / mL; the specific pathogen species is shown in Table 18, and the nucleic acid template was obtained by nucleic acid extraction;

[0206] The primers and probes in Table 1 and Table 2 above were mixed to prepare primer probe mixture 1, and the primers and probes in Table 17 were mixed to prepare primer probe mixture 2.

[0207] The corresponding PCR reaction systems (enzyme mixture, primer probe mixture 1 or primer probe mixture 2, DEPC-treated water, nucleic acid template) were prepared respectively, and the formulations are shown in Table 5.

[0208] PCR amplification was performed according to the procedure in Example 1.

[0209] Each sample at each concentration level was detected 3 times in two reaction systems, and the Ct value results are shown in Table 18. The corresponding Tm values of each target are shown in Table 19.

[0210] Table 18. Summary of comparative detection Ct values

[0211]

[0212]

[0213]

[0214] Table 19. Summary of comparative detection Tm values

[0215]

[0216] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer probe combination for detecting a respiratory pathogen, characterized in that, The target fragments include: an influenza B virus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 82; and / or a Mycoplasma pneumoniae target fragment having a nucleotide sequence as set forth in SEQ ID NO. 83; and / or an ORF1ab gene target fragment of a novel coronavirus having a nucleotide sequence as set forth in SEQ ID NO. 84; and / or a parainfluenza virus type 1 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 85; and / or a parainfluenza virus type 2 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 86; and / or a parainfluenza virus type 3 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 87; and / or a parainfluenza virus type 4 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 88; and / or a coronavirus 229E target fragment having a nucleotide sequence as set forth in SEQ ID NO. 89; and / or a coronavirus HKU1 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 90; and / or a coronavirus OC43 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 91; and / or a coronavirus NL63 target fragment having a nucleotide sequence as set forth in SEQ ID NO. 92; and / or a bocavirus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 93; and / or a Chlamydia pneumoniae target fragment having a nucleotide sequence as set forth in SEQ ID NO. 94; and / or a rhinovirus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 95; and / or a metapneumovirus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 96; and / or an adenovirus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 97; and / or a respiratory syncytial virus target fragment having a nucleotide sequence as set forth in SEQ ID NO. 98; and / or an N gene target fragment of a novel coronavirus having a nucleotide sequence as set forth in SEQ ID NO. 99; and / or an influenza A virus (IFA) target fragment having a nucleotide sequence as set forth in SEQ ID NO.

100.

2. The primer probe combination according to claim 1, characterized in that, The target fragments further include: an internal standard target fragment having a nucleotide sequence as set forth in SEQ ID NO.

101.

3. The primer probe combination of claim 2, wherein The primer probe combination includes: a primer set and a probe for detecting influenza B virus, the primer set and the probe for detecting influenza B virus comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 1, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 2, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 41 and / or SEQ ID NO. 42; and / or a primer set and a probe for detecting Mycoplasma pneumoniae, the primer set and the probe for detecting Mycoplasma pneumoniae comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 3, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 4, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 43 and / or SEQ ID NO. 44; and / or a primer set and a probe for detecting an ORF1ab gene of a novel coronavirus, the primer set and the probe for detecting an ORF1ab gene of a novel coronavirus comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 5, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 6, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 45 and / or SEQ ID NO. 46; and / or a primer set and a probe for detecting parainfluenza virus type 1, the primer set and the probe for detecting parainfluenza virus type 1 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 7, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 8, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 47 and / or SEQ ID NO. 48; and / or a primer set and a probe for detecting parainfluenza virus type 2, the primer set and the probe for detecting parainfluenza virus type 2 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 9, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 10, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 49 and / or SEQ ID NO. 50; and / or a primer set and a probe for detecting parainfluenza virus type 3, the primer set and the probe for detecting parainfluenza virus type 3 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 11, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 12, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 51 and / or SEQ ID NO. 52; and / or a primer set and a probe for detecting parainfluenza virus type 4, the primer set and the probe for detecting parainfluenza virus type 4 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 13, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 14, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 53 and / or SEQ ID NO. 54; and / or a primer set and a probe for detecting coronavirus 229E, the primer set and the probe for detecting coronavirus 229E comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 15, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 16, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 55 and / or SEQ ID NO. 56; and / or a primer set and a probe for detecting coronavirus HKU1, the primer set and the probe for detecting coronavirus HKU1 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 17, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 18, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 57 and / or SEQ ID NO. 58; and / or a primer set and a probe for detecting coronavirus OC43, the primer set and the probe for detecting coronavirus OC43 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 19, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 20, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 59 and / or SEQ ID NO. 60; and / or a primer set and a probe for detecting coronavirus NL63, the primer set and the probe for detecting coronavirus NL63 comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 21, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 22, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 61 and / or SEQ ID NO. 62; and / or a primer set and a probe for detecting bocavirus, the primer set and the probe for detecting bocavirus comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 23, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 24, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 63 and / or SEQ ID NO. 64; and / or a primer set and a probe for detecting Chlamydia pneumoniae, the primer set and the probe for detecting Chlamydia pneumoniae comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 25, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 26, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 65 and / or SEQ ID NO. 66; and / or a primer set and a probe for detecting rhinovirus, the primer set and the probe for detecting rhinovirus comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 27, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 28, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 67 and / or SEQ ID NO. 68; and / or a primer set and a probe for detecting metapneumovirus, the primer set and the probe for detecting metapneumovirus comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 29, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 30, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 69 and / or SEQ ID NO. 70; and / or a primer set and a probe for detecting adenov The primer set and probe for detecting Mycoplasma pneumoniae comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, and a probe with a nucleotide sequence as shown in SEQ ID NO. 43 and / or SEQ ID NO. 44; and / or The primer set and probe for detecting novel coronavirus comprise: primer set and probe combination 1 and primer set and probe combination 2; the primer set and probe combination 1 comprises: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, and a probe with a nucleotide sequence as shown in SEQ ID NO. 45 and / or SEQ ID NO. 46; the primer set and probe combination 2 comprises: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 35, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 36, and a probe with a nucleotide sequence as shown in SEQ ID NO. 75 and / or SEQ ID NO. 76; and / or The primer set and probe for detecting parainfluenza virus type 1 comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8, and a probe with a nucleotide sequence as shown in SEQ ID NO. 47 and / or SEQ ID NO. 48; and / or The primer set and probe for detecting parainfluenza virus type 2 comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 9, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, and a probe with a nucleotide sequence as shown in SEQ ID NO. 49 and / or SEQ ID NO. 50; and / or The primer set and probe for detecting parainfluenza virus type 3 comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 12, and a probe with a nucleotide sequence as shown in SEQ ID NO. 51 and / or SEQ ID NO. 52; and / or The primer set and probe for detecting parainfluenza virus type 4 comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and a probe with a nucleotide sequence as shown in SEQ ID NO. 53 and / or SEQ ID NO. 54; and / or The primer set and probe for detecting parainfluenza virus type 4 comprise: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and a probe with a nucleotide sequence as shown in SEQ ID NO. 53 and / or SEQ ID NO. 54; and / or A primer set and a probe for detecting coronavirus 229E, the primer set and the probe for detecting coronavirus 229E comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 15, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 16, and a probe with a nucleotide sequence as shown in SEQ ID NO. 55 and / or SEQ ID NO. 56; and / or A primer set and a probe for detecting coronavirus HKU, the primer set and the probe for detecting coronavirus HKU comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 17, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 18, and a probe with a nucleotide sequence as shown in SEQ ID NO. 57 and / or SEQ ID NO. 58; and / or A primer set and a probe for detecting coronavirus OC43, the primer set and the probe for detecting coronavirus OC43 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 19, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 20, and a probe with a nucleotide sequence as shown in SEQ ID NO. 59 and / or SEQ ID NO. 60; and / or A primer set and a probe for detecting coronavirus NL63, the primer set and the probe for detecting coronavirus NL63 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 21, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 22, and a probe with a nucleotide sequence as shown in SEQ ID NO. 61 and / or SEQ ID NO. 62; and / or A primer set and a probe for detecting bocavirus, the primer set and the probe for detecting bocavirus comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 23, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 24, and a probe with a nucleotide sequence as shown in SEQ ID NO. 63 and / or SEQ ID NO. 64; and / or A primer set and a probe for detecting chlamydia pneumoniae, the primer set and the probe for detecting chlamydia pneumoniae comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 25, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 26, and a probe with a nucleotide sequence as shown in SEQ ID NO. 65 and / or SEQ ID NO. 66; and / or A primer set and a probe for detecting rhinovirus, the primer set and the probe for detecting rhinovirus comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 27, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 28, and a probe with a nucleotide sequence as shown in SEQ ID NO. 67 and / or SEQ ID NO. 68; and / or a primer set and a probe for metapneumovirus detection, the primer set and the probe for metapneumovirus detection comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 29, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 30, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 69 and / or SEQ ID NO. 70; and / or a primer set and a probe for adenovirus detection, the primer set and the probe for adenovirus detection comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 31, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 32, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 71 and / or SEQ ID NO. 72; and / or a primer set and a probe for respiratory syncytial virus detection, the primer set and the probe for respiratory syncytial virus detection comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 33, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 34, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 73 and / or SEQ ID NO. 74; and / or a primer set and a probe for influenza A virus detection, the primer set and the probe comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 37, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 38, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 77 and / or SEQ ID NO.

78.

4. The primer probe combination of claim 3, wherein the primer probe combination further comprises a primer set and a probe for detecting an internal reference; the primer set and the probe for detecting an internal reference comprising: an upstream primer having a nucleotide sequence as set forth in SEQ ID NO. 39, a downstream primer having a nucleotide sequence as set forth in SEQ ID NO. 40, and a probe having a nucleotide sequence as set forth in SEQ ID NO. 79, SEQ ID NO. 80, and / or SEQ ID NO.

81.

5. A reagent characterized in that, The primer probe combination of any one of claims 1 to 4.

6. A kit characterized in that, The primer probe combination of any one of claims 1 to 4 and / or at least one of the reagent of claim 5 and a PCR reagent.

7. The kit of claim 6, wherein The PCR detection reagent comprises a DNA polymerase, a reverse transcriptase, an RNase inhibitor, and / or dNTPs.

8. A method for the detection of a respiratory pathogen for non-diagnostic purposes, characterized in that The detection of a respiratory sample using at least one of i) to iii) below: i) the primer probe combination of any one of claims 1 to 4; ii) the reagent of claim 5; iii) the kit of claim 6 or 7.

9. The detection method according to claim 8, characterized in that, The method comprising the steps of: mixing the sample, the reagent of claim 5, and the PCR reagent in the kit of any one of claims 6 to 8, and performing PCR detection.

10. The detection method of claim 9, wherein In the primer probe combination of the reagent, the concentration of the upstream primer is 100nM-300nM; the concentration of the downstream primer is 100nM-300nM; and the concentration of the probe is 50nM-200nM. In the PCR reagent of the kit, the concentration of the DNA polymerase is 1U / 20μL-3U / 20μL; the concentration of the reverse transcriptase is 1U / 20μL-4U / 20μL; the concentration of the RNase inhibitor is 5U / 20μL-20U / 20μL; and the concentration of the dNTPs is 100μM-300μM.

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