A primer probe composition for respiratory pathogen detection and application

Abstract

This application relates to a primer-probe composition for detecting respiratory pathogens and its application. The primer-probe composition includes a universally tagged primer and 24 sets of primers-probes, each specifically designed for detecting the respiratory pathogens to be detected. Each set of primers-probes includes an upstream primer, a downstream primer, and a probe. The 5' end of both the upstream and downstream primers is attached with the nucleotide sequence of the universally tagged primer, as shown in SEQ ID NO:76. Using this primer-probe composition or a kit containing the primer-probe composition, single-tube simultaneous detection of 23 respiratory pathogens can be achieved with high accuracy, sensitivity, and specificity. Furthermore, the entire detection process can be automated using fully automated equipment.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular biology, in particular to a primer probe composition for detection of respiratory tract pathogens and application. BACKGROUND

[0002] Respiratory tract pathogen infection is one of the common diseases in the world, and the pathogens causing respiratory tract infection include various viruses, bacteria, and other pathogens such as mycoplasma pneumoniae and chlamydia pneumoniae. Moreover, the symptoms of respiratory tract infection caused by various pathogens are similar, and even the symptoms of novel coronavirus infection are similar to those of infection of other respiratory tract pathogens. However, the infection of different types of pathogens has different treatment methods and effects, and therefore, from the perspective of clinical application, it is necessary to simultaneously detect the respiratory tract pathogens causing similar symptoms in order to accurately locate the type of pathogen causing infection and facilitate subsequent precise treatment.

[0003] DRG (Diagnosis Related Groups) is a method of grouping patients into different diagnostic groups for treatment after comprehensive analysis of the disease, so as to standardize the treatment process and control the treatment cost. In one sentence, it is a change from "paying by project" to "paying by disease" in medical insurance payment method.

[0004] Syndrome refers to clinical features, symptoms and phenomena that often appear simultaneously when some diseases occur, but the actual pathogen cannot be determined. Therefore, in order to confirm the cause, doctors generally use combined detection of multiple pathogens or one-by-one detection. Under the background of DRG implementation, the accumulated high charges limit the combined use of multiple detection items, and one-by-one detection will prolong the treatment period of the disease and also go against the original intention of DRG implementation.

[0005] Syndrome multiple detection, that is, combining pathogens causing the same or similar clinical symptoms in one detection unit, can obtain all detection results of related pathogens at one time, that is, it does not need to prolong the treatment period due to waiting for the sequential detection results of single pathogens, and does not need to increase the cumulative cost due to simultaneous detection of multiple items, and is a direct and effective solution that can simultaneously meet the needs of clinical diagnosis and cost control. Related prior art indicates that according to the research results, the infection of novel coronavirus is often accompanied by the infection of other respiratory tract pathogens, and identifying the type of infection pathogen plays an important guiding role in the effective implementation of treatment. Based on the needs of respiratory tract pathogen infection treatment and the guidance of national policy, there is an urgent need for a method that can detect multiple pathogens at one time.

[0006] Currently, the main methods for pathogen detection in clinical practice are culture and isolation identification, immunological detection, and nucleic acid detection. The culture and isolation identification method requires a long period, which cannot meet the needs of acute infection treatment. The immunological detection method is the main method for single-pathogen detection, but it has low sensitivity and cannot be used for early diagnosis. In addition, the cross-reaction of antigens and antibodies can also lead to false positives. Both of the above methods cannot be used for multi-pathogen detection. Compared with the above two methods, nucleic acid detection can avoid false positives due to the specificity of primers and probes. Gene chip and multiplex melting curve method can also achieve multi-pathogen detection. Therefore, nucleic acid detection has obvious technical advantages in the detection of respiratory infection syndromes. Compared with the chip method, the multiplex melting curve method still has the problem of limited number of fluorescence channels in real-time fluorescence PCR, which can only be improved to a certain extent. Therefore, it is still necessary to use separate holes to detect a large number of samples, and the cost increase caused by separate holes cannot be avoided. Due to the limitation of sensitivity, the existing gene chip technology is mainly used for screening in aspects that do not require high sensitivity, and is less used in pathogen detection that requires high sensitivity.

[0007] Microfluidics is a technology that integrates sample preparation, reaction, separation, and detection of biological, chemical, and medical analysis processes on a chip with a micron scale, and automatically completes the whole analysis process. Microfluidics has the advantages of small size, low sample and reagent consumption, fast reaction speed, large-scale parallel processing, and disposable use, and has great potential in the fields of biology, chemistry, medicine, and other fields.

[0008] The FilmArray system of Biofire Company is a representative product of molecular diagnostic syndromes multi-association detection. It was certified by FDA (Food and Drug Administration) as early as 2011, and can detect multiple pathogen targets that can cause respiratory infections. However, the high cost limits its application in China, and so far it has not been registered by NMPA (National Medical Products Administration) to realize popularization. The representative of NMPA certified multi-association detection products is the 13 respiratory pathogen multiplex detection kit (PCR capillary electrophoresis fragment analysis method) of Ningbo Haier Shki Gene Technology Co., Ltd. However, the complexity of its operation limits its expansion in clinical application. The novel coronavirus 2019-nCoV nucleic acid detection kit (isothermal amplification-real-time fluorescence method) of Hangzhou Yousida Biotechnology Co., Ltd. realizes the automation of the whole detection process, but it is difficult to realize multi-association detection, and the types of pathogens it can cover are limited.

[0009] Since the existing products cannot simultaneously consider multiple pathogen multiplex detection, simple operation (basilification), high sensitivity and specificity, and with the further sinking of medical institutions and needs, there is an urgent need for a product with simple operation (fully automatic, no manual intervention), covering a variety of pathogens related to respiratory tract infection symptoms, high sensitivity, accuracy and specificity, and cost-controllable comprehensive performance advantages to meet market needs. SUMMARY

[0010] In order to solve the defects that the existing products cannot simultaneously consider multiple pathogen multiplex detection, simple operation, high sensitivity and specificity, the present application provides a new primer probe composition for respiratory tract pathogen detection and a microfluidic chip technology platform and a matching kit for realizing full-automatic detection comprising the primer probe composition. The primer probe composition or kit can realize single-tube multiplex detection of 23 respiratory tract pathogens, which is enough to cover the related pathogens of syndromes, and has high detection accuracy, good sensitivity and specificity. At the same time, the whole detection process can be automatically realized by using a full-automatic device. The device can realize the automation of the whole detection process, and even the primary personnel can use it. The combination of various technologies ensures the convenience of operation, sensitivity, accuracy and specificity. The system has the ability of industrialization and transformation, and the cost is controllable.

[0011] Therefore, the first aspect of the present application provides a primer probe composition for respiratory tract pathogen detection, which comprises a universal tag primer and 24 groups of primer probe groups for detecting respiratory tract pathogens to be detected; each group of primer probe groups comprises an upstream primer, a downstream primer and a probe, and the 5' end of the upstream primer and the downstream primer is connected with the nucleotide sequence of the universal tag primer, and the nucleotide sequence of the universal tag primer is shown as SEQ ID NO: 76.

[0012] In some embodiments, the 5' end of the universal tag primer is modified with biotin.

[0013] For multiple systems, controlling the competition interference between different primers and inhibiting the formation of primer dimers are the key points to be overcome. Previous studies have shown that by introducing universal primers, the competition interference between different primers can be largely controlled, thereby effectively improving the amplification efficiency and sensitivity of the multiple system. A method for detecting 18 respiratory pathogens is disclosed in the related art, which uses the traditional design of multiple system and does not introduce universal primers. According to the final display results, the sensitivity is only 500 copies per reaction, and converted to the original sample, taking 0.2 mL sample as an example, the nucleic acid extraction elution volume is 50 μL, even if the nucleic acid extraction efficiency is 100%, the sensitivity based on the original sample can only reach 25,000 copies / mL, which is still at a low level. For the design of universal primers, the existing literature and patents mostly use the universal primer pair method, by adding a universal sequence to the 5' end of the forward and reverse specific primers, and the Tm value of the designed universal primer is generally higher than that of the specific primer, then two-step amplification is used to reduce the competition interference between multiple primers. This method still tests the amplification efficiency of the universal primer pair, and the universal primer pair still has the problem of unsatisfactory amplification efficiency due to primer dimer.

[0014] The existing literature reports a method of using a single tag to reduce primer dimer, and the related existing patent also uses a single universal primer (GTACGACTCACTATAGGGA) to achieve simultaneous detection of 16 respiratory pathogens, with a sensitivity of 1000 copies / mL, which is 1 order of magnitude higher than the detection sensitivity without using universal primers. However, analysis of the universal primer shows that the primer still has the risk of forming dimers, as shown below.

[0015]

[0016]

[0017] Therefore, in view of the above situation, the present application redesigns a universal tag primer (Tag), whose nucleotide sequence is ATACGACTCACTCTTGCGA (SEQ ID NO: 76), which does not bind to the genomic sequences of human genes, respiratory pathogens within the detection range, and cross pathogens tested and verified, and the sequence is less likely to form dimers compared to the previously reported ones, as shown below, which is more conducive to the amplification of multiple systems.

[0018] The sequence is connected to the 5' end of the designed forward and reverse specific primers to form a labeled specific primer. Among them, the specific primer labeled with Tag is purified by PAGE, and the 5' end of Tag is labeled with biotin and purified by HPLC. The detection results show that after using the universal tag primer, the detection effect is better than that of the product of the low fluorescent PCR platform of the conventional method, and although the pathogen coverage of the present application is more, the detection effect is better.

[0019] In some embodiments, the nucleotide sequences of the upstream primers, downstream primers and probes in the 24 groups of primer probe groups for detecting the respiratory tract pathogens to be detected are respectively as shown in SEQ ID NO: 1-72.

[0020] It is worth noting that the nucleotide sequences of the above-mentioned upstream and downstream primers and probes claimed in the present application are not limited to the above-mentioned range. Moving a few base positions before and after the primers and probes and or mismatching a few bases are also within the protection scope of the present application.

[0021] In the present application, the term "primer" means an oligonucleotide that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e. the 3'-end of the oligonucleotide provides a free 3'-OH group to which more "nucleotides" can be attached by a template-dependent DNA polymerase, establishing a 3' to 5' phosphodiester bond, thereby using deoxynucleoside triphosphates, and thereby releasing pyrophosphate.

[0022] In the present application, the term "upstream primer" is an oligonucleotide that proceeds uninterrupted elongation along the negative strand; the term "downstream primer" is an oligonucleotide that proceeds uninterrupted elongation along the positive strand. It should be understood that when the designation of the positive strand and the negative strand is interchanged, the naming of the corresponding upstream primer and the downstream primer can also be interchanged accordingly. That is, the upstream primer and the downstream primer in the present application are relative.

[0023] In the present application, the upstream and downstream primers and probes in the primer probe group are designed for the conserved regions of each pathogen, which guarantees the inclusivity and accuracy in the pathogen, and through comparison analysis and optimization design, the specificity between different pathogens is confirmed, and the specificity of target recognition is guaranteed through the double design of primers and probes. Further, the upstream and downstream primers in the primer probe group in the present application are modified by using universal tag primers, which reduces the competition and inhibition between primers in the multiplex system and the influence of primer dimers, and improves the amplification efficiency and detection sensitivity.

[0024] In some embodiments, the 5' end of the probe is modified with an amino group (NH2).

[0025] In some embodiments, the primer probe composition further comprises a set of primer probe combinations for an internal reference gene, which comprises an upstream primer for the internal reference gene, a downstream primer for the internal reference gene, and a probe for the internal reference gene.

[0026] In the present application, the internal reference gene is used to monitor the sampling and the whole detection process to ensure the accuracy of the detection process. In the present application, the internal reference gene used is RnaseP.

[0027] In some embodiments, the nucleotide sequences of the upstream primer, the downstream primer and the probe for the internal reference gene are shown in SEQ ID NOs: 73-75, respectively.

[0028] In some embodiments, the 5' end of the probe for the internal reference gene is modified with an amino group (NH2).

[0029] It should be noted that in the present application, only the universal tag primer is modified with biotin, and the 25 pairs of upstream and downstream primers for the internal reference gene and the respiratory pathogens to be detected do not need to be modified with biotin.

[0030] In the present application, the respiratory pathogens to be detected include 23 pathogens, which are SARS-CoV-2, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, enterovirus / nasovirus causing respiratory infection (enterovirus / nasovirus), metapneumovirus, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, bocavirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella pertussis, Legionella pneumophila, Haemophilus influenzae, Streptococcus pneumoniae, and Bordetella parapertussis.

[0031] The primer probe composition described in the present application can simultaneously detect the above-mentioned 23 respiratory pathogens, wherein a set of specific primer probe combinations is set for influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, enterovirus / nasovirus causing respiratory infection (enterovirus / nasovirus), metapneumovirus, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, bocavirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella pertussis, Legionella pneumophila, Haemophilus influenzae, Streptococcus pneumoniae, and Bordetella parapertussis, and two sets of specific primer probe combinations are set for SARS-CoV-2, one set for detecting the ORF1ab gene of SARS-CoV-2 and the other set for detecting the N gene of SARS-CoV-2.

[0032] The primer and probe composition described in this application covers the above 23 respiratory pathogens, including common viruses, bacteria, mycoplasma pneumoniae, and chlamydia that cause respiratory infection symptoms. This broad coverage can provide a more reliable basis for the effective implementation of clinical treatment.

[0033] The second aspect of this application provides a fully automated detection kit for detecting respiratory pathogens, comprising the primer and probe composition as described in the first aspect of this application.

[0034] In some embodiments, the kit includes nucleic acid extraction reagents, amplification reagents, and hybridization chromogenic reagents.

[0035] In this application, the nucleic acid extraction reagent is used to extract nucleic acids from the sample to be tested. The amplification reagent contains all the primers (universal tag primers and 25 pairs of upstream and downstream primers) in the primer-probe composition and is used for multiplex PCR amplification of the nucleic acids in the sample to be tested. The hybridization chromogenic reagent contains all the probes in the primer-probe composition. The probes are used to capture the target sequence in the multiplex PCR amplification product, thereby immobilizing the target sequence in the amplification product at a specific site on the hybridization membrane and using it for subsequent chromogenic development.

[0036] In some embodiments, the kit also includes positive and negative controls.

[0037] This application utilizes the aforementioned positive and negative control materials to effectively control the test results, avoid false positives and false negatives during the testing process, and further ensure the accuracy of the test results.

[0038] In some embodiments, the nucleic acid extraction reagent includes a lysis buffer, a lysis aid, a nucleic acid precipitation aid, magnetic beads, a binding buffer, a washing buffer, and an elution buffer.

[0039] In this application, the nucleic acid extraction reagent is used to obtain target nucleic acids from a sample. Specifically, the lysis buffer is used to disrupt the structure of pathogens in the sample, thereby fully releasing the nucleic acids within the sample; the lysis aid and nucleic acid precipitation aid are used to assist the lysis buffer, improving lysis efficiency and nucleic acid yield; the magnetic beads are used to adsorb the released nucleic acids; the binding solution is used to promote the binding of the released nucleic acids to the magnetic beads; the washing solution is used to purify the nucleic acids, removing impurities (proteins, etc.); and the elution solution is used to separate the magnetic beads from the nucleic acids, thereby releasing the purified nucleic acids, which are then dissolved in a liquid phase (e.g., water).

[0040] In some embodiments, the lysis solution comprises guanidine salt, surfactant and first buffer salt; the lysis aid is proteinase K; the nucleic acid precipitant is Carrier RNA; the magnetic beads are silicon hydroxyl magnetic beads; the binding solution is isopropanol; the rinsing solution comprises second buffer salt and organic alcohol; and the elution solution is water (e.g. purified water).

[0041] In some specific embodiments, the guanidine salt is guanidine isothiocyanate, the concentration of which in the lysis solution is 2-8 M; the surfactant is Triton X-100, the concentration of which in the lysis solution is 1-10 wt%; and the first buffer salt is MOPS (3-morpholinopropanesulfonic acid), the concentration of which in the lysis solution is 20-100 mM.

[0042] In some preferred specific embodiments, the lysis solution comprises 5 M guanidine isothiocyanate, 4 wt% Triton X-100 and 100 mM MOPS.

[0043] In some specific embodiments, the second buffer salt is MOPS, the concentration of which in the rinsing solution is 5-20 mM; and the organic alcohol is ethanol, the volume concentration of which is 40-70%.

[0044] In some preferred specific embodiments, the rinsing solution is a 70 v / v% ethanol solution comprising 10 mM MOPS.

[0045] The nucleic acid extraction reagent comprising the above components can be used to effectively obtain target nucleic acid from a sample to be tested.

[0046] In some embodiments, the amplification reagent comprises reverse transcriptase, Taq enzyme, UNG enzyme, dNTPs, specific primers and universal tag primers; wherein the specific primers are 25 upstream primers and 25 downstream primers for the internal reference gene and the respiratory pathogen to be detected in the primer probe composition.

[0047] In the present application, the amplification reagent is used to perform multiplex PCR amplification on the target nucleic acid obtained by lysis, so as to amplify the corresponding target sequence in the target nucleic acid.

[0048] In some embodiments, the content of reverse transcriptase in the amplification reagent is 1-10 U; the content of Taq enzyme is 1-4 U, the content of UNG enzyme is 0.03-1 U, the content of dNTPS is 0.2-0.3 mM, the content of each specific primer is independently 0.005-5 μM, and the content of universal tag primer is 0.1-5 μM.

[0049] In the present application, the reverse transcriptase is an RNA-dependent DNA polymerase that synthesizes complementary DNA (cDNA) from RNA template; the Taq enzyme is a heat-stable DNA polymerase; the UNG enzyme is uracil-N-glycosylase that selectively hydrolyzes and breaks the uracil glycosidic bond in double-stranded or single-stranded DNA containing dU to form a base-pairing DNA strand, which is further hydrolyzed and broken in alkaline medium and high temperature, thereby being eliminated. In order to facilitate the reverse transcription reaction, the UNG enzyme selected in the present system is a heat-sensitive UNG enzyme that can lose activity at 50°C. Since the kit of the present application is used for detecting pathogens including RNA viruses, the reverse transcriptase is added to the amplification reagent for effective amplification of the nucleic acid of the RNA virus; at the same time, the present application uses Taq enzyme and UNG enzyme to ensure the accuracy of the PCR result and prevent non-specific PCR amplification and contamination.

[0050] The present application reduces the competition interference and primer dimerization of the multiplex amplification system mainly by reducing the amount of specific primers and increasing the amount of universal tag primers. Therefore, in the design of the multiplex system, the content of each primer in the specific primers in the amplification reagent is 0.005-5 μM, and the content of the universal tag primer is 0.1-5 μM. By controlling the content of the universal tag primer in the amplification reagent and the content of each primer in the specific primers within the above range, the competition interference and primer dimerization of the multiplex amplification system can be significantly reduced, and the amplification effect can be improved.

[0051] In some embodiments, the content of reverse transcriptase in the amplification reagent is 5U, the content of Taq enzyme is 2U, the content of UNG enzyme is 0.1U, the content of dNTPS is 0.3mM, the content of each primer in the specific primers is 0.01 μM, and the content of the universal tag primer is 2 μM.

[0052] By controlling the content of each component in the amplification reagent within the above range, the present application can achieve the best amplification effect.

[0053] In some embodiments, the hybridization color developing reagent includes a hybridization membrane, a hybridization rinse solution, an enzyme binding solution, and a color developing solution, wherein the hybridization membrane is fixed with 25 probes in the primer probe composition for the internal reference gene and the respiratory tract pathogen to be detected.

[0054] The application discloses a hybridization color developing reagent for hybridization color development of multiplex PCR amplification products. The hybridization membrane is fixed with specific probes, the probes can be specifically combined with corresponding target sequences in the amplification products, and then the target sequences are fixed on specific positions of the hybridization membrane. The hybridization rinsing solution is used for providing a hybridization environment and removing interference of non-specific substances in the amplification products. The enzyme binding solution contains enzymes which can be fixed on the target sequences through biotin-streptavidin combination. The color developing solution can be combined with the enzymes in the enzyme binding solution, and then the target sequences are developed.

[0055] In some embodiments, the content of each probe on the hybridization membrane is 0.2-50 μM, preferably 50 μM.

[0056] In the application, the probes can be spotted on the hybridization membrane through nanoliter point membrane technology. The multiplex products amplified by a single tube are hybridized with the hybridization membrane covering the detected pathogens, and 23 respiratory tract pathogens are simultaneously detected by a single tube.

[0057] In some embodiments, the hybridization membrane is a nylon membrane; the hybridization rinsing solution contains sodium dodecyl sulfate and a third buffer salt; the enzyme binding solution contains streptavidin-labeled horseradish peroxidase; and the color developing solution contains urea peroxide, a fourth buffer salt and tetramethylbenzidine.

[0058] In some specific embodiments, the concentration of the sodium dodecyl sulfate in the hybridization rinsing solution is 5-15 wt%; the third buffer salt is phosphate, and the concentration of the phosphate in the hybridization rinsing solution is 10-100 mM; the concentration of the streptavidin-labeled horseradish peroxidase (HRP) in the enzyme binding solution is (0.1-1) μg / mL; the concentration of urea peroxide in the color developing solution is 0.01-0.1 wt%; the fourth buffer salt is citrate, and the concentration of the citrate in the color developing solution is 5-50 mM; and the concentration of tetramethylbenzidine (TMB) in the color developing solution is 0.01-0.05 wt%.

[0059] In some preferred specific embodiments, the hybridization rinsing solution contains 10 wt% SDS and 10 mM phosphate; the enzyme binding solution contains 0.4 μg / mL streptavidin-labeled horseradish peroxidase; and the color developing solution contains 0.03 wt% urea peroxide, 5 mM citrate and 0.015 wt% TMB.

[0060] In some embodiments, the hybridization membrane is further fixed with a color developing quality control probe (SP probe); preferably, the content of the color developing quality control probe on the hybridization membrane is 0.2-50 μM. In some specific embodiments, the color developing quality control probe is fixed on three positions of the hybridization membrane.

[0061] The SP probe described in the present application is used for quality control of hybridization color development to ensure the accuracy of the detection results. In addition, the SP probe is used as a positioning point for software automatic recognition and interpretation in addition to color development quality control. Through the design of three positioning points, the transition from manual visual interpretation to software automatic recognition is realized. After the experiment is completed, the automatic interpretation software searches for the three fixed positioning points (SP points) for positioning, and determines the position of other spots based on this; by setting a simulated circle range based on the size of the SP point, the average gray value of the pixel points in the simulated circle is counted as the spot brightness information, and the average gray value of the four position regions near the spot is counted as the background brightness. The gray difference between the spot brightness and the background brightness is the contrast reading value of the spot. The deeper the spot color development, the greater the gray difference between the spot position and the surrounding area, and the higher the contrast reading value. Therefore, the detection results of the spots can be reflected by the contrast reading value.

[0062] In some embodiments, the positive quality control is a false virus of influenza A virus and influenza B virus with positive detection results of both influenza A virus and influenza B virus; and the negative quality control is a sample preservation solution containing HEK293 cells with negative detection results of the respiratory pathogen to be detected.

[0063] In the present application, the principle of using the above-mentioned kit to detect the respiratory pathogen to be detected belongs to the nucleic acid extraction-amplification-reverse spot hybridization method, which specifically comprises: obtaining target nucleic acid from the sample by using nucleic acid extraction reagents; realizing amplification of the target sequence by using amplification reagents; and combining the amplified product to the probe on the hybridization membrane through hybridization. Since the primer is modified with biotin, the product combined to the hybridization membrane also has biotin modification. Through the interaction of biotin and avidin, the amplified product can be combined to the HRP modified with streptavidin. Then, the color development reaction of hydrogen peroxide and TMB catalyzed by HRP is used to realize the presentation of visual detection results. If the sample contains the target, the target enriched and amplified after extraction will be combined to the hybridization membrane containing the specific probe, and then the color development of HRP-TMB will present a blue spot at the position of the probe.

[0064] The kit described in the present application fixes the specific probes of each respiratory pathogen and the internal standard probe on the hybridization membrane through nanoliter point membrane technology, so that the kit described in the present application can realize the detection of 23 respiratory pathogens at one time. Whether the sample contains the corresponding respiratory pathogen is determined according to the color development of each probe position on the hybridization membrane. At the same time, the kit is combined with the automatic interpretation software to realize the automatic interpretation of the detection results. Figures 2-3 The device shown in the present application, such as Figures 4-5The chips are used together, can realize the automation of the whole detection process, avoid the operation error or pollution introduced by manual intervention, have good repeatability and high stability, and are suitable for screening and detection of primary institutions.

[0065] The third aspect of the application provides a method for detecting respiratory pathogens by using the kit of the second aspect of the application.

[0066] In some embodiments, the method comprises the following steps:

[0067] S1, obtaining target nucleic acid in a sample to be tested by using the nucleic acid extraction reagent;

[0068] S2, mixing the target nucleic acid with the amplification reagent, and then performing multiplex PCR amplification to obtain an amplification product;

[0069] S3, hybridizing and coloring the amplification product by using the hybridization and coloring reagent, and interpreting the coloring result.

[0070] In the application, the target nucleic acid in the sample to be tested in step S1 is obtained by a magnetic bead method. After the sample is lysed by a lysis solution, the released nucleic acid is captured by a magnetic bead. After washing and elution, the purified target nucleic acid is obtained.

[0071] In some embodiments, the multiplex PCR amplification in step S2 is completed in a single tube.

[0072] In some specific embodiments, the conditions of the multiplex PCR amplification are as follows:

[0073] 35℃ 5min;

[0074] 50℃ 10min;

[0075] 95℃ 2min;

[0076] 95℃ 15s, 55℃ 20s, 72℃ 20s, 45 cycles;

[0077] 72℃ 3min.

[0078] In some specific embodiments, the specific operation of step S3 is as follows: hybridizing the amplification product with a hybridization membrane at 30-50℃ for 10-15min, washing the hybridization membrane by adding a hybridization rinse solution, then adding an enzyme binding solution and reacting at 30-37℃ for 10-15min, then adding a coloring solution (urea peroxide+TMB) and reacting for 5-10min for coloring, and interpreting the coloring result.

[0079] In some embodiments, the method is automatically completed by a fully automated device.

[0080] The method described in the application can be automatically completed by a fully automated device, realizing the whole process without human intervention, thereby avoiding the operation errors or pollution introduced by human intervention, having good repeatability, high stability, and being suitable for screening and detection of primary institutions. In some specific embodiments, the fully automated device can use the microfluidic nucleic acid chip detection device of the applicant company.

[0081] Specifically, the characteristics of the detection method described in the application and other methods are compared and analyzed as shown in Table 1.

[0082] Table 1

[0083]

[0084] As can be seen from Table 1, compared with the existing products, the application has obvious comprehensive advantages in pathogen type coverage and typing ability, and automation degree. Compared with domestic multi-association detection products, in addition to more comprehensive pathogen type coverage, the implementation of automatic function avoids the possibility of pollution caused by human operation intervention. Compared with the leading enterprises of foreign multi-association detection, in addition to more comprehensive pathogen type coverage, the application has an advantage in cost control.

[0085] The application includes at least one of the following beneficial technical effects:

[0086] (1) Sensitivity: the sensitivity of the single-tube multiplex detection system after optimization of the application is consistent with or slightly higher than that of conventional real-time fluorescent PCR, indicating that the method in the application for detection not only covers a wider range of pathogens in single detection, but also does not cause obvious loss in sensitivity due to multiplexing, which can maximize the liberation of operating personnel and benefit patients.

[0087] (2) Accuracy: the results of the method described in the application and the conventional real-time fluorescent PCR detection show that the positive coincidence rate and the negative coincidence rate of the single-tube multiplex detection of the application and the conventional real-time fluorescent PCR detection are both 100%, indicating that the method of the application for detection not only covers a wider range of pathogens in single detection, but also has high accuracy.

[0088] (3) Specificity: the pathogens (such as varicella-zoster virus, Epstein-Barr virus, Escherichia coli, Stenotrophomonas maltophilia, Burkholderia cepacia, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus salivarius, Acinetobacter baumannii, Klebsiella pneumoniae, Cryptococcus, Serratia viscosa, Citrobacter, Aspergillus fumigatus, and Aspergillus flavus) outside the detection target range detected by the kit of the application are all negative, further confirming that the kit described in the application has high specificity.

[0089] (4) Interfering substance tolerance: The interfering substance tolerance test was performed using 5 times the detection limit concentration of the sample using a pseudo virus and inactivated pathogen culture. The interfering substances included: nasal spray or nasal drops (physiological seawater nasal care spray, 60 mg / L), nasal corticosteroids (triamcinolone acetonide econazole cream, 100 mg / L), throat lozenges and oral anesthetics and analgesics (compound menthol ointment, 1.7 mg / mL), antiviral drugs (ribavirin granules, 100 mg / L), antibiotics and nasal ointments (mupirocin ointment, 500 mg / L). The results showed that the above drugs had no interference on the detection results within the identification range.

[0090] (5) Operation convenience: During the detection implementation process, the operation involved in the present application is only to add the sample to the sample pool corresponding to the detection chip, and other operations are all completed by the equipment, thereby reducing the pollution caused by manual intervention and further improving the accuracy. Compared with non-automatic products involving a large number of operations, the present product has better user friendliness. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 Figure 1 is a dot array distribution diagram of the detection probes and color developing quality control probes for each respiratory pathogen and the internal reference gene on the hybridization membrane in Example 2; wherein the meanings of the various marks in the figure are as follows:

[0092] SP: color developing quality control probe; internal reference IC: detection probe for internal reference gene; the others are detection probes for each respiratory pathogen, wherein two detection target points of ORF1ab and N of the novel coronavirus are set;

[0093] The use rules for interpreting each mark are as follows: (1) the positive quality control in the kit must be interpreted by the software to show positive for influenza A virus and positive for influenza B virus (positive spots appear at the positions of influenza A virus, influenza B virus, internal reference IC and SP), otherwise the experiment is invalid; (2) the negative quality control in the kit must be interpreted by the software to show negative (only positive spots appear at the positions of internal reference IC and SP), otherwise the experiment is invalid.

[0094] Figure 2 Figure 2 is an appearance diagram of the automatic equipment used in Example 3.

[0095] Figure 3 Figure 3 is an internal structure diagram of the automatic equipment used in Example 3.

[0096] Figure 4 Figure 4 is a front view of the chip installed in the equipment described in Example 3.

[0097] Figure 5 Figure 5 is a back view of the chip installed in the equipment described in Example 3.

[0098] Figure 6 Figure 2 shows the electrophoresis results of the amplification products of Example 1 using three different primers under the same conditions. DETAILED DESCRIPTION

[0099] In order to make the present application more readily understood, reference will now be made to the following examples, which are intended to be illustrative only and not limiting of the scope of the application. The starting materials or components used in the present application can be obtained by commercial means or conventional methods unless otherwise indicated. Unless otherwise indicated, the nucleic acid sequences of the present application are written left to right in the 5' to 3' direction.

[0100] Example 1: Design of primer probe combination

[0101] The genomic sequences of the novel coronavirus, influenza A virus, influenza B virus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, metapneumovirus, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, enterovirus / nasovirus, bocavirus, Bordetella pertussis, Bordetella parapertussis, Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila were downloaded from the NCBI database respectively, sequence alignment was performed, and primers and probes were designed based on the ORF1ab and N genes of the novel coronavirus and the conserved gene regions of the above other pathogens. After the initial design was completed, the interaction between the primers was analyzed, and the designs with 5 or more bases matching at the 3' end were removed. The interaction between the probes and the amplification products of each target was analyzed, and the designs with continuous multiple bases matching were removed.

[0102] The primers and probes designed above were then analyzed with the cross-reactive pathogens to be studied to exclude the possibility of cross-false positive from the design level. The cross-reactive pathogens verified in the present application include varicella-zoster virus, Epstein-Barr virus, Escherichia coli, Stenotrophomonas maltophilia, Burkholderia cepacia, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus salivarius, Acinetobacter baumannii, Klebsiella pneumoniae, Cryptococcus, Serratia marcescens, Citrobacter, Aspergillus fumigatus, and Aspergillus flavus. After comparison and analysis, the present design does not have cross-reactivity with the above pathogens.

[0103] In order to control the competition interference between different primers in the multiplex system and inhibit the formation of primer dimers, a new universal tag primer (Tag) is designed in the application, and the nucleotide sequence of the tag primer is ATACGACTCACTCTTGCGA (SEQ ID NO: 76). The sequence does not bind to the genomic sequences of human genes, respiratory pathogens in the detection range and cross-reactive pathogens verified, and the sequence is less likely to form dimers compared to the previously reported sequences, thereby being more conducive to the amplification of the multiplex system. The sequence is connected to the 5' end of the designed forward and reverse specific primers to form a labeled specific primer, which can significantly reduce the competition interference between different primers in the multiplex system and improve the amplification efficiency. The specific primer labeled with the tag is purified by PAGE, and the 5' end of the tag is labeled with biotin and purified by HPLC.

[0104] In order to further verify the improvement of the universal tag primer (Tag) designed in the application on the amplification efficiency, the amplification efficiency of the primers labeled with non-tag, the existing single universal primer (GTACGACTCACTATAGGGA) and the universal tag primer designed in the application is compared under the same conditions, and the results are shown in Figure 6 Figure 6 In the figure, A is the electrophoresis result band of the amplification product of the primer labeled with the existing single universal primer, B is the electrophoresis result band of the amplification product of the primer labeled with the universal tag primer of the application, and C is the electrophoresis result band of the amplification product of the primer labeled with non-tag. From the results of the three groups of amplification product electrophoresis bands, Figure 6 it can be seen that compared with the conventional primer without Tag labeling, the Tag labeled primers of groups A and B can improve the amplification efficiency, and the improvement of the Tag provided in the application on the amplification efficiency is better.

[0105] In the application, RnaseP is selected as the internal reference gene (internal standard) for detecting the sampling condition and monitoring the whole process of the detection process.

[0106] After theoretical analysis and experimental verification, the nucleotide sequences of the primers and probes finally selected in the application are shown in Table 2.

[0107] Table 2: Nucleotide sequences of primers and probes in the primer and probe composition described in the application

[0108]

[0109]

[0110]

[0111] Example 2: Design of multiplex detection system​

[0112] 1. Nucleic acid extraction conditions

[0113] In this application, nucleic acid extraction is achieved by magnetic bead method. After the sample is lysed by lysis solution, the released nucleic acid is captured by magnetic beads. After washing and elution, purified nucleic acid is obtained.

[0114] In this embodiment, the preferred nucleic acid extraction conditions are as follows: add 0.2 mL sample, use 0.2 mL lysis solution containing 4% Triton X-100, 5M guanidine isothiocyanate, 100 mM MOPS, 0.2 mg proteinase k, 6 μg carrier RNA, 56°C lysis for 10 min; then add 0.2 mL isopropyl alcohol, 0.2 mg silicon hydroxyl magnetic beads, combine for 10 min; adsorb magnetic beads, then wash magnetic beads with 70 v / v% ethanol containing 10 mM MOPS; then add purified water, heat at 56°C for 5 min to elute nucleic acid.

[0115] 2. Amplification system

[0116] In this application, the use of one Tag to reduce the competition interference and primer dimer effect of multiplex amplification system is mainly achieved by reducing the specific primer and increasing the amount of Tag. Therefore, when designing the amplification system of the multiplex system, the content of each specific primer is 0.005-0.5 μM, and the content of Tag is 0.1-5 μM.

[0117] In this embodiment, the content of each specific primer in the preferred amplification system is 0.01 μM, the content of Tag is 1 μM, the content of reverse transcriptase is 5 U, the content of Taq enzyme is 2 U, the content of dNTPS is 0.3 mM, and the content of UNG enzyme is 0.1 U.

[0118] Using the primers designed in Example 1, the amplification system is prepared according to the content of each component in the amplification system in this embodiment, and the extracted nucleic acid is used for single-tube multiplex PCR amplification.

[0119] Among them, the amplification conditions are: 35°C for 5 min; 50°C for 10 min; 95°C for 2 min; (95°C for 15 s, 55°C for 20 s, 72°C for 20 s), 45 cycles; 72°C for 3 min.

[0120] 3. Hybridization system

[0121] In this application, the probes of the detected pathogens are spotted on the hybridization membrane by nanoliter dot membrane technology. The dot array distribution diagram of the detection probes of each pathogen and the internal reference gene on the hybridization membrane is shown in Figure 1 The multiplex product amplified by single-tube amplification is hybridized with the hybridization membrane covering the detected pathogens to achieve single-tube and simultaneous detection of multiple pathogens.

[0122] In this embodiment, the preferred hybridization system is: the concentration of each target probe on the hybridization membrane is 50 μM; the concentration of SDS in the hybridization rinse solution is 10 wt%, and the buffer salt is phosphate with a concentration of 10 mM; the concentration of HPR in the enzyme binding solution is 0.4 μg / mL; the concentration of urea peroxide in the color developing solution is 0.03 wt%; the buffer salt is citrate with a concentration of 5 mM; the concentration of TMB is 0.015%, and the buffer salt is citrate with a concentration of 5 mM.

[0123] After amplification is completed, the amplification product is hybridized to the hybridization membrane at 45°C for 15 min, the hybridization membrane is washed with the hybridization rinse solution, and then the enzyme binding solution is added, and the reaction is carried out at 37°C for 10 min. Then, the color developing solution (urea peroxide + TMB) is added, and the reaction is carried out for 5 min. According to the different templates added, different color spots can be seen on the hybridization membrane.

[0124] Example 3: Detection of 23 respiratory pathogens

[0125] The simulated samples of each respiratory pathogen were prepared by using the artificial synthesized pseudo-virus and inactivated pathogen culture of each respiratory pathogen. The simulated samples were diluted to 1000 copies / mL, wherein the new coronavirus pseudo-virus was diluted to 500 copies / mL. The nucleic acid extraction, amplification and hybridization of the simulated samples were carried out according to the conditions of Example 2, and the detection results are shown in Table 3.

[0126] The above detection process is automatically carried out by an automatic device. The automatic device used is a nucleic acid chip detector produced by Beijing Bohui Innovation Biotechnology Group Co., Ltd., and the appearance and internal structure diagrams thereof are shown in Figure 2 and 3 The front and back views of the chip installed in the device are shown in Figure 4 and 5 The chip used in this embodiment has been patented (CN201110235199.2, CN201110235234.0, CN201210121023.9, CN201220175387.0).

[0127] Table 3: Detection of 23 respiratory pathogens by the multiplex system of the present application

[0128]

[0129]

[0130] As can be seen from Table 3, each pathogen can be normally detected by the present application, that is, the system can achieve a sensitivity level of 1000 copies / mL (500 copies / mL for the new coronavirus).

[0131] Example 4: Performance evaluation

[0132] The automated device used in this example is the same as that of Example 3.

[0133] 1. Accuracy detection

[0134] For the clinical residual samples (40 cases), parallel comparison detection was performed by real-time fluorescent PCR (qPCR) and the method described in this application (using the kit system determined in Example 2 combined with an automated device). The results are shown in Table 4. The automated device used in this example is the nucleic acid chip detector produced by Beijing Bohui Innovation Biotechnology Group Co., Ltd., the appearance and internal structure diagrams of which are shown in Figure 2 and 3 respectively; the front and back views of the chip installed in the device are shown in Figure 4 and 5 respectively. The chip used in this example has been patented (CN201110235199.2, CN201110235234.0, CN201210121023.9, CN201220175387.0).

[0135] Table 4: Comparison results of the method described in this application and real-time fluorescent PCR

[0136]

[0137]

[0138]

[0139] From Table 4, it can be seen that the positive coincidence rate and negative coincidence rate of the single-tube multiplex detection of this application and the detection results of conventional real-time fluorescent PCR are both 100%, indicating that the accuracy of the detection method described in this application is high.

[0140] 2. Sensitivity detection

[0141] Gradient dilution was performed on 5 of the above-mentioned 40 clinical residual samples, and then parallel comparison detection was performed by real-time fluorescent PCR (qPCR) and the method described in this application (using the kit system determined in Example 2 combined with an automated device). The results are shown in Table 5.

[0142] Table 5: Comparison results of the method described in this application and real-time fluorescent PCR

[0143]

[0144]

[0145] 3. Interferent tolerance verification

[0146] Using the system determined in Example 2 combined with automated equipment, the interferent tolerance test was performed using 5 times the limit of detection concentration with pseudovirus and inactivated pathogen culture as samples. The interferents included: nasal spray or nasal drops (physiological seawater nasal care spray, 60 mg / L), nasal corticosteroids (triamcinolone acetonide econazole cream, 100 mg / L), throat lozenges and oral anesthetics and analgesics (compound menthol ointment, 1.7 mg / mL), antiviral drugs (ribavirin granules, 100 mg / L), antibiotics and nasal ointments (mupirocin ointment, 500 mg / L). The results are shown in Table 6.

[0147] Table 6: Interferent tolerance test results

[0148]

[0149]

[0150] As can be seen from Table 6, the above-mentioned drugs do not interfere with the test results within the identified range, indicating that the method described in the application has good interferent tolerance.

[0151] 4. Specificity detection

[0152] Using the system determined in Example 2 combined with automated equipment, the cross-interference of the pathogen outside the detection target range (pox zoster virus, EB virus, Escherichia coli, Stenotrophomonas maltophilia, Burkholderia cepacia, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus salivarius, Acinetobacter baumannii, Klebsiella pneumoniae, Cryptococcus, Serratia viscosa, Citrobacter, Aspergillus fumigatus and Aspergillus flavus) was tested, and the results are shown in Table 7.

[0153] Table 7: Specificity detection results

[0154]

[0155] As can be seen from Table 7, the method of the application detects pathogen outside the detection target range, and the results are all negative. It shows that the above-mentioned pathogen outside the detection target range does not interfere with the test results, and the detection method described in the application has good specificity.

[0156] In summary, under the premise of realizing pathogen multi-coverage and fully automated detection, the performance is not lost, and the sensitivity, accuracy, specificity and interferent tolerance are all better than those of the products of the conventional platform.

[0157] It should be noted that the foregoing examples have been provided merely for the purposes of explanation and are in no way to be construed as limiting. The examples have been described with reference to illustrative rather than limiting examples. Modifications can be made to the examples as well as equivalents to the examples that fall within the scope of the application. Although the present application has been described with reference to particular means, materials and embodiments, from the foregoing description, one skilled in the art can effect a wide variety of modifications to the preferred embodiments of the application without departing from the scope of the application.

Claims

1. A primer probe composition for respiratory pathogen detection, characterized in that, The primer probe composition comprises one universal tag primer and 24 groups of primer probe groups respectively used for detecting respiratory pathogens to be detected; each of the primer probe groups comprises one upstream primer, one downstream primer and one probe, and the 5' ends of the upstream primer and the downstream primer are connected with the nucleotide sequence of the universal tag primer, and the nucleotide sequence of the universal tag primer is shown as SEQ ID NO: 76; the nucleotide sequences of the upstream primer, the downstream primer and the probe in the 24 groups of primer probe groups respectively used for detecting respiratory pathogens to be detected are shown as SEQ ID NO: 1-72.

2. The primer probe composition of claim 1, wherein, The 5' end of the universal tag primer is modified with biotin.

3. The primer probe composition of claim 1, wherein, The 5' end of the probe is modified with amino.

4. The primer probe composition according to claim 1 or 2, characterized in that, The primer probe composition further comprises one group of primer probe groups for the internal reference gene, which comprises one upstream primer for the internal reference gene, one downstream primer for the internal reference gene and one probe for the internal reference gene.

5. The primer probe composition of claim 4, wherein, The nucleotide sequences of the upstream primer, the downstream primer and the probe for the internal reference gene are shown as SEQ ID NO: 73-75; and the 5' end of the probe for the internal reference gene is modified with amino.

6. The primer probe composition according to claim 1 or 2, characterized in that, The respiratory pathogens to be detected comprise 23 pathogens, which are novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, enterovirus / nasovirus of respiratory infection, metapneumovirus, coronavirus 229E, coronavirus OC43, coronavirus NL63, coronavirus HKU1, bocavirus, mycoplasma pneumoniae, chlamydia pneumoniae, bordetella pertussis, legionella pneumophila, haemophilus influenzae, streptococcus pneumoniae and bordetella parapertussis.

7. A fully-automatic detection kit for respiratory pathogens, comprising the primer probe composition according to any one of claims 1-6.

8. The kit of claim 7, wherein The kit comprises nucleic acid extraction reagents, amplification reagents and hybridization color developing reagents.

9. The kit of claim 8, wherein The kit further comprises positive and negative quality control samples.

10. The kit of claim 8, wherein The nucleic acid extraction reagents comprise a lysis solution, a lysis aid, a nucleic acid aid, magnetic beads, a binding solution, a rinsing solution and an elution solution.

11. The kit of claim 10, wherein The lysis solution comprises guanidinium salt, a surfactant and a first buffer salt; the lysis aid is proteinase K; the nucleic acid aid is Carrier RNA; the magnetic beads are silicon hydroxyl magnetic beads; the binding solution is isopropanol; the rinsing solution comprises a second buffer salt and an organic alcohol; and the elution solution is water.

12. The kit of claim 11, wherein The guanidinium salt is guanidinium isothiocyanate, and the concentration of the guanidinium isothiocyanate in the lysis solution is 2-8 M; the surfactant is Triton X-100, and the concentration of the Triton X-100 in the lysis solution is 1-10 wt%; and the first buffer salt is MOPS, and the concentration of the MOPS in the lysis solution is 20-100 mM.

13. The kit of claim 11, wherein The second buffer salt is MOPS, and the concentration of the MOPS in the rinsing solution is 5-20 mM; the organic alcohol is ethanol, and the volume concentration of the ethanol is 40-70%.

14. The kit of claim 8, wherein The amplification reagent comprises reverse transcriptase, Taq enzyme, UNG enzyme, dNTPs, specific primers and universal tag primers; wherein the specific primers are 25 upstream primers and 25 downstream primers in the primer probe composition for the internal reference gene and the respiratory pathogen to be detected.

15. The kit of claim 14, wherein The content of the reverse transcriptase in the amplification reagent is 1-10 U; the content of the Taq enzyme is 1-4 U, the content of the UNG enzyme is 0.03-1 U, the content of the dNTPS is 0.2-0.3 mM, the content of each of the specific primers is independently 0.005-5 μM, and the content of the universal tag primer is 0.1-5 μM.

16. The kit of claim 8, wherein The hybridization chromogenic reagent comprises a hybridization membrane, a hybridization rinsing solution, an enzyme binding solution and a chromogenic solution, wherein the hybridization membrane is fixed with 25 probes in the primer probe composition for the internal reference gene and the respiratory pathogen to be detected.

17. The kit of claim 16, wherein The content of each probe on the hybridization membrane is 0.2-50 μM.

18. The kit of claim 16, wherein The hybridization membrane is a nylon membrane; the hybridization rinsing solution comprises sodium dodecyl sulfate and a third buffer salt; the enzyme binding solution comprises streptavidin-labeled horseradish peroxidase; and the chromogenic solution comprises urea peroxide, a fourth buffer salt and tetramethylbenzidine.

19. The kit of claim 18, wherein The concentration of the sodium dodecyl sulfate in the hybridization rinsing solution is 5-15 wt%; the third buffer salt is phosphate, and the concentration of the phosphate in the hybridization rinsing solution is 10-100 mM; the concentration of the streptavidin-labeled horseradish peroxidase in the enzyme binding solution is (0.1-1) μg / mL; the concentration of urea peroxide in the chromogenic solution is 0.01-0.1 wt%; the fourth buffer salt is citrate, and the concentration of the citrate in the chromogenic solution is 5-50 mM; and the concentration of tetramethylbenzidine in the chromogenic solution is 0.01-0.05 wt%.

20. The kit of claim 16, wherein The hybridization membrane is further fixed with a chromogenic quality control probe.

21. The kit of claim 20, wherein The content of the chromogenic quality control probe on the hybridization membrane is 0.2-50 μM.

22. The kit of claim 9, wherein The positive quality control is a false virus of the influenza A virus and the influenza B virus with the detection results of the influenza A virus and the influenza B virus being positive; and the negative quality control is a sample storage solution containing HEK293 cells with the detection results of the respiratory pathogen to be detected being negative.

23. A method for detecting a respiratory pathogen for non-disease diagnosis purposes by using the kit according to any one of claims 8-22.

24. The method of claim 23, wherein, The method comprises the following steps: S1, obtaining target nucleic acid in a sample to be detected by using the nucleic acid extraction reagent; S2, mixing the target nucleic acid with the amplification reagent to perform multiplex PCR amplification to obtain an amplification product; S3, performing hybridization and chromogenic reaction on the amplification product by using the hybridization chromogenic reagent, and interpreting the chromogenic result.

25. The method according to claim 24, in step S2, the multiplex PCR amplification is completed in a single tube.

26. The method of any of claims 23-25, wherein, The method is automatically completed by a fully automatic device. The method is automatically completed by a fully automatic device.

Images