Primer probe group, kit and method for detecting respiratory pathogens based on fluorescent quantitative PCR (Polymerase Chain Reaction)

Abstract

The invention belongs to the technical field of pathogen detection, and particularly relates to a primer probe group, a kit and a method for detecting respiratory tract pathogens based on fluorescent quantitative PCR (Polymerase Chain Reaction). According to the invention, nine key respiratory pathogens (Flu A, Flu B, RSV, HRV, MP, ADV, PIV I, PIV II and PIV III) are creatively integrated into one detection system for detection, and the purpose of'one-time detection and comprehensive screening 'is achieved. The primer probe group and the kit have high sensitivity and specificity, the detection steps are simple, the clinical diagnosis efficiency is greatly improved, unprecedented comprehensive etiological evidence can be provided for medical staff, precise treatment is assisted, and resource waste caused by multiple times of sampling and multiple times of detection is avoided.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, and specifically relates to a primer and probe set, kit and method for detecting respiratory pathogens based on quantitative real-time PCR. Background Technology

[0002] Upper respiratory tract infection (URT) is one of the infectious diseases with the highest incidence and disease burden worldwide. It is caused by a wide variety of pathogens, including influenza virus, respiratory syncytial virus, human rhinovirus, Mycoplasma pneumoniae, adenovirus, and parainfluenza virus. These pathogens cause highly overlapping clinical symptoms, but treatment and prevention strategies differ significantly. Therefore, achieving rapid and accurate etiological differential diagnosis is crucial for improving diagnostic and treatment efficiency, promoting the rational use of antibiotics, and effectively controlling the spread of infection.

[0003] Currently, the diagnosis of the above-mentioned respiratory pathogens in clinical practice mainly relies on techniques such as rapid antigen detection, viral culture, and molecular diagnostics. However, these methods all have inherent limitations: (1) Although rapid antigen detection is simple to operate, it has low sensitivity and is prone to false negative results in the early stage of infection or when the viral load is low; (2) Viral culture is the "gold standard" with good specificity, but the process is time-consuming (usually several days to several weeks), complicated to operate, and has strict requirements on sample activity, making it difficult to meet the urgent clinical need for rapid diagnosis.

[0004] Molecular diagnostic technologies based on polymerase chain reaction (PCR), especially quantitative real-time PCR (qPCR), have become the gold standard for accurate pathogen detection due to their high sensitivity, high specificity, and rapid turnaround time. However, existing PCR detection methods in this field still face the following serious challenges: ① Limited throughput and low clinical diagnostic efficiency: Most commercially available kits can only detect a single or a small number (3-5) pathogens. Faced with similar respiratory symptoms caused by multiple pathogens, clinicians often need to request multiple tests sequentially, resulting in cumbersome procedures, long total testing cycles, large sample consumption, and high overall costs, failing to achieve comprehensive and efficient pathogen screening. ② Poor reagent stability and high storage and transportation costs and risks: Conventional liquid PCR reagents are highly sensitive to temperature and must rely on a -20°C deep cold chain system for storage and transportation. This not only significantly increases the costs at each stage of production and use, but also makes the reagents prone to inactivation during repeated freeze-thaw cycles, directly threatening the reliability of the test results. ③ The operation steps are cumbersome and the risk of cross-contamination is prominent: Traditional multiplex PCR testing requires laboratory personnel to dispense, prepare and mix multiple liquid reagents on site. The steps are cumbersome, which not only increases the operation time and labor costs, but also greatly increases the risk of aerosol contamination caused by frequent opening of the lid, which may lead to false positive results and affect the accuracy of diagnosis.

[0005] Therefore, there is an urgent need in this field for an innovative, integrated solution that can simultaneously cover multiple key respiratory pathogens and fundamentally overcome the shortcomings of existing technologies in terms of throughput, stability, and operability. Developing a solution with... High throughput, high stability, easy operation and controllable cost The lyophilized multiplex PCR reagents and kits developed are of great practical significance and application value for improving the diagnosis and treatment of respiratory infectious diseases and the public health emergency response capabilities. Summary of the Invention

[0006] The purpose of this invention is to provide a primer and probe set, kit, and method for detecting respiratory pathogens based on real-time quantitative PCR, which enables the preparation of lyophilized multiplex PCR reagents and kits with high throughput, high stability, simple operation, and controllable cost. This has significant practical significance and application value for improving the diagnosis and treatment of respiratory infectious diseases and the public health emergency response capabilities.

[0007] Therefore, the present invention provides the following technical solution.

[0008] A first aspect of the present invention provides a primer and probe set for detecting respiratory pathogens based on quantitative real-time PCR, the primer and probe set comprising at least one of the following primer and probe sets: A first primer-probe set for detecting influenza A virus, comprising an upstream primer with nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2, a downstream primer with nucleotide sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4, and a probe with nucleotide sequences as shown in SEQ ID NO. 5; A second primer and probe set for detecting influenza B virus, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, and a probe with a nucleotide sequence as shown in SEQ ID NO. 8; A third primer-probe set for detecting respiratory syncytial virus, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 9, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 10, and a probe with the nucleotide sequence shown in SEQ ID NO. 11; A fourth primer-probe set for detecting human rhinovirus, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 12, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 13, and a probe with the nucleotide sequence shown in SEQ ID NO. 14; The fifth primer-probe set for detecting Mycoplasma pneumoniae comprises an upstream primer with the nucleotide sequence shown in SEQ ID NO. 15, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 16, and a probe with the nucleotide sequence shown in SEQ ID NO. 17. A sixth primer-probe set for detecting adenovirus, comprising an upstream primer with nucleotide sequences as shown in SEQ ID NO. 18 and SEQ ID NO. 21, a downstream primer with nucleotide sequences as shown in SEQ ID NO. 19 and SEQ ID NO. 22, and probes with nucleotide sequences as shown in SEQ ID NO. 20 and SEQ ID NO. 23; The seventh primer and probe set for detecting parainfluenza virus type I comprises an upstream primer with the nucleotide sequence shown in SEQ ID NO. 24, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 25, and a probe with the nucleotide sequence shown in SEQ ID NO. 26; The eighth primer-probe set for detecting parainfluenza virus type II comprises an upstream primer with the nucleotide sequence shown in SEQ ID NO. 27, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 28, and a probe with the nucleotide sequence shown in SEQ ID NO. 29; The ninth primer and probe set for detecting parainfluenza virus type III comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 30, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 31, and a probe with a nucleotide sequence as shown in SEQ ID NO. 32.

[0009] In a preferred embodiment of the present invention, a tenth primer-probe set for detecting an internal standard is further included, the tenth primer-probe set comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 33, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 34, and a probe with a nucleotide sequence as shown in SEQ ID NO. 35.

[0010] In a preferred embodiment of the present invention, the 5' end of the probe is labeled with a fluorescent reporter group and the 3' end is labeled with a fluorescent quencher group; the fluorescent reporter group is selected from ROX, FAM, HEX, VIC or CY5, and the fluorescent quencher group is selected from TAMRA, MGB, BHQ1 or BHQ2.

[0011] A second aspect of the present invention provides a kit for detecting respiratory pathogens based on real-time quantitative PCR, the kit comprising the primer and probe set as described above.

[0012] In a preferred embodiment of the present invention, the kit further includes qPCR premix, a positive control, and a negative control.

[0013] In a preferred embodiment of the present invention, the qPCR premix comprises SuperFast Lyo Probe OneStep RT-qPCR U + Mix, freeze-drying protectant.

[0014] In a preferred embodiment of the present invention, the positive control comprises a pseudovirus containing specific fragments of FluA, FluB, RSV, HRV, MP, ADV, PIV I, PIV II, and PIV III, and a pseudovirus containing a specific fragment of the human RNaseP gene, as well as Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus is 10. 5 copies / mL.

[0015] In a preferred embodiment of the present invention, the negative control is a pseudovirus containing a specific fragment of the human RNase P gene, along with Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus is 10. 5 copies / mL.

[0016] In a preferred embodiment of the present invention, the concentration of each primer in the kit is independently 0.2 ~ 0.4 μM, and the concentration of each probe is independently 0.1 ~ 0.2 μM.

[0017] In a preferred embodiment of the present invention, the final concentration of the primers for detecting influenza A virus is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0018] In a preferred embodiment of the present invention, the final concentration of the primers used for influenza B virus detection is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0019] In a preferred embodiment of the present invention, the final concentration of the primers for respiratory syncytial virus detection is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0020] In a preferred embodiment of the present invention, the final concentration of the primers for human rhinovirus detection is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0021] In a preferred embodiment of the present invention, the final concentration of the primers used for Mycoplasma pneumoniae detection is independently 0.2 μM, and the final concentration of the probe is 0.1 μM.

[0022] In a preferred embodiment of the present invention, the final concentration of the primers used for adenovirus detection is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0023] In a preferred embodiment of the present invention, the final concentration of the primers used for detecting parainfluenza virus type I is independently 0.2 μM, and the final concentration of the probe is 0.1 μM.

[0024] In a preferred embodiment of the present invention, the final concentration of the primers used for detecting parainfluenza virus type II is independently 0.2 μM, and the final concentration of the probe is 0.1 μM.

[0025] In a preferred embodiment of the present invention, the final concentration of the primers used for detecting parainfluenza virus type III is independently 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0026] In a preferred embodiment of the present invention, the final concentration of the primers used for internal standard detection is independently 0.2 μM, and the final concentration of the probe is 0.1 μM.

[0027] In a preferred embodiment of the present invention, the kit is used for the joint detection of influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, mycoplasma pneumoniae, adenovirus, parainfluenza virus type I, parainfluenza virus type II and parainfluenza virus type III.

[0028] In a preferred embodiment of the present invention, the primer and probe set in the kit is in the form of lyophilized powder, that is, the qPCR premix and the primer and probe set are mixed and then prepared into a reaction lyophilized powder by the following lyophilization program: 0℃, 30min; -45℃, 240min; -30℃, 210min, 14bar; -10℃, 180min, 14bar; 0℃, 150min, 14bar; 30℃, 240min, 14bar.

[0029] A third aspect of the present invention provides a method for detecting respiratory pathogens for non-diagnostic purposes, comprising: 1) Extract pathogen nucleic acid from the sample; 2) Perform a real-time PCR reaction on the pathogen nucleic acid using the primer and probe set or kit described above; 3) Obtain and analyze the results.

[0030] In a preferred embodiment of the present invention, in step 2), the fluorescence quantitative PCR reaction program is as follows: reverse transcription at 55℃ for 30s, pre-denaturation at 95℃ for 10s, amplification for 40 cycles (denaturation at 95℃ for 10s, annealing extension at 58℃ for 30s fluorescence collection), wherein the FAM channel detects FluA, HRV, and PIV I, the ROX channel detects FluB, MP, and PIV II, the CY5 channel detects RSV, ADV, and PIVIII, and the VIC channel detects the internal standard.

[0031] By employing the above technical solution, the present invention has at least the following advantages: (1) This invention creatively integrates nine key respiratory pathogens (Flu A, Flu B, RSV, HRV, MP, ADV, PIV I, PIV II, PIV III) into a single detection system, achieving the goal of "one test, comprehensive screening". The primer and probe set and kit of this invention have high sensitivity and specificity, and the detection steps are simple, which greatly improves the efficiency of clinical diagnosis. It can provide medical staff with unprecedented comprehensive etiological evidence, assist in precision treatment, and avoid the waste of resources caused by multiple sampling and testing.

[0032] (2) This invention uses advanced freeze-drying technology to prepare all PCR reaction components into freeze-dried reagent powder. The resulting freeze-dried reagent can be stored stably for a long time at 2~8℃, with excellent stability, which completely reduces the dependence on cold chain logistics, greatly reduces the warehousing and logistics costs of production enterprises and the usage costs of end users, while ensuring the uniformity of reagent efficacy and the reliability of results.

[0033] (3) This invention premixes and freeze-dries all primers, probes, enzymes, and other components required for respiratory pathogen detection in the same reaction tube. The detection steps are simple; users only need to add purified nucleic acid samples for testing, significantly reducing operational steps and human error. This freeze-drying method minimizes the risk of aerosol contamination, ensuring high reliability and consistency of detection results. Through sophisticated primer and probe design and reaction system optimization, efficient and specific detection of nine targets is achieved within a limited fluorescence channel. It also ensures the accuracy and sensitivity of pathogen signal interpretation, maximizing resource utilization without sacrificing the detection performance of individual indicators while maintaining high throughput.

[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0035] Figure 1This is a physical image of the lyophilized PCR reagent prepared according to the present invention; Figure 2 The images show the PCR amplification curves of the lyophilized PCR reagent and liquid PCR reagent prepared in this invention for detecting the test samples. Detailed Implementation

[0036] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0037] Unless otherwise specified, the primers, probes, and pseudoviruses used in the embodiments of this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The qPCR premix (containing SuperFast Lyo Probe One Step RT-qPCR U) + The mix and lyophilization protectant were purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., product model: CW3378. The remaining components in the kit were selected from commercially available products of Jiangsu Kangwei Century Biotechnology Co., Ltd., or other similar commercially available products meeting the specifications can be used as substitutes.

[0038] Example 1: Primer and probe design This invention is based on the genome sequences of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus (RSV), human rhinovirus (HRV), mycoplasma pneumoniae (MP), adenovirus (ADV), parainfluenza virus type I (PIV I), parainfluenza virus type II (PIV II), and parainfluenza virus type III (PIV III) published in databases such as GISAID, NCBI, and BV-BRC over the past 10 years. It also incorporates genome sequences of other respiratory viruses (human metapneumovirus (hMPV) and coronavirus (CoV)). Specifically, it targets the M gene on the influenza A virus (Flu A) genome, the NS gene on the influenza B virus (Flu B) genome, the M gene on the respiratory syncytial virus (RSV) genome, the 5'UTR region of human rhinovirus (HRV), the P1 gene on the mycoplasma pneumoniae (MP) genome, the Hexo gene on the adenovirus (ADV) genome, and the genome sequences of parainfluenza virus type I (PIV I) and parainfluenza virus type II (PIV III). Specific primers and probes were designed for the conserved regions of the HN gene in the genomes of influenza virus type II and parainfluenza virus type III (PIV III), with the human RNase P gene used as an internal control gene. The sequences of each primer and probe are shown in Table 1 below.

[0039] Table 1 Primer and probe sequences Example 2: Preparation of lyophilized PCR reagents and kits (1) Prepare the lyophilized powder reaction solution according to the ingredients shown in Table 2, then vortex to mix well, and dispense 25 μL / tube into 8-tube arrays. Then perform lyophilization, that is: place the 8-tube array containing the reaction solution into a lyophilizer without capping, and perform lyophilization according to the following lyophilization procedure. Finally, collect and package: after lyophilization, cap the tubes in time, seal them in aluminum foil bags, and vacuum them to obtain PCR lyophilized powder.

[0040] Table 2 Freeze-dried powder formulation The reaction solution was prepared into a lyophilized reaction powder by the following lyophilization procedure: freeze control (0℃, 30 min; -45℃, 240 min); single drying (-30℃, 210 min, 14 bar; -10℃, 180 min, 14 bar; 0℃, 150 min, 14 bar); and desorption drying (30℃, 240 min, 14 bar).

[0041] (2) Preparation of positive controls: Pseudoviruses containing specific fragments of the M gene of influenza A virus, pseudoviruses containing specific fragments of the NS gene of influenza B virus, pseudoviruses containing specific fragments of the M gene of respiratory syncytial virus, pseudoviruses containing specific fragments of the 5'UTR region of human rhinovirus, pseudoviruses containing specific fragments of the P1 gene of Mycoplasma pneumoniae, pseudoviruses containing specific fragments of the Hexo gene of adenovirus, pseudoviruses containing specific fragments of the HN gene of parainfluenza virus type I, pseudoviruses containing specific fragments of the HN gene of parainfluenza virus type II, pseudoviruses containing specific fragments of the HN gene of parainfluenza virus type III, and pseudoviruses containing specific fragments of the human RNase P gene, Tris buffer, EDTA, Triton X-100, and gelatin are mixed evenly to obtain positive controls.

[0042] The concentration of each pseudovirus was 10. 5 copies / mL.

[0043] (3) Negative control: Pseudovirus containing a specific fragment of the human RNase P gene, Tris buffer, EDTA, Triton X-100, and gelatin are mixed evenly.

[0044] The concentration of each pseudovirus was 10. 5 copies / mL.

[0045] The components of the kit (48 doses / box) are shown in Table 3 below.

[0046] Table 3 Reagent Kit Components Example 3: Detection Procedure of Lyophilized PCR Reagents and Kits for Respiratory Pathogen Detection 1) Sample processing (performed in the sample processing area) Take 200 μL of the sample to be tested, positive control and negative control, and use the nucleic acid extraction or purification reagent (Su Tai Medical Device Registration No. 20200126) produced by Jiangsu Kangwei Century Biotechnology Co., Ltd. to extract the nucleic acid of the sample according to its instructions.

[0047] 2) Sample addition (performed in the sample processing area) 2.1 Preparation of reaction solution: Calculate the required number of reactions based on the number of samples to be tested. If the number of samples is n, then the total number of reactions N = n + 2. Take N tubes of lyophilized reaction powder, centrifuge for 30 seconds to ensure that the lyophilized reaction powder reaches the bottom of the tube, and set aside for later use.

[0048] 2.2 Sample addition: Gently open the cap of the eight-tube containing the lyophilized powder, and transfer 25 μL of the extracted test sample, positive control and negative control nucleic acid into the eight-tube respectively, and then tighten the cap.

[0049] 2.3 Vortex for 1 min until completely dissolved and the solution is clear with no white lyophilized residue. Remove air bubbles and centrifuge briefly for 30 seconds. Set aside for use.

[0050] 3) PCR amplification detection (performed in the amplification detection area) After adding the samples, the eight-tube strips were transferred to a real-time PCR instrument (ABI7500) for PCR amplification. The reaction program settings are shown in Table 5 below, where: the Reporter channels were selected as FAM, ROX, CY5, and VIC, the Quencher group was selected as "None", and the Passive Reference was selected as "None". The detection targets for each channel in each reaction well are shown in Table 4 below.

[0051] Table 4 Detection targets for each channel of each reaction well Table 5 PCR reaction procedure Note: Due to limitations of the ABI7500 instrument, the time cannot be set to 30 seconds. You can set it to 31 seconds or 32 seconds.

[0052] 4) Results Analysis Settings 4.1 Baseline Setting The baseline setting principle is to use the region where the fluorescence signal is stable before exponential amplification of all samples in this experiment. The setting can be adjusted according to the actual situation (generally, the starting point cycle number is 3-5 cycles, and the ending point cycle number is generally selected between 15-20 cycles), or it can be automatically interpreted by the instrument.

[0053] 4.2 Threshold Setting The target threshold should be set based on the highest point of the normal negative control curve. The internal standard threshold should be set based on the exponential amplification phase of the internal control amplification curve in the normal negative control. Thresholds may be expressed differently by different instruments; refer to the instrument software for settings.

[0054] 4.3 Quality Control Positive control: The target Ct value for the FAM, ROX, CY5 and VIC channels should be ≤38.

[0055] Negative control: No amplification curve (No Ct) or Ct value > 38 for FAM, ROX and CY5 channels, and the Ct value of the target for VIC channel should be ≤ 38.

[0056] Note: All of the above requirements must be met simultaneously in the same experiment; otherwise, the experiment will be invalid and a retest will be required.

[0057] 4.4 Positive cutoff value Clinical samples were tested, and the receiver operating characteristic (ROC) curve was used to determine that the positive cutoff value for each target and internal standard in this kit was ≤38.

[0058] 4.5 Interpretation of Test Results The sample test results are shown in Table 6 below: Table 6 Result Judgment For samples that test positive for the target, no requirement is placed on the internal standard test result; for negative samples, the internal standard test should be positive. If the internal standard test is negative, the test result of the sample is invalid, the cause should be found and eliminated, and the sample should be retested.

[0059] Example 4: Comparison of detection performance between lyophilized PCR reagents and liquid PCR reagents The lyophilized PCR reagent used in this embodiment is the lyophilized PCR reagent for respiratory pathogen detection prepared in Example 2. The control reagent used is a liquid PCR reagent (with a formula basically the same as the lyophilized PCR reagent). The positive control, negative control and normal human pharyngeal swab samples prepared in Example 2 are used as test samples, and PCR amplification reaction is performed according to the detection method described in Example 3.

[0060] The detection reaction systems are as follows: The lyophilized PCR reagent amplification reaction system is as follows: lyophilized reaction powder (prepared in Example 2) + 5 μL of the sample to be tested + 20 μL of LNase-Free Water.

[0061] Liquid PCR amplification reaction system: Prepare the liquid PCR amplification reaction system as shown in Table 7, vortex to mix, and dispense 20 μL / well. Then add 5 μL of the sample to be tested and perform PCR amplification reaction according to the detection method described in Example 3.

[0062] Table 7 Liquid PCR Amplification Reaction System Reaction lyophilized powder, such as Figure 1 As shown, the test results are as follows. Figure 2 .

[0063] like Figure 2 As shown: The lyophilized PCR reagent for detecting respiratory pathogens prepared in this invention has essentially the same detection performance as the liquid PCR reagent (with the same formulation as the lyophilized PCR reagent).

[0064] Example 5: Detection limit test of lyophilized PCR reagents and kits for respiratory pathogen detection To fully evaluate the detection limits of the lyophilized PCR reagents and kits of the present invention for influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, mycoplasma pneumoniae, adenovirus, parainfluenza virus type I, parainfluenza virus type II, and parainfluenza virus type III, this embodiment uses cultures of each target virus as templates for sensitivity detection, specifically including: First, the target virus cultures were diluted to the following concentrations: 1000 copies / mL, 750 copies / mL, 500 copies / mL, 250 copies / mL, and 150 copies / mL, as test samples, with RNase-Free Water as a template-free control. The lyophilized PCR reagents and kits for respiratory pathogen detection prepared in Example 2 were used for detection, with 20 repeated tests. The detection method was the same as in Example 3. The concentration value corresponding to a detection rate of ≥95% was selected as the limit of detection for the pathogen. The results are shown in Table 8.

[0065] Table 8 Detection results for each pathogen As shown in Table 8 above, the lyophilized PCR reagents and kits of the present invention have a detection sensitivity of 500 copies / mL for influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, mycoplasma pneumoniae, adenovirus, parainfluenza virus type I, parainfluenza virus type II and parainfluenza virus type III.

[0066] Example 6: Specificity test of lyophilized PCR reagents and kits for respiratory pathogen detection To evaluate the specificity of the lyophilized PCR reagents and kits of the present invention, this embodiment assesses the specificity of the lyophilized PCR reagents and kits by detecting other pathogens, specifically including: Using the pathogens shown in Table 9 as test samples, the lyophilized PCR reagents or kits for respiratory pathogen detection prepared in Example 2 were used for detection. The detection method was the same as in Example 3, detecting the cross-reactivity of the kit with other common respiratory pathogens during PCR amplification. The detection results are shown in Table 9 below.

[0067] Table 9 Detection results for each pathogen As shown in the table above, the lyophilized PCR reagents and kits of the present invention did not cross-react with other common respiratory pathogens, proving that the lyophilized PCR reagents and kits of the present invention have high specificity.

[0068] Example 7: Inclusivity test of lyophilized PCR reagents and kits for respiratory pathogen detection Clinical pharyngeal swab samples and viral strains from different regions were used as test samples, including 12 cases of influenza A virus, 6 cases of influenza B virus, 6 cases of respiratory syncytial virus, 9 cases of human rhinovirus, 6 cases of mycoplasma pneumoniae, 21 cases of adenovirus, 3 cases of parainfluenza virus type I, 3 cases of parainfluenza virus type II, and 3 cases of parainfluenza virus type III. Limit of detection and repeatability validation were performed, specifically including: Each target pathogen was diluted to 500 copies / mL as the test sample. The lyophilized PCR reagent and kit for respiratory pathogen detection prepared in Example 2 were used for detection, with 20 repeated tests. The detection method was the same as in Example 3 to verify the kit's coverage at the limit of detection concentration. CV was calculated to verify its repeatability. The results are shown in Table 10.

[0069] Table 10 Detection results for each pathogen As shown in Table 10 above, by using the lyophilized PCR reagent and kit of the present invention to repeatedly test for influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, mycoplasma pneumoniae, adenovirus, parainfluenza virus type I, parainfluenza virus type II, and parainfluenza virus type III at the detection limit concentration 20 times, at least 19 were positive, indicating that the kit has good tolerance to the detection limit concentration; at the same time, the calculated CV values ​​were all less than 5%, indicating that the kit of the present invention also has good tolerance to nine pathogens from different regions.

[0070] Example 8: Application of lyophilized PCR reagents and kits for respiratory pathogen detection in clinical sample testing The reagents used in this embodiment are the lyophilized PCR reagents and kits for respiratory pathogen detection prepared in Example 2. The samples to be tested consist of 10 pharyngeal swab samples each of influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, mycoplasma pneumoniae, adenovirus, parainfluenza virus type I, parainfluenza virus type II, and parainfluenza virus type III, and 20 negative pharyngeal swab samples remaining from the hospital's laboratory. The detection method is the same as in Example 3, and the detection results are shown in Table 11 below.

[0071] Table 11 Detection results of clinical samples Note: Un. means Undetermined.

[0072] According to the test results in Table 11, the lyophilized PCR reagents and kits of the present invention are consistent with the standard diagnostic results of hospitals.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A primer and probe set for detecting respiratory pathogens based on real-time quantitative PCR, characterized in that, The primer-probe set includes at least one of the following primer-probe sets: A first primer-probe set for detecting influenza A virus, comprising an upstream primer with nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2, a downstream primer with nucleotide sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4, and a probe with nucleotide sequences as shown in SEQ ID NO. 5; A second primer and probe set for detecting influenza B virus, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, and a probe with a nucleotide sequence as shown in SEQ ID NO. 8; A third primer-probe set for detecting respiratory syncytial virus, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 9, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 10, and a probe with the nucleotide sequence shown in SEQ ID NO. 11; A fourth primer-probe set for detecting human rhinovirus, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 12, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 13, and a probe with the nucleotide sequence shown in SEQ ID NO. 14; The fifth primer-probe set for detecting Mycoplasma pneumoniae comprises 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. 17; A sixth primer-probe set for detecting adenovirus, comprising an upstream primer with nucleotide sequences as shown in SEQ ID NO. 18 and SEQ ID NO. 21, a downstream primer with nucleotide sequences as shown in SEQ ID NO. 19 and SEQ ID NO. 22, and probes with nucleotide sequences as shown in SEQ ID NO. 20 and SEQ ID NO. 23; The seventh primer and probe set for detecting parainfluenza virus type I comprises an upstream primer with the nucleotide sequence shown in SEQ ID NO. 24, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 25, and a probe with the nucleotide sequence shown in SEQ ID NO. 26; The eighth primer-probe set for detecting parainfluenza virus type II comprises an upstream primer with the nucleotide sequence shown in SEQ ID NO. 27, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 28, and a probe with the nucleotide sequence shown in SEQ ID NO. 29; The ninth primer and probe set for detecting parainfluenza virus type III comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 30, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 31, and a probe with a nucleotide sequence as shown in SEQ ID NO.

32.

2. The primer-probe set according to claim 1, characterized in that, It also includes a tenth primer-probe set for detecting the internal standard, the tenth primer-probe set comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO. 33, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 34, and a probe with the nucleotide sequence shown in SEQ ID NO.

35.

3. The primer-probe set according to claim 1 or 2, characterized in that, The probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; the fluorescent reporter group is selected from ROX, FAM, HEX, VIC or CY5, and the fluorescent quencher group is selected from TAMRA, MGB, BHQ1 or BHQ2.

4. A kit for detecting respiratory pathogens based on real-time quantitative PCR, characterized in that, The kit comprises the primer and probe set as described in any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes qPCR premix, positive control, and negative control.

6. The reagent kit according to claim 5, characterized in that, The qPCR premix includes SuperFast LyoProbe One Step RT-qPCR U + Mix, freeze-drying protectant.

7. The reagent kit according to claim 5, characterized in that, The positive controls consisted of pseudoviruses containing specific fragments of FluA, FluB, RSV, HRV, MP, ADV, PIV I, PIV II, and PIV III, as well as pseudoviruses containing specific fragments of the human RNaseP gene, along with Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudoviruses was 10. 5 copies / mL; The negative control consisted of a pseudovirus containing a specific fragment of the human RNase P gene, as well as Tris buffer, EDTA, Triton X-100, and gelatin; the concentration of the pseudovirus was 10. 5 copies / mL.

8. The reagent kit according to claim 4, characterized in that, The concentration of each primer in the kit is independently 0.2 ~ 0.4 μM, and the concentration of each probe is independently 0.1 ~ 0.2 μM.

9. A method for detecting respiratory pathogens for non-diagnostic purposes, characterized in that, include: 1) Extract pathogen nucleic acid from the sample; 2) Perform a real-time PCR reaction on the nucleic acid of the pathogen using the primer and probe set according to any one of claims 1-3 or the kit according to any one of claims 4-8; 3) Obtain and analyze the results.

10. The detection method according to claim 9, characterized in that, In step 2), the fluorescence quantitative PCR reaction program is as follows: reverse transcription at 55℃ for 30s, pre-denaturation at 95℃ for 10s, and amplification for 40 cycles (denaturation at 95℃ for 10s, annealing and extension at 58℃ for 30s fluorescence collection).

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