Fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for 30 respiratory pathogens and application thereof

Abstract

The invention provides a primer and probe combination for detecting respiratory tract infection pathogens in a sample through a fluorescent quantitative PCR method, and application of the primer and probe combination in preparation of a reagent for detecting the respiratory tract infection pathogens in the sample.

Description

Technical Field

[0001] The present invention relates to the field of PCR detection, in particular to a fluorescence quantitative PCR detection method and its application in the field of detecting human severe acute respiratory pathogens. Background Art

[0002] At present, infectious diseases remain a major threat to human health, especially severe acute respiratory infectious diseases. Due to their characteristics such as a wide variety of types, rapid transmission, and extensive prevalence, they pose a major threat to human health, social stability, and economic development, and are the main causes of morbidity and mortality worldwide. The Global Burden of Diseases, Injuries, and Risk Factors (GBD) study found that in 2016, lower respiratory tract infections caused the deaths of 650,000 children under 5 years old and 1.08 million adults over 70 years old, and approximately 2.38 million people of all ages worldwide died from lower respiratory tract infections, making lower respiratory tract infections the sixth leading cause of death across all ages and the leading cause of death in children under 5 years old, posing a major threat to public health, social stability, and economic development (see: GBD 2016 Lower Respiratory Infection Collaborators. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990 - 2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Infect Dis. 2018, 18(11): 1191 - 1210 and Murdoch DR, Howie SRC. The global burden of lower respiratory infections: making progress, but we need to do better. Lancet Infect Dis. 2018, 18(11): 1162 - 1163). At the same time, due to the lack of early and clear pathogen diagnosis, the misuse and overuse of antimicrobial drugs have accelerated the emergence of antimicrobial drug resistance. Antimicrobial drug resistance can affect anyone of any age in any country. Therefore, more and more infectious diseases are becoming increasingly difficult to treat, prevent, and control.The World Health Organization (WHO) has warned that antimicrobial resistance is one of the greatest threats to global health, food security, and development today (see https: / / www.who.int / news-room / fact-sheets / detail / antibiotic-resistance.), and it leads to longer hospital stays, increased medical costs, and higher mortality rates (see Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. PT. 2015 Apr; 40(4): 277-83). Therefore, there is an urgent need to develop sensitive, rapid, specific, stable, low-cost, and easy-to-use diagnostic methods that can provide a basis for identifying pathogens, early diagnosis, and understanding the epidemiological trends of pathogens in clinical applications, promote early rational treatment, reduce the resistance burden caused by the increased use of empirical antimicrobial agents, and enable the early use of the correct drugs for targeted pathogens to control the condition in a timely manner.

[0003] Since the advent of PCR technology, nucleic acid testing has played an important role in the diagnosis and treatment of infectious diseases. In particular, multiplex real-time PCR systems can detect multiple viruses and bacteria simultaneously and have a decisive guiding role in the diagnosis of diseases in areas such as respiratory tract infections (see Brittain-Long R, Nord S, Olofsson S, Westin J, Anderson LM, Lindh M. Multiplex real-time PCR for detection of respiratory tract infections. J Clin Virol. 2008, 41(1): 53-6.). However, the nucleic acid extraction and the configuration of the reaction system in the PCR technology process require professional operations. Therefore, strict laboratory conditions and high personnel technical requirements are the main factors restricting the implementation of real-time fluorescence quantitative PCR technology at the grass-roots level. The point-of-care testing (POCT) of PCR, with its characteristics of being portable, rapid, pollution-free, and not requiring professional personnel to operate, has begun to quickly occupy the market in the detection fields at home and abroad. However, the high cost of its supporting equipment and reagents limits its popularization and application in less economically developed regions. Therefore, in domestic laboratories or hospital inspection departments, the most widely used method at present is still fluorescence quantitative PCR.

[0004] The object of the present invention is to develop 8 sets of multiplex fluorescence PCR detection systems for the identification of respiratory tract infection pathogens for severe acute lower respiratory tract infections, covering a total of 30 targets such as viruses, bacteria, mycoplasmas, and chlamydiae, and pre-drying and prefabricating specific multiplex detection primer-probes on a PCR well plate. On this basis, a rapid detection system suitable for common fluorescence PCR instruments is established, making it applicable to the rapid early diagnosis of clinical severe acute lower respiratory tract samples, and providing support for the early reasonable treatment of clinical severe acute lower respiratory tract infection patients and the research and development of clinical drugs. Summary of the Invention

[0005] The object of the present invention is to provide a rapid detection method for fluorescence quantitative PCR for detecting respiratory tract infection pathogens. The present invention designs a combination of fluorescence PCR primers and probes (Taqman probes) for a total of 30 targets covering viruses, bacteria, mycoplasmas, chlamydiae, etc. in respiratory tract infection pathogens, and divides them into 8 sets of multiplex detection systems.

[0006] Due to the large number of primer-probes in the respiratory multi-pathogen system, the conventional laboratory operation steps are cumbersome, and it is easy to make mistakes and cross-contaminate during the system configuration process. The prefabricated detection system provided by the present invention can greatly reduce the operation time of conventional fluorescence PCR detection, shorten the process, and quickly load the machine without affecting the detection effect. The primers and probes in the detection system have good sensitivity and specificity, and the detection system has good repeatability.

[0007] The respiratory tract infection pathogens that can be detected by the present invention are selected from one or more of the following pathogens: PIV1-4 (human parainfluenza virus types 1-4), NL63, 229E, OC43, HKU1 (human coronaviruses NL63, 229E, OC43, HKU1), RSVA / B (respiratory syncytial virus), hMPV (human metapneumovirus), BOV (human bocavirus), RV (human rhinovirus), EV (human enterovirus), FLUA (influenza A virus), FLUB (influenza B virus), ADV (adenovirus), CPN (Chlamydia pneumoniae), MP (Mycoplasma pneumoniae), GAPDH (housekeeping gene), Mca (Moraxella catarrhalis), Hin (Haemophilus influenzae), HinB (Haemophilus influenzae type B), Aba (Acinetobacter baumannii), Paer (Pseudomonas aeruginosa), Spn (Streptococcus pneumoniae), Sau (Staphylococcus aureus), Kpn (Klebsiella pneumoniae), Bssp (Bordetella spp.), Pji (Pneumocystis jirovecii), Lssp (Legionella spp.).

[0008] In a first aspect, the present invention provides a primer and probe combination for simultaneously detecting PIV1, NL63, RSVA, and 229E in a sample by a fluorescence quantitative PCR method.

[0009] In a second aspect, the present invention provides a primer and probe combination for simultaneously detecting OC43, PIV2, HKU1, and RSVB in a sample by a fluorescence quantitative PCR method.

[0010] In a third aspect, the present invention provides a primer and probe combination for simultaneously detecting hMPV, BOV, PIV4, and RV in a sample by a fluorescence quantitative PCR method.

[0011] In a fourth aspect, the present invention provides a primer and probe combination for simultaneously detecting EV, GAPDH, FLUB, and PIV3 in a sample by a fluorescence quantitative PCR method.

[0012] Fifth aspect, the present invention provides a primer and probe combination for simultaneously detecting ADV, FLUA, CPN, and MP in a sample by fluorescence quantitative PCR method.

[0013] Sixth aspect, the present invention provides a primer and probe combination for simultaneously detecting Mca, Spn, Aba, and Bssp in a sample by fluorescence quantitative PCR method.

[0014] Seventh aspect, the present invention provides a primer and probe combination for simultaneously detecting Lssp, Pji, and Paer in a sample by fluorescence quantitative PCR method.

[0015] Eighth aspect, the present invention provides a primer and probe combination for simultaneously detecting HinB, Hin, Sau, and Kpn in a sample by fluorescence quantitative PCR method.

[0016] Ninth aspect, the present invention provides a fluorescence quantitative PCR kit, and the kit includes the primer and probe combinations of the first to eighth aspects.

[0017] Tenth aspect, the present invention provides the use of the primer and probe combinations of the first to eighth aspects for preparing a reagent for detecting respiratory tract infection pathogens in a sample. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only specific examples or their results recorded in the present application. For those of ordinary skill in the art, other specific implementation schemes and their results can also be obtained based on these drawings.

[0019] Figure 1 shows the amplification curve of detecting respiratory tract infection pathogens by fluorescence quantitative PCR method. The abscissa in the figure is the cycle number, and the ordinate is the relative fluorescence unit (RFU, relative fluorescence units).

[0020] Figure 2 shows the amplification efficiency results of detecting respiratory tract infection pathogens by fluorescence quantitative PCR method. In the left figure, the abscissa is the cycle number, and the ordinate is the relative fluorescence unit (RFU, relative fluorescence units).

[0021] Figure 3 shows the sensitivity results of detecting respiratory tract infection pathogens by fluorescence quantitative PCR method. The abscissa in the figure is the copy number, the ordinate is the amplification ratio, and LOD (The limits of detection) is the detection limit (95% confidence level).

[0022] Figure 4 shows the actual amplification curve of the fluorescence quantitative PCR method for detecting clinical samples in the virus detection system. Detailed implementation mode

[0023] The present invention provides a primer and probe combination for detecting respiratory tract infection pathogens in a sample by a fluorescence quantitative PCR method, wherein the primer is used for specifically amplifying the gene sequence of the respiratory tract infection pathogen, and the probe specifically binds to the internal sequence of the respiratory tract infection pathogen gene.

[0024] In a specific embodiment, the primer and probe combination is used for simultaneously detecting PIV1, NL63, RSVA, and 229E in a sample.

[0025] In a specific embodiment, the primer and probe combination is used for simultaneously detecting OC43, PIV2, HKU1, and RSVB in a sample.

[0026] In a specific embodiment, the primer and probe combination is used for simultaneously detecting hMPV, BOV, PIV4, and RV in a sample.

[0027] In a specific embodiment, the primer and probe combination is used for simultaneously detecting EV, GAPDH, FLUB, and PIV3 in a sample.

[0028] In a specific embodiment, the primer and probe combination is used for simultaneously detecting ADV, FLUA, CPN, and MP in a sample.

[0029] In a specific embodiment, the primer and probe combination is used for simultaneously detecting Mca, Spn, Aba, and Bssp in a sample.

[0030] In a specific embodiment, the primer and probe combination is used for simultaneously detecting Lssp, Pji, and Paer in a sample.

[0031] In a specific embodiment, the primer and probe combination is used for simultaneously detecting HinB, Hin, Sau, and Kpn in a sample.

[0032] The present invention also provides a fluorescence quantitative PCR kit, which includes the above-mentioned primer and probe combination for respiratory tract infection pathogens.

[0033] In a specific embodiment, the kit further includes an enzyme mixture, a PCR buffer, and nuclease-free water.

[0034] The present invention also provides the use of the above-mentioned primer and probe combination for respiratory tract infection pathogens for preparing a reagent for detecting respiratory tract infection pathogens in a sample.

[0035] In a specific embodiment, the sample is a clinical sample of a patient with a respiratory tract infection. Preferably, the sample includes sputum, nasopharyngeal swabs, and bronchoalveolar lavage fluid.

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the specific experimental methods not specified in the following embodiments, they are generally carried out according to conventional conditions and methods, such as the methods described in the Molecular Cloning Laboratory Manual (Sambrook, et al. New York: Cold Spring Harbor Laboratory Press, 1989) or the methods provided by the reagent manufacturers. The 96-well PCR plates and fluorescence PCR instruments used in the embodiments of the present invention can be selected from the conventional commercially available models in the art.

[0037] Example 1 Design and synthesis of probes and primers

[0038] According to the conserved region sequences of respiratory tract infection pathogens, real-time fluorescence quantitative PCR primers and TaqMan probes were designed using software Primer3 and blast. The design method is as follows: For the targeted target gene, the TaqMan probe was designed using the sliding window method, with the window width set to 120 nucleotides and the step size to 1 nucleotide, and all possible probe sequences were exhausted. The following conditions were used to screen the probes: 1) The probe sequence length is 20-35 nucleotides; 2) The Tm value of the probe sequence is 70±2°C; 3) The GC content of the probe sequence is 40%-70%. After determining the TaqMan probe, primers matching it were designed using software, and the primers needed to meet the following conditions: 1) The length is 18-35 nucleotides, and the lengths of the upstream and downstream primers differ by no more than 4 nucleotides; 2) The Tm value of the primers is 60±2°C, and the Tm difference between the upstream and downstream primers is no more than 2°C; 3) The primers do not form a circular hairpin structure by themselves; 4) The primers do not form dimers by themselves; 5) The amplicon size of the upstream and downstream primers is 100-220 nucleotides. The designed multiple pairs of primers and probes were subjected to blast online alignment and matching experiments, and the primer pairs with better specificity and lower CT values (cycle threshold) were selected.

[0039] The designed primers and probes were synthesized by conventional methods, and the primer and probe sequences for amplification finally determined are as follows.

[0040]

[0041]

[0042]

[0043]

[0044] Example 2 Preparation of a Precast Detection System 96-Well PCR Plate

[0045] Dilute the synthesized primers and probes with nuclease-free water. Dilute the forward primer and reverse primer to a concentration of 10 μM, and dilute the fluorescent probe to a concentration of 5 μM. Take 1 μl each of the primers and probes and mix them separately in 8 systems, and add them to the bottom of the PCR plate wells according to the diagram. Place the PCR plate in a vacuum dryer. After drying, take out the PCR plate, stick on an aluminum foil film, and it can be stored at 4°C for 6 months and at -20°C for 1 year. The precast detection system 96-well PCR plate can detect 10 nucleic acid samples, as well as one negative control and one positive control at a time. When in use, take out the PCR plate with precast primers and probes, add a commercial one-step multiplex pre-mixed reaction solution for fluorescent PCR, and the nucleic acid template to be detected. After mixing, run the program on a fluorescent PCR instrument.

[0046] The targets in the 8 precast systems are as follows:

[0047] System FAM HEX ROX CY5 Set 1 PIV1 NL63 RSVA 229E Set 2 OC43 PIV2 HKU1 RSVB Set 3 hMPV BOV PIV4 RV Set 4 EV GAPDH FLUB PIV3 Set 5 ADV FLUA CPN MP Set 6 Mca Spn Aba Bssp Set 7 Lssp Pji - PaeR Set 8 HinB Hin Sau Kpn

[0048] The precast systems on the 96-well plate are set as follows:

[0049]

[0050] Example 3 Extraction of Nucleic Acids

[0051] Take clinical samples from patients with acute respiratory infections, such as sputum, nasopharyngeal swabs, bronchoalveolar lavage fluid, etc. Extract nucleic acids according to a conventional commercial kit (magnetic bead method or purification column method), and dissolve the obtained nucleic acids in 50 μl of eluent (such as EB buffer from Qiagen). Quantify and detect the purity of the extracted nucleic acids according to conventional methods. Aliquot the extracted nucleic acids into small portions and store them in an -80°C refrigerator.

[0052] Example 4 Detection of Respiratory Infection Viruses and Bacterial Pathogens by Fluorescent Quantitative PCR Method

[0053] 4.1 Preparation of the Fluorescent Quantitative PCR System

[0054] Take out the primers and probes from the refrigerator, dissolve them and place them on ice. Prepare the reaction system in a PCR tube:

[0055] Composition 1×25 (μl) Nuclease-free water 9.5 2×RT-PCR buffer 12.5 Primer-probe mixture - 25×RT-PCR enzyme mixture 1 Total 23

[0056] Take out the nucleic acids from the -80°C refrigerator, adjust the nucleic acid concentration, and add 2 μl (nucleic acid content 1 - 50 ng) of the nucleic acid solution to the above PCR tube.

[0057] 4.2 Fluorescent Quantitative PCR Reaction Program

[0058] Put the prepared fluorescence quantitative PCR reaction system into a fluorescence quantitative PCR instrument and execute the following program:

[0059] 50°C for 15 min, 95°C for 3 min, and then 95°C for 15 s, 60°C for 30 s + plate Read, for a total of 40 cycles.

[0060] 4.3 Result determination

[0061] Determination criteria: No amplification curve for the negative control; CT of the sample < 35 and a typical "S"-shaped amplification curve.

[0062] The results showed that respiratory infection viruses and bacterial pathogens positive samples could be specifically detected in acute respiratory infection samples, such as Figure 1A -H shows.

[0063] Example 5 Analysis of amplification efficiency of fluorescence quantitative PCR method for detecting respiratory infection pathogens

[0064] Use pure water to perform 10-fold serial dilutions of the positive plasmid samples (inserted with target gene fragments) corresponding to each pathogen, and perform qPCR amplification on each diluted concentration sample. The system configuration and procedure are the same as in Example 4. After the program runs to completion, directly calculate the amplification efficiency of the serial dilutions in a fluorescence PCR instrument such as the Cooboo q225 software.

[0065] The detection results of the amplification efficiency of each pathogen target are shown in Figure 2. The amplification efficiency of Staphylococcus aureus is 87%, and the amplification efficiencies of the other bacterial targets are all above 90%, among which the amplification efficiency of the virus target is above 98%. The overall amplification efficiency of the multiplex pathogen detection system is relatively high.

[0066] Example 6 Sensitivity analysis of fluorescence quantitative PCR method for detecting respiratory infection pathogens

[0067] Using pure water, plasmid samples of each pathogen target gene were serially diluted 2-fold in the low-concentration range (within 10E0 - 10E2 copies / Reaction), and qPCR amplification was performed on each diluted sample with 5 parallel wells for each concentration. The system configuration and procedure were the same as in Example 4. After the experiment, the amplification percentage of each concentration was calculated, and Matlab was used to fit the amplification ratio and the detected copy number, and the abscissa value when the fitted curve y = 0.95 was taken. The primer sensitivity test results of each pathogen target are shown in Figure 3. The LOD of the virus detection system varies from 1.4 - 6.8 copies / Reaction, and the LOD of the bacterial detection system varies from 1.3 - 22.3 copies / Reaction. The LOD concentration of most commercial single-nucleic acid detection kits is generally in the range of 1E2 - 1E4 copies / mL, and that of multiplex nucleic acid detection kits is generally in the range of 5E2 - 5E4 copies / mL. According to the actual operation process from sample to nucleic acid extraction to nucleic acid input for PCR detection, generally the sample volume corresponding to the finally detected nucleic acid input is 1 / 10 - 1 / 20 of the original sample volume. Assuming a sample of 1E3 / ml, it is equivalent to 50 - 100 copies / Reaction. Obviously, the LOD of each target of the virus and bacterial systems in this patent application is lower than 50 copies / Reaction, and the sensitivity is higher than that of most commercially available multiplex nucleic acid detection kits.

[0068] Example 7 Repeatability (Precision) Analysis of Fluorescent Quantitative PCR Detection Method for Respiratory Tract Infection Pathogens

[0069] For both viruses and bacteria, three types of samples were selected: medium-low level positive samples (2 - 4 times the LOD concentration); low level positive samples (1 - 2 times the LOD concentration); negative samples: pure water. 10 7 copies / reaction were used, and two batches of reagents were used to perform fluorescent quantitative PCR analysis on two Corbett Research Quantagene q225MX fluorescent quantitative PCR instruments on the same test day respectively, and the cycle threshold (CT value) was detected, and each sample was detected twice on each instrument. The average and standard deviation were calculated respectively, and the within-batch and between-batch coefficient of variation (CV) was calculated according to standard deviation / average. The within-batch and between-batch CV values obtained by detecting pathogen samples through the present invention are both less than 5%, indicating good repeatability. The detection rates of each pathogen sample are shown in Table 1, the detection results of virus pathogens are shown in Table 2, and the detection results of bacterial pathogens are shown in Table 3 (Avg: average value; SD: standard deviation; CV: coefficient of variation).

[0070] Table 1. Detection Rates of Pathogen Samples in Each System

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[0072]

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[0075] Example 8 Analysis of Clinical Samples of Respiratory Tract Infection Pathogens

[0076] Using the methods in Examples 4 and 5, clinical samples of various respiratory tract infection pathogens were detected and analyzed, and the results are shown in Table 4. An example of the measured amplification curve of the clinical sample in the virus detection system is shown in Figure 4. After the first detection of the clinical sample was determined to be positive nucleic acid using the commercially available FTD Respiratory Pathogen Screening Kit from Fast-tRack diagnostics, it was then screened for comparison using the system established in this patent, and the compliance of the positive results was highly consistent.

[0077] Table 4. Analysis Results of Clinical Samples

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[0080]

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[0082]

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[0084]

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[0086]

[0087] Example 9 Comparison of the Respiratory Virus Detection System with Other PCR Methods

[0088] Compared with the nested PCR method of the nucleic acid detection scheme for febrile respiratory syndrome viruses in the network laboratory of the National Science and Technology Major Project for Infectious Disease Surveillance Platform and the commercially available FTD Respiratory Pathogen Screening Kit from Fast-tRack diagnostics5, taking the samples that were detected positive by the nested PCR method and had correct sequencing as the positive reference, and at the same time randomly selecting 500 negative samples for analysis, the specificity, sensitivity, and result compliance rate of the detection system established in this patent all reached

[0089] 98 - 100%, achieving good detection results (Table 5, Table 6). Sensitivity = the number of positive samples detected by this kit / the number of positive samples verified by PCR sequencing × 100%, Specificity = the number of negative samples detected by this kit / the total number of negative samples detected by PCR × 100%.

[0090] Positive number / Positive number verified by PCR sequencing × 100%, Specificity = Number of negative samples detected by this kit 10 / Total number of negative samples detected by PCR × 100%.

[0091] Table 5. Comparison between the respiratory virus detection system and the ordinary nested PCR method

[0092]

[0093] Table 6. Comparison between the respiratory virus detection system and other commercially available kits

[0094]

[0095]

[0096] Note: The FTD kit is the Fast-tRack diagnostics Respiratory pathogens detection kit from Luxembourg. The Xunbida kit cannot distinguish between OC43 and NL63, 229E and HKU1.

Claims

1. A primer and probe combination for simultaneously detecting PIV1, NL63, RSVA, and 229E in a sample by real-time fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting PIV1 are shown in SEQ ID NOs: 1-3. The nucleotide sequences of the primers and probes for detecting NL63 are shown in SEQ ID NOs: 4-6. The nucleotide sequences of the primers and probes for detecting RSVA are shown in SEQ ID NOs: 7-9. The nucleotide sequences of the primers and probes for detecting 229E are shown in SEQ ID NOs: 10-12.

2. A primer and probe combination for simultaneously detecting OC43, PIV2, HKU1, and RSVB in a sample by real-time fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting OC43 are shown in SEQ ID NOs: 13-15. The nucleotide sequences of the primers and probes for detecting PIV2 are shown in SEQ ID NOs: 16-18. The nucleotide sequences of the primers and probes for detecting HKU1 are shown in SEQ ID NOs: 19-21. The nucleotide sequences of the primers and probes for detecting RSVB are shown in SEQ ID NOs: 22-24.

3. A primer and probe combination for simultaneously detecting hMPV, BOV, PIV4, and RV in a sample by real-time fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting hMPV are shown in SEQ ID NOs: 25-28. The nucleotide sequences of the primers and probes for detecting BOV are shown in SEQ ID NOs: 29-31. The nucleotide sequences of the primers and probes for detecting PIV4 are shown in SEQ ID NOs: 32-34. The nucleotide sequences of the primers and probes for detecting RV are shown in SEQ ID NOs: 35-37.

4. A primer and probe combination for simultaneously detecting EV, GAPDH, FLUB, and PIV3 in a sample by real-time fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting EV are shown in SEQ ID NOs: 38-40. The nucleotide sequences of the primers and probes for detecting GAPDH are shown in SEQ ID NOs: 41-43. The nucleotide sequences of the primers and probes for detecting FLUB are shown in SEQ ID NOs: 44-46. The nucleotide sequences of the primers and probes for detecting PIV3 are shown in SEQ ID NOs: 47-49.

5. A primer and probe combination for simultaneously detecting ADV, FLUA, CPN, and MP in a sample by real-time fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting ADV are shown in SEQ ID NOs: 50-52. The nucleotide sequences of the primers and probes for detecting FLUA are shown in SEQ ID NOs: 53-55. The nucleotide sequences of the primers and probes for detecting CPN are shown in SEQ ID NOs: 56-58. The nucleotide sequences of the primers and probes for detecting MP are shown in SEQ ID NOs: 59-61.

6. A primer and probe combination for simultaneously detecting Mca, Spn, Aba, and Bssp in a sample by fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting Mca are shown in SEQ ID NOs: 62-64, The nucleotide sequences of the primers and probes for detecting Spn are shown in SEQ ID NOs: 65-67, The nucleotide sequences of the primers and probes for detecting Aba are shown in SEQ ID NOs: 71-73, The nucleotide sequences of the primers and probes for detecting Bssp are shown in SEQ ID NOs: 68-70.

7. A primer and probe combination for simultaneously detecting Lssp, Pji, and PaeR in a sample by fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting Lssp are shown in SEQ ID NOs: 74-76, The nucleotide sequences of the primers and probes for detecting Pji are shown in SEQ ID NOs: 77-79, The nucleotide sequences of the primers and probes for detecting PaeR are shown in SEQ ID NOs: 80-82.

8. A primer and probe combination for simultaneously detecting HinB, Hin, Sau, and Kpn in a sample by fluorescence quantitative PCR method, wherein: The nucleotide sequences of the primers and probes for detecting HinB are shown in SEQ ID NOs: 83-85, The nucleotide sequences of the primers and probes for detecting Hin are shown in SEQ ID NOs: 86-88, The nucleotide sequences of the primers and probes for detecting Sau are shown in SEQ ID NOs: 89-91, The nucleotide sequences of the primers and probes for detecting Kpn are shown in SEQ ID NOs: 92-94.

9. A fluorescence quantitative PCR kit, which includes the primer and probe combination described in any one of claims 1-8.

10. The kit according to claim 9, further comprising an enzyme mixture, a PCR buffer, and nuclease-free water.

11. Use of the primer and probe combination described in any one of claims 1-8 for preparing a reagent for detecting respiratory tract infection pathogens in a sample.

12. The use according to claim 11, wherein the sample is a clinical sample of a respiratory tract infection patient.

13. The use according to claim 12, wherein the sample includes sputum, nasopharyngeal swab, and bronchoalveolar lavage fluid.

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