Method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR

Abstract

The application discloses a method for synchronously detecting multiple respiratory viruses by multiplex fluorescent quantitative PCR, and belongs to the technical field of virus detection in clinical medicine. The method is based on TaqMan multiplex fluorescent quantitative PCR technology, and a primer and probe combination specific to common respiratory viruses such as influenza virus A, influenza virus B, respiratory syncytial virus, adenovirus and rhinovirus is designed. By optimizing the components of the multiplex PCR reaction system, the annealing temperature and the cycle parameters, the non-specific binding and the superimposed interference of the fluorescence signal between different primers and probes are avoided, and an internal reference gene is introduced to calibrate the sample extraction and amplification error. The application can realize the qualitative and quantitative detection of multiple respiratory viruses in a single tube and a single PCR reaction, greatly reducing the detection time and the detection cost, and has high specificity, high sensitivity and high repeatability. The application is suitable for large-scale clinical sample screening and epidemic tracing monitoring scenes in the disease control center, effectively improves the rapid diagnosis and early warning and prevention and control efficiency of respiratory viral diseases, and has high clinical application and public health monitoring value.

Description

Technical Field

[0001] This invention relates to the fields of clinical medical testing and molecular biology virus detection technology, specifically to a method for simultaneous detection of multiple respiratory viruses using multiplex quantitative PCR, which is suitable for rapid detection of large batches of respiratory clinical samples, epidemiological monitoring, and pathogen tracing in disease control centers. Background Technology

[0002] Respiratory viral infections are a major cause of respiratory illnesses such as fever, cough, and pneumonia. They are characterized by rapid transmission, wide spread, and seasonal outbreaks, making them a key disease for public health surveillance by disease control centers. Common pathogenic respiratory viruses include influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, and rhinovirus. The clinical symptoms of these various viral infections are highly similar and cannot be distinguished by clinical symptoms alone; laboratory molecular testing is essential for diagnosis.

[0003] Currently, the mainstream testing methods in clinical and disease control fields are singleton quantitative PCR and colloidal gold rapid detection. Among them, colloidal gold detection has low sensitivity and high false positive rate, and cannot achieve quantitative detection of viruses; singleton quantitative PCR can only detect one virus at a time, and multiple amplifications and batch testing are required for batches of samples, which is cumbersome, time-consuming, and costly in terms of reagents and consumables, and cannot meet the needs of rapid screening of large numbers of samples during the peak season of respiratory viruses.

[0004] Current multiplex PCR detection technologies generally suffer from problems such as primer-probe cross-interference, overlapping fluorescence spectra, and uneven amplification efficiency, which easily lead to distorted test results and decreased sensitivity. Furthermore, the lack of a standardized internal control calibration system makes it impossible to avoid errors during sample processing and amplification, resulting in poor detection stability and repeatability, making it difficult to meet the stringent requirements of disease control systems for precise testing and epidemiological tracing. Currently, some commercially available multiplex respiratory virus detection kits are designed only for single RNA or DNA viruses, failing to simultaneously cover both RNA and DNA respiratory viruses, resulting in a significant limitation in detection coverage. This necessitates two amplification tests, still failing to completely solve the problem of low detection efficiency. Simultaneously, commercially available kits often use generic reaction systems without sequence optimization and system adaptation for respiratory virus strains prevalent in my country. When dealing with low-frequency mutant strains or clinical samples with low viral loads, insufficient amplification efficiency and false negatives are highly likely. In addition, existing technologies lack standardized sample quality control and amplification parameter adaptation schemes for batch sample testing. They are significantly affected by fluctuations in ambient temperature, reagent batches, and instrument parameters, resulting in poor repeatability of test data. This hinders disease control centers from conducting long-term epidemiological data statistics, monitoring viral mutations, and assessing epidemic trends. Conventional testing methods also generally suffer from insufficient quantitative accuracy, failing to accurately reflect the viral load level in patients. This makes it difficult to assist clinicians in judging the severity of infection, evaluating treatment effectiveness, and assessing the time to negative conversion, thus failing to meet the dual needs of public health monitoring and clinical diagnosis.

[0005] Therefore, there is an urgent need to develop a method for simultaneous detection of multiple respiratory viruses that is highly specific, highly sensitive, highly efficient, and adapted to local strains. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for simultaneous detection of multiple respiratory viruses by multiplex quantitative PCR, which solves the problems of existing technologies such as single detection type, low efficiency, large interference, poor accuracy and inability to perform batch detection, and meets the clinical testing and public health monitoring needs of disease control centers.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for simultaneous detection of multiple respiratory viruses using multiplex quantitative PCR includes the following steps: S1. Preprocessing and nucleic acid extraction of respiratory samples to be tested to obtain total nucleic acid template of the sample; S2. Configure a multiplex real-time PCR amplification reaction system, wherein the reaction system contains specific primer and probe combinations corresponding to multiple respiratory viruses, PCR buffer, dNTPs, Taq enzyme, internal control primer and probe and enzyme-free water; S3. Set up the optimized PCR amplification program and perform multiplex real-time PCR amplification of the nucleic acid template. S4. Collect real-time signals from different fluorescence channels during the amplification process, and combine them with internal reference calibration data to interpret the positive, negative, and viral load quantification results of various respiratory viruses.

[0008] Furthermore, the respiratory samples to be tested mentioned in S1 include nasopharyngeal swabs, pharyngeal swabs, sputum, and bronchoalveolar lavage fluid. After collection, the samples are placed in a virus preservation solution and transported under low temperature refrigeration. Total RNA / DNA nucleic acid extraction is completed using a magnetic bead nucleic acid extraction kit. After extraction, the nucleic acid template is stored at -20°C for later use.

[0009] Furthermore, the various respiratory viruses mentioned in S2 include five common respiratory pathogenic viruses: influenza A virus, influenza B virus, respiratory syncytial virus, human adenovirus, and human rhinovirus. The primers and probes corresponding to each virus do not have cross-homology.

[0010] Furthermore, the total volume of the multiplex real-time PCR amplification reaction system described in S2 is 20 μL, and the specific components include: 10 μL of 2×PCR premix, 2 μL of multiplex specific primer mixture, 1 μL of probe mixture, 0.5 μL of internal control primer and probe, 2 μL of nucleic acid template, and 4.5 μL of enzyme-free water.

[0011] Furthermore, in S2, each virus probe is labeled with a different fluorescent group. The influenza A virus probe is labeled with the FAM fluorescent group, the influenza B virus probe is labeled with the HEX fluorescent group, the respiratory syncytial virus probe is labeled with the ROX fluorescent group, the adenovirus probe is labeled with the CY5 fluorescent group, and the rhinovirus probe is labeled with the VIC fluorescent group. There is no spectral overlap or interference between the fluorescent channels.

[0012] Furthermore, the PCR amplification program described in S3 is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 58℃ annealing extension for 30 s, 40 cycles; each fluorescence channel signal is collected simultaneously during the annealing extension stage of each cycle.

[0013] Furthermore, the internal reference gene is the human β-actin gene, which is used to monitor the integrity of nucleic acid extraction from samples and the inhibition of PCR amplification, thereby avoiding false negative test results.

[0014] Furthermore, the result interpretation criteria in S4 are as follows: if the internal reference amplification curve is normal and the Ct value is ≤32, the sample test result is valid; if the fluorescence channel corresponding to the virus to be tested shows a typical amplification curve and the Ct value is ≤35, it is judged as positive; if there is no amplification curve or the Ct value is >35, it is judged as negative.

[0015] Furthermore, the method has a virus detection sensitivity of up to 10 copies / mL, 100% specificity for each virus primer and probe, no cross-reactivity with other respiratory pathogens, and intra- and inter-batch coefficients of variation of ≤3%.

[0016] Furthermore, the aforementioned method for simultaneous detection of multiple respiratory viruses using multiplex quantitative PCR is applied in clinical testing at disease control centers, epidemiological screening of respiratory viruses, source tracing and monitoring of viral respiratory diseases, and rapid clinical diagnosis.

[0017] The beneficial effects of this invention are as follows: 1. High detection efficiency: A single tube and a single reaction can simultaneously detect five core respiratory viruses, replacing the traditional single tube and single test mode, increasing detection efficiency by more than 5 times, significantly saving consumables and labor costs, and making it suitable for disease control and prevention batch screening scenarios.

[0018] 2. Excellent specificity and sensitivity: The primers and probes have no cross-homology, the fluorescence channels do not overlap or interfere, the detection specificity is 100%, and the limit of detection is as low as 10 copies / mL, which can accurately detect low-load latent infection samples.

[0019] 3. Accurate and stable test results: The built-in internal reference gene calibration system effectively avoids false negatives and false positives caused by sample degradation, amplification inhibition, and operational errors, and provides good repeatability within and between batches.

[0020] 4. Easy to operate and highly adaptable: The reaction system and amplification procedure are standardized, requiring no complex debugging. It is compatible with various mainstream real-time PCR instruments and can be widely used in routine respiratory virus testing and epidemiological tracing and monitoring in disease control centers and hospital laboratories. Attached Figure Description

[0021] Figure 1 This is an overall flowchart of the method for simultaneous detection of multiple respiratory viruses using multiplex real-time PCR according to the present invention; Figure 2 This is a block diagram of the multiple fluorescence detection principle and internal reference quality control system of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0023] The present invention proposes a method for simultaneous detection of multiple respiratory viruses using multiplex quantitative PCR, which includes four core steps: sample pretreatment and nucleic acid extraction, configuration of multiplex PCR reaction system, gradient-optimized PCR amplification, and fluorescence signal acquisition and result interpretation.

[0024] This invention specifically designs primers and probes for five common respiratory viruses, distinguishes detection channels by using differentiated fluorescent group labeling, optimizes the reaction system ratio and amplification temperature parameters, completely avoids the problems of non-specific binding of primers and probes and fluorescence interference, and introduces human β-actin internal reference gene to calibrate detection errors throughout the process.

[0025] Furthermore, this invention optimizes the reaction system to an optimal 20 μL. By precisely proportioning the amounts of premixed solution, primers, probes, and nucleic acid templates, the amplification efficiency of multiple viruses is balanced, avoiding the masking effect of a single virus amplification advantage on low-load virus signals, and significantly improving the sensitivity and accuracy of simultaneous detection of multiple viruses.

[0026] Furthermore, this invention optimizes the PCR amplification procedure, adopts a uniform annealing and extension temperature, adapts to the amplification requirements of all target viruses, simplifies the operation process, shortens the detection cycle, and requires only 60 minutes for a single detection, enabling rapid screening of large batches of samples.

[0027] To address the amplification imbalance caused by the coexistence of multiple primer and probe sets in multiplex PCR systems, this invention specifically optimizes and screens primer length, GC content, and Tm value for each target virus, strictly controlling the Tm value difference of all primers and probes within ±1℃. This completely avoids the problem of low amplification efficiency and weak signal of some viruses due to differences in annealing temperature adaptability. Simultaneously, this invention performs gradient optimization of the concentration ratio of multiplex primers and probes, abandoning the crude design of fixed concentration ratios in traditional commercial kits. Through multiple rounds of concentration gradient comparison experiments, the amplification efficiency of five viruses is balanced, effectively solving the technical challenge of suppressing the signal of low-abundance viruses due to the amplification advantage of high-abundance viruses, and significantly improving the detection accuracy of mixed-infection samples and low viral load samples.

[0028] In addition, this invention is adapted to the complex sample testing scenarios of disease control centers. It optimizes the amount of Taq enzyme and the concentration of buffer ions in the reaction system for PCR inhibitors such as hemoglobin, mucus, and polysaccharides commonly found in clinical samples, thereby improving the anti-interference ability of the amplification system and avoiding problems such as amplification failure and result deviation caused by sample impurities.

[0029] This invention innovatively integrates a simultaneous amplification scheme for DNA and RNA viruses. Through a one-step reverse transcription-amplification integrated system, it eliminates the need for separate processing and batch testing of RNA and DNA virus samples. A single system configuration is sufficient to simultaneously detect two different nucleic acid types of viruses, completely overcoming the shortcomings of existing technologies that cannot simultaneously detect two types of viruses. Simultaneously, this invention establishes a standardized quality control system. Through negative controls, positive controls, blank controls, and internal reference genes, it provides four-fold quality control, comprehensively monitoring the entire process of sample extraction, system configuration, and amplification reaction. This eliminates detection errors caused by experimental operations, reagents, and instruments, ensuring the authenticity, accuracy, and comparability of batch detection data. It can provide accurate, continuous, and standardized experimental data support for disease control departments in analyzing virus epidemic trends, monitoring strains, and tracing the source of outbreaks.

[0030] Compared to commercially available reagent kits that often use fixed reaction parameters and cannot be fine-tuned according to the characteristics of local circulating strains, this invention designs primers and probes based on conserved gene segments of mainstream strains such as influenza A, influenza B, and respiratory syncytial virus that have been prevalent in China in recent years. This effectively avoids primer binding failure caused by minor viral mutations and significantly improves the detection suitability of local clinical samples. At the same time, it establishes corresponding parameter calibration and batch quality control rules to address common problems in large-scale screening by disease control, such as batch processing of samples, parameter drift of instruments over long periods of operation, and batch differences of reagents. These rules can effectively offset systematic errors and ensure the continuity and comparability of monitoring data from multiple batches over long periods of time.

[0031] In addition, this invention distinguishes the amplification characteristics of single infection and multiple mixed infection samples. By optimizing the probe fluorescence signal threshold and baseline correction algorithm, it solves the problems of fluorescence baseline shift and weak signal masking when multiple viruses coexist, which greatly improves the detection accuracy of common multi-virus mixed infection cases in clinical practice and makes up for the industry shortcoming of low detection rate of mixed infection in existing multiplex PCR technology.

[0032] The present invention will be further described in detail below with reference to specific embodiments.

[0033] 1. Sample collection and preprocessing Nasopharyngeal swab samples were collected from patients suspected of having respiratory viral infections. The swabs were placed in a special virus preservation solution and transported under refrigeration at 2-8°C. Nucleic acid extraction was completed within 24 hours. A commercially available magnetic bead-based nucleic acid extraction kit was used, and the kit was operated strictly according to the instructions. Total nucleic acid was extracted from the samples, and 50 μL of nucleic acid template was obtained by elution for later use.

[0034] 2. Preparation of multiplex PCR reaction system (20μL system) This invention, through multiple rounds of gradient concentration optimization, has determined the optimal 20μL multiplex real-time PCR reaction system suitable for the simultaneous amplification of five viruses. The proportions of each group are precise, and the amplification efficiency is balanced, effectively avoiding the problem of single-virus amplification advantage suppressing low-load target signals. The specific system proportions are shown in the table below:

[0035] The entire system preparation process was performed on ice in a sterile laminar flow hood. After adding each component sequentially, the mixture was gently blown and stirred until homogenized. Low-speed, momentary centrifugation was then used to remove air bubbles from the tubes, preventing interference with fluorescence signal acquisition and amplification reaction stability. In this invention, each target virus probe is labeled with a differentiated, independent fluorescent group. There is no spectral overlap or signal crosstalk between channels. Specific fluorescence channel matching parameters are shown in the table below.

[0036] Add the following to a sterile PCR reaction tube in sequence: 10 μL of 2×TaqMan PCR premix, 2 μL of specific primer mixture for influenza A / influenza B / respiratory syncytial virus / adenovirus / rhinovirus, 1 μL of corresponding specific probe mixture, 0.5 μL of β-actin internal control primer probe, 2 μL of nucleic acid template to be tested, and 4.5 μL of enzyme-free water. Mix thoroughly, centrifuge at low speed to remove air bubbles from the tube, and avoid affecting the acquisition of fluorescence signals.

[0037] 3. PCR amplification program settings The configured PCR reaction tubes were placed in a real-time PCR instrument. After multiple rounds of optimization of annealing temperature gradients and cycling parameters, a unified standardized procedure suitable for the simultaneous amplification of five viruses was determined, completely resolving the problems of mismatched amplification parameters and unbalanced amplification efficiency for multiple targets. The specific amplification procedure parameters are shown in the table below:

[0038] This program is compatible with the Tm value characteristics of all target virus primers and probes. The uniform annealing temperature can avoid the problem of compatibility differences among multiple sets of primers and probes. The entire amplification process takes no more than 60 minutes, which greatly improves the efficiency of batch sample detection.

[0039] Place the prepared PCR reaction tubes in a real-time PCR instrument and set the amplification program: 95℃ pre-denaturation for 3 min, activate Taq enzyme and complete template pre-denaturation; then perform 40 cycles of amplification, with each cycle parameters being 95℃ denaturation for 10 s and 58℃ annealing extension for 30 s. Acquire the signals of each fluorescence channel in real time during each annealing extension stage.

[0040] 4. Result Interpretation and Data Analysis After amplification, the amplification curves and Ct values ​​for each channel were read using the PCR instrument's accompanying analysis software. First, the internal control result was determined. If the internal control amplification curve was complete and the Ct value was ≤32, the test was considered valid. If the fluorescence channel corresponding to the virus to be tested showed a typical S-shaped amplification curve and the Ct value was ≤35, the virus was considered positive; no amplification curve or a Ct value >35 indicated a negative result. Simultaneously, the viral copy number could be calculated based on the standard curve to achieve quantitative detection of viral load.

[0041] 5. Performance verification experiments and system quality control tests To fully verify the accuracy, stability, and applicability of the detection method of this invention, and to meet the requirements of disease control centers for large-scale, high-precision, and traceable testing, this invention sets up multiple control experiments, covering sensitivity, repeatability, specificity, anti-interference, and clinical sample verification. All experiments are equipped with a four-level quality control system and strictly follow the disease control testing standards.

[0042] 5.1 Sensitivity Verification Experiment Five viral standards were serially diluted 10-fold to prepare standard samples with different concentration gradients. Six replicates were set for each concentration group to verify the limit of detection and stability of this method in low-load samples. The specific detection results are shown in the table below:

[0043] Experimental results show that the lowest stable detection limit for the five target viruses in this invention can reach 10 copies / mL, with no missed detection in low-load occult infection samples, and the sensitivity is significantly better than that of existing commercial kits.

[0044] 5.2 Repeatability Verification Experiment Mixed positive samples with high, medium, and low viral loads were selected, and intra-batch and inter-batch repeatability tests were conducted. The coefficient of variation (Ct) was calculated to verify the stability of the method. The specific data are shown in the table below:

[0045] The overall coefficient of variation is ≤3%, and the detection fluctuation is minimal, meeting the data accuracy requirements for disease control virus tracing and epidemiological monitoring.

[0046] 5.3 Specificity, anti-interference and clinical validation experiments Specificity experiments were conducted using parallel detection of 20 common respiratory interfering pathogens. All interfering samples showed no specific amplification, no false positives, and no cross-reactions, demonstrating extremely strong primer and probe targeting. Anti-interference experiments simulated complex sample scenarios such as hemolysis, mucus, and residual preservation solutions. The amplification curves of the samples remained intact, and the Ct values ​​showed no significant shift, indicating strong anti-interference capabilities. Storage stability experiments confirmed that the reagents showed no performance degradation after 6 months of low-temperature storage. Large-scale clinical sample validation involved 300 suspected samples from different populations. Comparison with the national standard single-pair PCR method showed a 100% positive match rate and an increase in detection efficiency of over 70%. It is particularly suitable for detecting low-viral-load and multi-virus mixed infection samples, fully meeting the needs of disease control monitoring and tracing the source of outbreaks.

[0047] To comprehensively verify the accuracy, stability, and applicability of the detection method of this invention, and to meet the requirements of large-scale, high-precision, and traceable testing work in disease control centers, this invention sets up multiple control experiments, including specificity experiments, sensitivity gradient experiments, repeatability experiments, anti-interference experiments, storage stability experiments, and large-scale clinical sample validation experiments. All experiments employ a four-level quality control system: blank control group, negative control group, weakly positive control group, and strongly positive control group, strictly adhering to the "Clinical Molecular Biology Laboratory Technical Specifications" and the disease control pathogen screening quality control standards. Specificity experiments: Commonly encountered respiratory commensal bacteria, opportunistic pathogens, and other respiratory viruses were selected as cross-interference samples, including Mycoplasma pneumoniae, Chlamydia pneumoniae, novel coronavirus, parainfluenza virus, Bordetella pertussis, enteroviruses other than respiratory adenovirus, and Haemophilus influenzae, totaling 20 common pathogens. Parallel detection was performed using the method of this invention, with each sample group tested three times.

[0048] Experimental results showed that no specific amplification curves were observed in any of the interfering pathogen samples. Only five target respiratory viruses showed specific positive amplification, with no non-specific amplification or false positive results. This demonstrates that the primers and probes of this invention have extremely strong targeting, excellent compatibility with multiplex systems, and superior anti-interference capabilities. Sensitivity gradient experiment: Standard plasmids and standard RNA samples of the five viruses were serially diluted 10-fold to precisely prepare standard samples with concentration gradients ranging from 10 copies / mL, 50 copies / mL, 100 copies / mL, and 10-10 copies / mL. Six replicates were set for each concentration group for detection and verification. The results showed that the detection limit of this invention consistently reached 10 copies / mL for all five target viruses. The detection rate of low-concentration weakly positive samples was 100%. The amplification curves of high, medium, and low concentration samples were regular, and the Ct values ​​were stable. There were no missed detections in low-load occult infection samples. The detection sensitivity is significantly better than that of existing commercial multiplex PCR kits.

[0049] Repeatability experiment: Multi-virus mixed positive samples with high, medium and low viral loads were selected and subjected to intra-batch and inter-batch repeatability tests. Intra-batch repeatability tests were performed 20 times within the same batch, and inter-batch repeatability tests were performed over 5 consecutive working days. The standard deviation and coefficient of variation of Ct values ​​for each group were calculated. The final intra-batch coefficient of variation was ≤2.1%, and the inter-batch coefficient of variation was ≤2.8%. The overall test fluctuation was very small, and the repeatability and stability met the data accuracy requirements of disease control and source tracing monitoring.

[0050] Anti-interference experiment: Simulating the scenario of unqualified and complex clinical samples, trace amounts of common PCR inhibitors such as hemolyzed hemoglobin, sputum mucus, saliva impurities, and residual preservation solution were added to the nucleic acid samples to be tested. After mixing, the samples were tested on the instrument. The results showed that the amplification curve was smooth and complete, the Ct value did not shift significantly, there was no amplification inhibition, and there were no false negatives. It can be adapted to various samples with varying quality from primary disease control and clinical samples.

[0051] Storage stability test: The prepared multiple primer-probe mixture and reaction system reagents were stored at -20℃ for 1 month, 3 months and 6 months respectively. Samples were taken for testing. The reagent performance did not decrease and the test results were the same. The reagent has excellent stability and is suitable for long-term use in bulk by disease control.

[0052] Large-scale clinical sample validation: 300 suspected respiratory virus infection clinical samples from the annual monitoring of the CDC were randomly selected, covering different populations including children, adults, and the elderly. The method of this invention and the national standard single-pair PCR detection method were used for double-blind parallel comparison testing. The positive detection matching rate of this invention was 100%, with no missed or false detections. The single batch detection efficiency was improved by more than 70%, especially in low-viral-load infection and mixed virus infection samples, which showed significant advantages. It fully meets the stringent standards of CDC clinical testing, epidemiological monitoring, and tracing of sudden outbreaks.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneous detection of multiple respiratory viruses using multiplex real-time PCR, characterized in that, Includes the following steps: S1. Preprocessing and nucleic acid extraction of respiratory samples to be tested to obtain total nucleic acid template of the sample; S2. Configure a multiplex real-time PCR amplification reaction system, wherein the reaction system contains specific primer and probe combinations corresponding to multiple respiratory viruses, PCR buffer, dNTPs, Taq enzyme, internal control primer and probe and enzyme-free water; S3. Set up the optimized PCR amplification program and perform multiplex real-time PCR amplification of the nucleic acid template. S4. Collect real-time signals from different fluorescence channels during the amplification process, and combine them with internal reference calibration data to interpret the positive, negative, and viral load quantification results of various respiratory viruses.

2. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The respiratory samples to be tested described in S1 include nasopharyngeal swabs, pharyngeal swabs, sputum, and bronchoalveolar lavage fluid. After collection, the samples are placed in a virus preservation solution and transported under low temperature. Total RNA / DNA nucleic acid is extracted using a magnetic bead nucleic acid extraction kit. After extraction, the nucleic acid template is stored at -20°C for later use.

3. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The respiratory viruses described in S2 include five common respiratory pathogenic viruses: influenza A virus, influenza B virus, respiratory syncytial virus, human adenovirus, and human rhinovirus. The primers and probes corresponding to each virus have no cross-homology.

4. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The total volume of the multiplex real-time PCR amplification reaction system described in S2 is 20 μL, and the specific components include: 10 μL of 2×PCR premix, 2 μL of multiplex specific primer mixture, 1 μL of probe mixture, 0.5 μL of internal control primer and probe, 2 μL of nucleic acid template, and 4.5 μL of enzyme-free water.

5. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, In S2, each virus probe is labeled with a different fluorescent group. The influenza A virus probe is labeled with the FAM fluorescent group, the influenza B virus probe is labeled with the HEX fluorescent group, the respiratory syncytial virus probe is labeled with the ROX fluorescent group, the adenovirus probe is labeled with the CY5 fluorescent group, and the rhinovirus probe is labeled with the VIC fluorescent group. There is no spectral overlap or interference between the fluorescent channels.

6. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The PCR amplification program described in S3 is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 58℃ annealing and extension for 30 s, 40 cycles; each fluorescence channel signal is collected simultaneously during the annealing and extension phase of each cycle.

7. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The internal reference gene is the human β-actin gene, which is used to monitor the integrity of nucleic acid extraction from samples and the inhibition of PCR amplification, thereby avoiding false negative test results.

8. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The result interpretation criteria in S4 are as follows: if the internal reference amplification curve is normal and the Ct value is ≤32, the sample test result is valid; if the fluorescence channel corresponding to the virus to be tested shows a typical amplification curve and the Ct value is ≤35, it is judged as positive; if there is no amplification curve or the Ct value is >35, it is judged as negative.

9. The method for simultaneous detection of multiple respiratory viruses by multiplex real-time PCR according to claim 1, characterized in that, The method described above has a virus detection sensitivity of up to 10 copies / mL, 100% specificity for each virus primer and probe, no cross-reactivity with other respiratory pathogens, and intra- and inter-batch coefficients of variation of ≤3%.

10. The application of the multiplex real-time PCR method for simultaneous detection of multiple respiratory viruses according to any one of claims 1-9 in clinical testing, respiratory virus epidemiological screening, source tracing and monitoring of viral respiratory diseases, and rapid clinical diagnosis in disease control centers.

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