Fluorescent PCR kit and method for detecting respiratory virus

Abstract

The present application relates to the field of molecular biology detection, in particular to a fluorescent PCR kit and a detection method for respiratory virus detection. The present application constructs specific primer groups and probes for detecting respiratory viruses, including influenza A virus, influenza B virus, human parainfluenza virus type I, human parainfluenza virus type II, human parainfluenza virus type III, respiratory syncytial virus and metapneumovirus. Through a matching PCR system, the simultaneous detection of multiple respiratory pathogens is realized in a single-color detection channel by using the melting curve and the characteristic peak of the melting temperature as indicators. The present application at least doubles the existing fluorescent PCR detection throughput, has low cost, short time consumption, greatly improves the detection efficiency, and can be widely used for mixed detection and analysis of multiple pathogens. The detection results can be further used for assisting clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biochemical detection, and in particular to a fluorescent PCR kit and a detection method for respiratory virus detection. Background Art

[0002] Respiratory tract infections (RTIs) are the most common human illness, occurring in all genders, ages, and geographic regions. They are one of the leading causes of morbidity and mortality worldwide. The clinical symptoms and signs of RTIs are relatively similar, primarily manifesting as rhinitis, pharyngitis, laryngitis, and tonsillitis. Severe cases can cause tracheitis, bronchitis, and pneumonia. However, treatment, efficacy, and course of disease vary depending on the infection caused by the different pathogens. It has been demonstrated that the majority of respiratory diseases are caused by pathogens other than bacteria, with respiratory viruses being the most common. Common respiratory viruses include influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, adenovirus, respiratory enterovirus, coronavirus, and bocavirus.

[0003] Traditional testing often requires a blood draw, but routine clinical biochemical analysis cannot accurately identify the pathogen causing the infection. Most clinical treatments remain empirical and often lack the necessary context. The gold standard for respiratory virus detection is isolation and culture, which typically requires a long time and rigorous laboratory environment. Furthermore, some highly contagious and pathogenic respiratory viruses, such as Zika and human metapneumovirus, cannot be cultured in general hospitals and require specialized facilities. This method, with its long lead times and demanding environmental requirements, is not only labor-intensive and resource-intensive, but can also delay treatment for patients with severe respiratory infections. Therefore, a rapid, sensitive, and accurate respiratory virus detection method is urgently needed to provide an effective tool for rapid detection and targeted treatment. While several test kits for respiratory pathogens are currently available on the market, these primarily rely on single-target or multi-target testing, unable to detect multiple pathogens simultaneously. Detecting multiple targets requires multiple tests, which is time-consuming, with inconsistent testing procedures and complex procedures.

[0004] At present, the mainstream technology used in the field of molecular in vitro detection is real-time fluorescence PCR technology. However, with the development of the detection industry, the detection of multiple genes and multiple pathogens has become more and more urgent. Faced with the increasingly complex multi-target detection needs of complex pathogen infections, multiplex PCR amplification has become one of the most ideal and fastest molecular detection methods due to its high throughput, high efficiency, low cost and low time consumption. However, when conducting multiplex PCR amplification, experiments also have to face new problems. For example, the existing commonly used fluorescence quantitative instruments have 4 to 5 detection channels, and the number of target genes detected in a single tube is generally 4 to 5. At the same time, with the increase in the number of primer probes in the system, higher requirements are placed on the design of primer probes. These factors have greatly affected the application and promotion of multiplex real-time quantitative PCR detection technology in the detection field. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a fluorescent PCR kit and a detection method for respiratory virus detection.

[0006] The present invention provides a primer-probe combination comprising:

[0007] A probe A and a primer set for detecting influenza A virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2; and / or

[0008] A probe B and a primer set for detecting influenza B virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; and / or

[0009] A probe C and a primer set for detecting respiratory syncytial virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 6; and / or

[0010] A probe D and a primer set for detecting parainfluenza virus type 1, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 8; and / or

[0011] A probe E and a primer set for detecting parainfluenza virus type 2, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 10; and / or

[0012] A probe F and a primer set for detecting parainfluenza virus type 3, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 12; and / or

[0013] A probe G and primer set for detecting human metapneumovirus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 14.

[0014] Furthermore, the primer-probe combination of the present invention also includes a probe H for detecting an internal standard and a primer set, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 22, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 23.

[0015] In the primer-probe combination of the present invention,

[0016] The 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group;

[0017] The fluorescent reporter group is selected from any one of FAM, HEX, ROX or CY5, and the fluorescent quencher group is selected from any one of BHQ1 or BHQ2;

[0018] Among the probes A to H, any two of them form a group, and the 5'-end fluorescent reporter groups of the two probes in the same group are the same. In some specific embodiments of the present invention, the 5'-end fluorescent reporter group and 3'-end fluorescent quencher group of the probes for detecting influenza A virus and influenza B virus are FAM and BHQ1, respectively; the 5'-end fluorescent reporter group and 3'-end fluorescent quencher group of the probes for detecting respiratory syncytial virus and parainfluenza virus type 1 are ROX and BHQ2, respectively; the 5'-end fluorescent reporter group and 3'-end fluorescent quencher group of the probes for detecting parainfluenza virus type 2 and parainfluenza virus type 3 are HEX and BHQ2, respectively; the fluorescent reporter group and fluorescent quencher group of the probes for detecting human metapneumovirus and internal standard are CY5 and BHQ2, respectively.

[0019] Furthermore, of the two probes in the same group of probes A to H, the TM of any one is 60-75° C., and the Tm value of the other probe is 0-30° C. lower than the Tm value of the primer set for detecting the same respiratory pathogen.

[0020] In the present application, two pathogenic bacteria are detected simultaneously in a single light channel, and the Tm values of the probes for detecting the two pathogenic bacteria are different, one of which has a higher Tm value, preferably a Tm value of 60-75℃, and the other has a Tm value that is 0-30℃ lower than that of the primer set for detecting the same respiratory pathogenic bacteria. The primer sets and probes for detecting different pathogenic bacteria in a single light detection channel have been tried many times in the present application, and in the comparative examples of the present application, the primer sets and probes for the pathogenic bacteria in the same detection channel are also set according to this requirement. The results show that a successful multiplex detection system cannot be obtained simply by combining single target point primers and probes.

[0021] In the present application, the nucleotide sequence of probe A is as shown in SEQ ID NO: 15.

[0022] The nucleotide sequence of probe B is as shown in SEQ ID NO: 16.

[0023] The nucleotide sequence of probe C is as shown in SEQ ID NO: 17.

[0024] The nucleotide sequence of probe D is as shown in SEQ ID NO: 18.

[0025] The nucleotide sequence of probe E is as shown in SEQ ID NO: 19.

[0026] The nucleotide sequence of probe F is as shown in SEQ ID NO: 20.

[0027] The nucleotide sequence of probe G is as shown in SEQ ID NO: 21.

[0028] The nucleotide sequence of probe H is as shown in SEQ ID NO: 24.

[0029] The nucleotide sequence of probe H is as shown in SEQ ID NO: 24.

[0030] The present application provides the use of the primer probe combination in the preparation of a kit for detecting respiratory pathogenic bacteria.

[0031] The present application provides a kit for detecting respiratory pathogenic bacteria, which comprises the primer probe combination of the present application.

[0032] Further, the kit further comprises at least one of a PCR reaction buffer, a Mn 2+ solution, a dNTPs solution and a rTth polymerase.

[0033] In the present application, the nucleotide sequence of probe A is as shown in SEQ ID NO: 15.

[0034] The components of the PCR reaction buffer include 100mM Tricine, 200mM KOAC and 0.1‰-1‰ (volume fraction) NaN3.

[0035] The Mn 2+ The solvent of the solution is water, in which Mn 2+ The concentration used was 3 mM;

[0036] The solvent of the dNTPs solution is water, in which the concentration of each dNTP is 0.2 mM;

[0037] The concentration of rTth polymerase used was 15 U / 80 μL.

[0038] The present invention provides a method for detecting respiratory pathogens, which uses the primer-probe combination of the present invention or the kit of the present invention to detect respiratory pathogens in a sample.

[0039] Furthermore, the detection method comprises the following steps: performing real-time fluorescence PCR amplification after obtaining the template, detecting the fluorescence signal during the amplification process, and obtaining the detection result.

[0040] The template in the steps of the detection method can be purified RNA, mixed RNA, or isolated and extracted RNA, which is not limited in the present invention; in some specific embodiments of the present invention, the template is isolated and extracted RNA, which is obtained by magnetic bead extraction.

[0041] The present invention obtains the detection result by detecting the fluorescent signal in the real-time fluorescent PCR amplification process.

[0042] In some specific embodiments of the present invention, a monochromatic light channel can simultaneously detect two pathogens. During detection, a fluorescent probe is used to detect one target in the monochromatic fluorescence channel, and the Ct value, which is the number of cycles required for the fluorescence signal to reach the set threshold, is used as the criterion for determining whether the test is positive or negative. A Ct value of less than or equal to 40 is positive, and a Ct value greater than 40 is negative. The detection of another target is carried out using a melting curve method, and the presence or absence of a characteristic peak at Tm by the melting curve method is used as the criterion for determining whether the test is positive or negative. If there is a characteristic peak at a specific Tm temperature, it is positive; otherwise, it is negative.

[0043] The detection method described in the present invention solves the bottleneck problem that fluorescent PCR detection is limited by the detection channel. The above-mentioned real-time fluorescent quantitative RT-PCR detection system, kit and detection method for jointly detecting multiple respiratory viruses are based on two technical principles: rTth enzyme hydrolyzes the fluorescent probe to produce a fluorescent signal; and the low Tm value probe does not bind to the target sequence in the amplification stage, thereby avoiding hydrolysis by the rTth enzyme, and the low Tm value fluorescent probe hybridizes with the amplified product to produce a fluorescent signal in the dissolution stage. A fluorescent probe is used for the detection of one target in a monochrome fluorescent channel, and a low Tm value fluorescent probe and the melting curve of the amplified product are used for the detection of another target, thereby realizing the simultaneous detection and analysis of two targets in a monochrome fluorescent channel, thereby enabling the joint detection of multiple respiratory viruses, and doubling the existing fluorescent PCR detection throughput, and can detect multiple pathogens at a time, which is beneficial to solving the problem that the pathogen test kits on the market currently have a single detection target and cannot obtain information on multiple targets at the same time.

[0044] Furthermore, the detection methods described herein include both diagnostic and non-diagnostic detection methods, which are not limited in the present invention. Non-diagnostic detection methods include testing of environmental samples or testing for scientific research purposes, where the environmental samples include, but are not limited to, food, drinking water, domestic water, domestic wastewater, or surface swabs. Diagnostic detection methods include testing of human or animal bodies or in vitro samples thereof.

[0045] The present invention is based on the rTth enzyme hydrolysis fluorescent probe to produce fluorescent signal and low Tm value probe does not bind to the target sequence in the amplification stage, thereby avoiding the hydrolysis of rTth enzyme, and in the dissolution stage low Tm value fluorescent probe hybridizes with the amplified product to produce two technical principles of fluorescent signal, targeted to the monochromatic light detection channel of the pathogen primer probe combination is differentially designed, the test result does not have mutual interference, while the monochromatic light probe TM value difference is large, the low Tm probe melting curve method adopts the nucleic acid hybridization method, reduces the difficulty of probe design, especially for the sequence with low GC content of the target gene, broadens the scope of application of PCR fluorescent probe method. And the specially designed primer set and probe supporting PCR detection reagent and program together, complete the monochromatic light channel to detect two pathogens simultaneously, further improve the detection flux, greatly improve the detection efficiency, save manpower and material resources. In addition, the use of multiple fluorescent PCR amplification detection technology compared with traditional culture and immunological detection has higher sensitivity, avoids cross reaction, shortens the detection window period and effectively improves sample turnover efficiency.

[0046] In the present invention, the sample includes but is not limited to at least one of a throat swab, sputum, alveolar lavage fluid or blood.

[0047] Furthermore, when developing a real-time multiplex PCR detection platform, the primer-probe combination, kit, and detection method are compatible with the hardware facilities of common quantitative PCR instruments available on the market, thereby solving the bottleneck problem of multiple detection channels of existing fluorescent quantitative PCR instruments and achieving the purpose of multiplex real-time quantitative PCR detection.

[0048] The present invention constructs specific primer sets and probes for detecting respiratory viruses including influenza A virus, influenza B virus, human parainfluenza virus type I, human parainfluenza virus type II, human parainfluenza virus type III, respiratory syncytial virus, and metapneumovirus. Through a matching PCR system, a single-color detection channel is used to simultaneously detect multiple respiratory pathogens using melting curves and melting temperature characteristic peaks as indicators, thereby increasing the existing fluorescence PCR detection throughput by at least one-fold. The method is low in cost, saves time, greatly improves detection efficiency, and can be widely used for mixed detection and analysis of multiple pathogens. The detection results can be further used to assist clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a positive test result of influenza A virus in the FAM channel;

[0050] Figure 2 A positive test result for respiratory syncytial virus in the ROX channel;

[0051] Figure 3 A positive test result for parainfluenza virus type 2 in the HEX channel;

[0052] Figure 4 This is the internal standard detection result of CY5 channel;

[0053] Figure 5 It is a positive test result for influenza B virus in the FAM channel;

[0054] Figure 6 This is the result of parainfluenza virus type 1 detection in the ROX channel;

[0055] Figure 7 A positive test result for parainfluenza virus type 3 in the HEX channel;

[0056] Figure 8 This is the human metapneumovirus detection result of the CY5 channel;

[0057] Figure 9 Negative sample test results in all channels;

[0058] Figure 10 is the detection result of FAM influenza A virus in sensitivity analysis;

[0059] Figure 11Detection result of respiratory syncytial virus in ROX channel for sensitivity analysis;

[0060] Figure 12 Detection result of parainfluenza virus 2 in HEX channel for sensitivity analysis;

[0061] Figure 13 Detection result of influenza B virus in FAM channel for sensitivity analysis;

[0062] Figure 14 Detection result of parainfluenza virus 1 in ROX channel for sensitivity analysis;

[0063] Figure 15 Detection result of parainfluenza virus 3 in HEX channel for sensitivity analysis;

[0064] Figure 16 Detection result of human metapneumovirus in CY5 channel for sensitivity analysis;

[0065] Figure 17 Ct value is about 25 when the primer probe combination of the application is used for detection of influenza A virus;

[0066] Figure 18 Ct value is about 29 when the primer probe combination of the comparative example is used for detection of influenza A virus;

[0067] Figure 19 No characteristic peak when the primer probe combination of the comparative example is used for detection of influenza B virus;

[0068] Figure 20 Ct value is about 25 when the primer probe combination of the application is used for detection of respiratory syncytial virus;

[0069] Figure 21 Ct value is about 32 when the primer probe combination of the comparative example is used for detection of respiratory syncytial virus;

[0070] Figure 22 No characteristic peak when the primer probe combination of the comparative example is used for detection of parainfluenza virus 1;

[0071] Figure 23 Ct value is about 24 when the primer probe combination of the application is used for detection of parainfluenza virus 2;

[0072] Figure 24 Ct value is about 33 when the primer probe combination of the comparative example is used for detection of parainfluenza virus 2;

[0073] Figure 25 No characteristic peak when the primer probe combination of the comparative example is used for detection of parainfluenza virus 3;

[0074] Figure 26When the comparative primer-probe combination was used to detect human metapneumovirus, the characteristic peaks were not obvious;

[0075] Figure 27 When the primer-probe combination of the present invention is used as an internal standard for detection, the Ct value is around 24;

[0076] Figure 28 When the comparative primer-probe combination was used as the internal standard for detection, the Ct value was around 32; DETAILED DESCRIPTION

[0077] The present invention provides a fluorescent PCR kit and detection method for respiratory virus detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0078] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0079] The present invention will be further described below in conjunction with the embodiments:

[0080] Example 1 Preparation of the kit

[0081] 1. Primer and probe design

[0082] The kit provided in this embodiment includes a fluorescent PCR detection system with the following sequences: SEQ ID NO. 1 to SEQ ID NO. 24, as shown in Table 1 below. This kit can specifically detect a variety of respiratory viruses, including influenza A virus (IFV A), influenza B virus (IFV B), respiratory syncytial virus (RSV), parainfluenza virus type 1 (PIV1), parainfluenza virus type 2 (PIV2), parainfluenza virus type 3 (PIV3), and human metapneumovirus (hMPV), with high specificity, convenient detection, and a wide range of clinical applications.

[0083] Table 1. Primers

[0084]

[0085] RNase PF, RNase PR, and RNase PP were used as internal standard primer probes.

[0086] It is understood that in other embodiments, the fluorescent PCR detection system in the kit may include a combination of any two or more of influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, and human metapneumovirus, as long as the two detection targets in the same fluorescent channel correspond.

[0087] 2. Experimental steps

[0088] 1. Extraction of sample RNA

[0089] In this embodiment, the detection sample source can be a throat swab, a nasopharyngeal swab, etc., and RNA is extracted using a magnetic bead method. The following operations are performed in the sample processing room:

[0090] 1.1 Take an appropriate amount of 1.5 mL sterile centrifuge tubes, mark the negative control (nuclease-free water) and the sample to be tested respectively, add 30 μL of proteinase K, 600 μL of negative control and sample, 100 μL of magnetic bead suspension (mix the magnetic beads before aspiration), and 1.2 mL of lysate to the 1.5 mL sterile centrifuge tube, mix well, and incubate in a 37°C incubator for 2 minutes;

[0091] 1.2 After incubation, magnetically aspirate the mixed solution for 1 min 30 s and discard the waste liquid;

[0092] 1.3 Demagnetize, add 2 mL of washing solution A, mix well, magnetically absorb for 1 min 30 s, and discard the waste liquid;

[0093] 1.4 Demagnetize by adding 2 mL of washing solution B, mixing, magnetically aspirating for 1 min 30 s, and discarding the waste liquid (minimize the residual amount);

[0094] 1.5 Add 100-300 μL (according to the requirements of subsequent experiments) of eluent, mix well, and dissociate in an 80°C dry-bed thermostat for 5 min;

[0095] 1.6 After 2 minutes of magnetic attraction, the extracted or purified product was used for subsequent experiments.

[0096] 2. Real-time fluorescence PCR amplification

[0097] Take a 0.2mL PCR tube, add 30μL PCR reaction solution and 50μL of the above elution solution, cover the tube, and transfer it to the amplification detection area.

[0098] The reaction system for real-time fluorescence PCR amplification provided in this embodiment is shown in Table 2 below, and the real-time fluorescence quantitative RT-PCR amplification procedure is shown in Table 3 below.

[0099] Table 2. Reaction system for real-time fluorescence PCR amplification

[0100] Component name Volume or concentration in each reaction <![CDATA[Mn 2+ ]]> 4mM dNTPs (100 mM) 0.2mM Tth enzyme (5 U / μl) 15U SEQ ID NO: 1-14 200nM SEQ ID NO: 15-21 100nM SEQ ID NO: 22-23 100nM SEQ ID NO:24 50nM Template amount 50 μl PCR buffer (1×) Up to 80μL

[0101] Table 3. Real-time fluorescence quantitative RT-PCR amplification program

[0102]

[0103] 3. Results Analysis

[0104] The principle of the TaqMan probe-based fluorescent quantitative PCR used in this example is as follows: During PCR amplification, a pair of primers and a specific fluorescent detection probe are simultaneously added. The probe is an oligonucleotide labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end. When the probe is intact, the 5'-end fluorescent group absorbs energy and transfers it to the adjacent 3'-end fluorescent quencher group (fluorescence resonance energy transfer, FRET), making the fluorescence emitted by the 5'-end fluorescent reporter group undetectable. However, during PCR amplification, during annealing of the PCR reaction, the 5'-3' exonuclease activity of Taq DNA polymerase cleaves the fluorescent reporter group attached to the 5' end of the probe, freeing it from the shielding of the 3'-end fluorescent quencher group and allowing it to emit a fluorescent signal upon light stimulation. That is, a fluorescent molecule is formed for each DNA strand amplified, achieving complete synchronization of fluorescent signal accumulation and PCR product formation.

[0105] The principle of the fluorescence melting curve method is: first, a large number of target sequences are generated by PCR amplification. Since the Tm value of the probe used in the melting curve is 5 to 30°C lower than the Tm value of the probe in the same channel, it cannot bind to the template during the PCR reaction stage, so no fluorescent signal and Ct value are generated. In the melting curve reaction stage, the target sequence generated hybridizes with the probe, and the hybridization product has a specific melting point. When the temperature is lower than the designed Tm value of the amplification product, the amplification product is in a double-stranded DNA state that hybridizes with each other in the system. At this time, the probe is in a free state. At this time, due to the molecular flexibility of the oligonucleotide, the fluorescent reporter group and the fluorescent quencher group are The physical positions of the groups are close, and the fluorescence emitted by the fluorescent reporter group will be quenched by the fluorescent quenching group, and the fluorescent signal detected by the system will be weakened accordingly; when the temperature is higher than the designed Tm value of the amplification product, the amplification product exists in the system as a single-stranded DNA. At this time, the fluorescent probe binds to the single-stranded template, and the fluorescent group of the probe is far away from the quenching group. Therefore, the fluorescence generated by the fluorescent reporter group cannot be quenched by the fluorescent quenching group, and the system can detect a stronger fluorescent signal. Therefore, by monitoring the changes in the intensity of the fluorescent signal, it is possible to detect whether the target sequence is amplified, and then it is possible to detect whether the target pathogen is present.

[0106] The technical principle of this embodiment is that one target in a channel uses the TaqMan probe method to determine the positive or negative detection by the Ct value, while the other target in the same channel uses the melting curve analysis method to determine the positive or negative detection by whether there is a characteristic peak of the melting curve.

[0107] Determine the baseline and threshold for each channel: The baseline is generally set at 3 to 15 cycles, which can be adjusted according to actual conditions. Threshold setting: The threshold line should just exceed the highest point of the normal negative control.

[0108] First, analyze whether the internal reference detects the amplification curve in the HEX (VIC) channel and whether Ct ≤ 35. If so, it means that this test is valid and subsequent analysis can continue.

[0109] 1) If the FAM channel detects an amplification curve and Ct ≤ 40, it indicates that the influenza B virus test result is positive; if the FAM channel detects a Tm (49-51°C) characteristic peak, it indicates that the influenza A virus test result is positive;

[0110] 2) If the ROX channel detects an amplification curve and Ct ≤ 40, it indicates that the respiratory syncytial virus test result is positive; if the ROX channel detects a Tm (56-58°C) characteristic peak, it indicates that the parainfluenza virus type 1 test result is positive;

[0111] 3) If the HEX (VIC) channel detects an amplification curve and Ct ≤ 40, it indicates that the test result for human parainfluenza virus type 2 is positive; if the HEX (VIC) channel detects a Tm (50-52°C) characteristic peak, it indicates that the test result for human parainfluenza virus type 3 is positive;

[0112] 4) If the CY5 channel detects a characteristic peak at Tm (50-52°C), it indicates that the human metapneumovirus test result is positive;

[0113] 5) If an amplification curve is detected in the CY5 channel and Ct>35, the internal standard test result is invalid, indicating that the sample concentration is too low or there are interfering substances that inhibit the reaction or there is a problem with sample collection, and retesting is required.

[0114] 2. Results

[0115] Using the positive pseudovirus of each target as a template to simulate the experimental sample, multiple PCR detection was performed on a Macrostone fluorescent quantitative PCR instrument (model: SLAN-96P). The results are as follows Figures 1 to 8 As shown, Figure 1 It is a positive test result of influenza A virus in the FAM channel; Figure 2 A positive test result for respiratory syncytial virus in the ROX channel; Figure 3 A positive test result for parainfluenza virus type 2 in the HEX channel; Figure 4 This is the internal standard detection result of CY5 channel; Figure 5 It is a positive test result for influenza B virus in the FAM channel; Figure 6This is the result of parainfluenza virus type 1 detection in the ROX channel; Figure 7 A positive test result for parainfluenza virus type 3 in the HEX channel; Figure 8 This is the human metapneumovirus detection result of the CY5 channel.

[0116] Example 2 Detection sensitivity, accuracy and specificity of the kit

[0117] (1) Detection sensitivity of the kit of the present invention

[0118] For 7 pathogens, samples of different concentrations were set up and nucleic acid amplification was performed using the real-time fluorescence quantitative RT-PCR method through this detection kit.

[0119] Extract nucleic acids from each pathogen, determine and calculate the nucleic acid template concentration, and dilute to 5×10 4 copies / mL, as the initial concentration, and a 10-fold serial dilution was performed to obtain 5×10 3 copies / mL, 5×10 2 A total of 4 concentration gradients were set, including 50 copies / mL, 50 copies / mL, and 5 copies / mL. These samples were used as reaction templates and real-time fluorescence quantitative RT-PCR amplification was performed according to the sample addition method of the kit. The test results of the kit are shown in Table 4 and Figures 9 to 16 As shown:

[0120] Table 4. Sensitivity test of various pathogens

[0121]

[0122] The results showed that the primer-probe combination designed by the present invention has strong sensitivity. The detection sensitivity of the primer probe for influenza A virus reached 50 copies / mL, the detection sensitivity of the primer probe for influenza B virus reached 50 copies / mL, the detection sensitivity of the primer probe for respiratory syncytial virus reached 50 copies / mL, the detection sensitivity of the primer probe for parainfluenza virus type 1 reached 50 copies / mL, the detection sensitivity of the primer probe for parainfluenza virus type 2 reached 50 copies / mL, the detection sensitivity of the primer probe for parainfluenza virus type 3 reached 50 copies / mL, and the detection sensitivity of the primer probe for human metapneumovirus reached 500 copies / mL.

[0123] (2) Detection accuracy and specificity of the kit of the present invention

[0124] The kit of the present invention was used to detect 15 pathogens, including 7 pathogens including influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza types 1, 2, and 3 (HPIV1, HPIV2, HPIV3), human metapneumovirus (hMPV), and 8 other pathogens including measles virus, mumps virus, rubella virus, pertussis virus, bocavirus, human parainfluenza virus type 4, Candida albicans, and Staphylococcus aureus.

[0125] The nucleic acids of each pathogen were extracted for later use. The reaction system of the fluorescent PCR detection kit was used, and the nucleic acid template of the sample to be tested was added. The real-time fluorescent quantitative RT-PCR amplification was performed using the reaction program recommended by the detection kit. The accuracy and specificity results of the kit are shown in Table 5:

[0126] Table 5. Accuracy and specificity tests for each pathogen

[0127]

[0128]

[0129] The results showed that seven pathogens displayed fluorescence in their respective channels, including influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza types 1, 2, and 3 (PIV1, PIV2, and PIV3), human metapneumovirus (hMPV), and Legionella. No amplification or melting curves were observed for measles virus, mumps virus, rubella virus, pertussis virus, bocavirus, human parainfluenza virus type 4, Candida albicans, Staphylococcus aureus, or the negative control.

[0130] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0131] During the primer and probe design process, the inventors also designed other primers and probes (numbered 2 in Table 2 constitutes a combination) for detecting the virus. The specific sequences are shown in Table 6 below. PCR detection was performed using a positive control on a Macrostone fluorescent quantitative PCR instrument. The results are shown in Table 6. Figures 5 to 8 As shown in Figures 17 to 28. It can be seen from the figure that:

[0132] When influenza A virus is detected using the composition of the present invention, the Ct value is about 25. Figure 17 When the influenza A virus 2 in the comparative example table 2 is detected, the Ct value is about 29, such as Figure 18 .

[0133] When influenza B virus is detected using the composition of the present invention, there are obvious characteristic peaks, such as Figure 5 When the influenza B virus 2 in the comparative example table 2 is detected, there is no characteristic peak, such as Figure 19.

[0134] When respiratory syncytial virus is detected using the composition of the present invention, the Ct value is about 25. Figure 20 When the respiratory syncytial virus 2 in comparative example table 2 is detected, the Ct value is about 32, as Figure 21 .

[0135] When parainfluenza virus type 1 is detected using the composition of the present invention, there are obvious characteristic peaks, such as Figure 6 When the parainfluenza virus type 1 2 in comparative example table 2 is detected, there is no characteristic peak, such as Figure 22 .

[0136] When parainfluenza virus type 2 is detected using the composition of the present invention, the Ct value is around 24. Figure 23 When the parainfluenza virus type 2 in comparative example table 2 is detected, the Ct value is about 33, as shown in FIG. Figure 24 .

[0137] When parainfluenza virus type 3 is detected using the composition of the present invention, there are obvious characteristic peaks, such as Figure 7 When detecting with parainfluenza virus type 3 2 in comparative example table 2, there is no characteristic peak, such as Figure 25 .

[0138] When human metapneumovirus is detected using the composition of the present invention, there are obvious characteristic peaks such as Figure 8 When the human metapneumovirus 2 in Table 2 is detected, the characteristic peak is not obvious, such as Figure 26 .

[0139] When the internal standard is detected with the composition of the present invention, the Ct value is about 24. Figure 27 When the internal standard 2 in Comparative Example Table 2 is used for detection, the Ct value is around 32. Figure 28 .

[0140] This shows that in the construction of a multiplex fluorescent PCR system, a successful multiplex detection system cannot be obtained by simply combining single-target primers and probes. The design and construction of a stable multiplex system requires multiple adjustments and optimizations.

[0141] Table 6. Comparative Example Primers

[0142]

[0143]

[0144] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A kit for detecting respiratory pathogens, characterized in that: Includes primer probe combinations and rTth polymerase for detecting respiratory pathogens: The primer-probe combination for detecting respiratory pathogens includes: A probe A and a primer set for detecting influenza A virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2; and A probe B and a primer set for detecting influenza B virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; and A probe C and a primer set for detecting respiratory syncytial virus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 6; and A probe D and a primer set for detecting parainfluenza virus type 1, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 8; and A probe E and a primer set for detecting parainfluenza virus type 2, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 10; and A probe F and a primer set for detecting parainfluenza virus type 3, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 12; and A probe G and a primer set for detecting human metapneumovirus, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 14; and A probe H and a primer set for detecting an internal standard, wherein the nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO: 22, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 23; The nucleotide sequence of probe A is shown in SEQ ID NO: 15; The nucleotide sequence of probe B is shown in SEQ ID NO: 16; The nucleotide sequence of probe C is shown in SEQ ID NO: 17; The nucleotide sequence of the probe D is shown in SEQ ID NO: 18; The nucleotide sequence of the probe E is shown in SEQ ID NO: 19; The nucleotide sequence of the probe F is shown in SEQ ID NO: 20; The nucleotide sequence of the probe G is shown in SEQ ID NO: 21; The nucleotide sequence of the probe H is shown in SEQ ID NO: 24; Among the probes A to H, any two of them form a group, and the 5'-end fluorescent reporter groups of the two probes in the same group are the same; Furthermore, among the two probes in the same set, the Tm of one of them was 60-75°C, and the Tm value of the other probe was 0-30°C lower than the Tm value of the primer set for detecting the same respiratory pathogen.

2. The kit according to claim 1, wherein The 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a fluorescent quencher group; The fluorescent reporter group is selected from any one of FAM, HEX, ROX or CY5, and the fluorescent quencher group is selected from any one of BHQ1 or BHQ2.

3. The kit according to claim 2, wherein The kit also includes PCR reaction buffer, Mn 2+ At least one of a solution and a dNTPs solution.

4. The kit according to claim 3, wherein The components of the PCR reaction buffer include 100mM Tricine, 200mM KOAC and NaN3 with a volume fraction of 0.1‰~1‰; The Mn 2+ The solvent of the solution is water, in which Mn 2+ The concentration used was 3 mM; The solvent of the dNTPs solution is water, in which the concentration of each dNTP is 0.2 mM; The concentration of rTth polymerase used was 15 U / 80 μL.

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