Nucleic acid combination product for detecting multiple respiratory pathogens, kit containing nucleic acid combination product and method

Abstract

The invention discloses a nucleic acid combination product for detecting various respiratory pathogens, a kit containing the nucleic acid combination product and a method. The nucleic acid combination product comprises a primer pair and a probe, wherein the nucleotide sequences of the primer pair and the probe are respectively shown as SEQ ID NO: 1-21. According to the nucleic acid combination product, on the technical basis of Taqman fluorescent quantification, seven targets can be detected by one tube, so that the time and energy of operators are greatly shortened, and the nucleic acid combination product is convenient and rapid, is not easy to make mistakes, and is simpler and easier to understand in result interpretation.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to nucleic acid combination products for detecting multiple respiratory pathogens, kits containing such products, and methods thereof. Background Technology

[0002] The novel coronavirus can cause severe acute respiratory syndrome (SERS). Variant strains of this virus, such as Alpha, Beta, Gamma, Delta, and Omicron, are highly contagious and spread rapidly globally. All individuals can be infected with the novel coronavirus, but the mortality rate is higher among the elderly and those with underlying chronic diseases. Furthermore, vaccination against the novel coronavirus can provide some immunity. The main clinical symptoms of novel coronavirus infection are fever, dry cough, and fatigue; nasal congestion, runny nose, sore throat, and decreased or lost sense of smell and taste may also occur. Some patients may not show any clinical symptoms during the course of the disease; these are called asymptomatic carriers. Severe cases may develop acute respiratory distress syndrome (ARDS) or septic shock, and even death. This disease is contagious; the main source of infection is infected patients, and the virus is infectious during the incubation period.

[0003] Influenza A virus is a common influenza virus. It is most prone to mutation. Avian influenza (AH) is an acute respiratory infectious disease caused by certain strains of certain subtypes of avian influenza A virus. After viral gene mutation, it can infect humans. Symptoms after infection mainly include high fever, cough, runny nose, and muscle pain. Most cases are accompanied by severe pneumonia. In severe cases, multiple organ failure, such as heart and kidney failure, can lead to death, resulting in a high mortality rate.

[0004] Human rhinovirus (HRV) is a species of rhinovirus belonging to the family Picornaviridae and the genus Rhinovirus. It is the main pathogen causing the common cold in humans. HRV infection often manifests as cold symptoms. Approximately 10%–20% of colds in adults are caused by HRV infection, while 15%–30% of colds in infants and young children are caused by HRV. HRV infection is self-limiting, but it can sometimes cause serious complications such as asthma, congestive heart failure, bronchiectasis, and cystic fibrosis. Furthermore, HRV often co-infects with other respiratory viruses, such as respiratory syncytial virus (RSV), adenovirus, parainfluenza virus, coronavirus, and enterovirus.

[0005] Influenza B virus, short for influenza virus, is a highly contagious acute respiratory infectious disease caused by the influenza virus. Influenza B virus often causes localized epidemics. The outer layer of the influenza virus has two different glycoproteins forming radial projections: hemagglutinin (H) and neuraminidase (N). Antigenic variation of the influenza virus refers to changes in the structure of the H and N antigens. Antigenic variation of the influenza B virus is very slow. If both H and N undergo significant changes, a new subtype (qualitative change) is created, such as H2N2 (A2 subtype) in 1957 and H3N2 (A3 subtype) after 1968. Influenza B viruses also mutate among themselves, but these changes are not classified as subtype transitions.

[0006] Respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus of the family Pneumoviridae. It is an enveloped, non-segmented, negative-sense RNA virus. It primarily causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months of age, and upper respiratory tract infections such as rhinitis and colds in older children and adults. RSV infection is the leading cause of hospitalization for viral respiratory infections in infants and young children, seriously endangering their health, especially premature infants and infants with congenital heart disease or primary immunodeficiency.

[0007] Mycoplasma pneumoniae (M. pneumoniae) is the pathogen causing mycoplasma pneumonia in humans. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchopneumonia, known as primary atypical pneumonia. It is mainly transmitted through droplets, with an incubation period of 2-3 weeks, and the highest incidence is in adolescents. Clinical symptoms are usually mild, or even asymptomatic; if present, they are only common respiratory symptoms such as headache, sore throat, fever, and cough, although there have been isolated reports of death. It can occur in all seasons, but is more common in autumn and winter.

[0008] Adenovirus is a non-enveloped particle with a diameter of 70-90 nm, composed of 252 capsomeres arranged in an icosahedral pattern. Each capsomere has a diameter of 7-9 nm. The capsid contains a linear double-stranded DNA molecule, approximately 30-47 kb, with inverted repeat sequences of about 100 bp at each end. Because the 5' end of each DNA strand is covalently bound to a protein molecule with a relative molecular mass of 55 x 10³ Da, a circular structure of the double-stranded DNA can occur.

[0009] Currently, multiplex nucleic acid detection technologies for respiratory pathogens have made some progress. For example, some reports utilize multiple fluorescent channels to label corresponding detection targets, and monitor the amplification of different targets by monitoring changes in fluorescence signals within different channels, thus enabling the detection of multiple pathogenic microorganisms. However, its limitation is that a single system can only detect a maximum of 5 respiratory pathogens, and it cannot detect combinations of more than 5 pathogens in one tube. Summary of the Invention

[0010] Therefore, it is necessary to provide nucleic acid combination products for detecting multiple respiratory pathogens, as well as kits and methods containing them.

[0011] The first aspect of this application provides a nucleic acid combination product for detecting multiple respiratory pathogens, comprising the following primer and probe set:

[0012] Primer and probe set 1: The sequences are shown in SEQ ID NO: 1~3, which are primer pairs and probes used to detect the novel coronavirus;

[0013] Primer and probe set 2: The sequences are shown in SEQ ID NO: 4~6, which are primer pairs and probes used to detect influenza A virus;

[0014] Primer and probe set 3: The sequences are shown in SEQ ID NO: 7~9, and the primer pairs and probes are used to detect human rhinovirus.

[0015] Primer and probe set 4: The sequences are shown in SEQ ID NO: 10~12, which are primer pairs and probes used to detect influenza B virus;

[0016] Primer and probe set 5: The sequences are shown in SEQ ID NO: 13~15, which are primer pairs and probes used to detect respiratory syncytial virus;

[0017] Primer-probe set 6: The sequences are shown in SEQ ID NO: 16~18, representing primer pairs and probes for detecting Mycoplasma pneumoniae; and,

[0018] Primer and probe set 7: The sequences are shown in SEQ ID NO: 19~21, which are primer pairs and probes used to detect adenovirus.

[0019] In some implementations, primer pairs and probes with sequences such as SEQ ID NO: 22-24 are also included.

[0020] In some embodiments, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0021] In some embodiments, the fluorescent group includes one or more of FAM, HEX, ROX, CY5, QUSAR705, ATTO425, CY7, and AF405.

[0022] In some embodiments, the quenching group includes one or more of BHQ1, BHQ2, and TAMRA.

[0023] The second aspect of this application provides a kit for detecting multiple respiratory pathogens, including the nucleic acid combination product described in the first aspect of this application.

[0024] In some implementations, one or more of nucleic acid extraction reagents and PCR amplification reagents are also included.

[0025] In some embodiments, the PCR amplification reagent includes PCR buffer, DNA polymerase, reverse transcriptase, dNTPs, dUTPs, and Mg. 2+ One or more of them.

[0026] In some embodiments, the nucleic acid combination product and the PCR amplification reagent each independently include a lyophilization protectant and / or a lyophilization excipient, and the nucleic acid combination product and the PCR amplification reagent are packaged in the form of lyophilized reagents.

[0027] In some embodiments, the freeze-drying protectant includes trehalose.

[0028] In some embodiments, the lyophilization excipient includes polyethylene glycol.

[0029] In some implementations, one or more of positive and negative control samples are also included.

[0030] The third aspect of this application provides a method for detecting multiple respiratory pathogens, the method comprising the following operations in a closed reaction tube: pretreating a sample to be tested with a nucleic acid release reagent, and quantitatively mixing the resulting pretreatment product with the nucleic acid combination product described in the first aspect of this application to achieve detection of nucleic acids in the sample to be tested.

[0031] In some embodiments, in the mixed reaction system, the working concentration of each primer is 200 nM to 400 nM, and the working concentration of each probe is 100 nM to 200 nM.

[0032] In some implementations, the sample to be tested is a swab sample; alternatively, the swab sample is a throat swab or a nasal swab.

[0033] The nucleic acid combination product provided in this application, based on TaqMan fluorescence quantitative PCR technology, enables the detection of seven targets in a single tube, significantly reducing the time and effort required by operators. It is convenient, quick, and less prone to errors, and the results are easier to interpret. The introduction of lyophilization technology improves reagent stability to a certain extent and allows for storage and transportation at room temperature, greatly reducing reagent costs. Furthermore, lyophilized reagents are simpler and easier to handle during use. In addition, it exhibits strong anti-interference capabilities and high sensitivity. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0035] Figure 1 This is a schematic diagram illustrating a novel coronavirus positive result in one embodiment of this application;

[0036] Figure 2 This is a schematic diagram illustrating a positive influenza A virus in one embodiment of this application;

[0037] Figure 3 This is a schematic diagram illustrating a positive human rhinovirus test result in one embodiment of this application;

[0038] Figure 4 This is a schematic diagram illustrating a positive influenza B virus in one embodiment of this application;

[0039] Figure 5 This is a schematic diagram illustrating a respiratory syncytial virus (RSV) positive result in one embodiment of this application.

[0040] Figure 6 This is a schematic diagram illustrating a positive result for Chlamydia pneumoniae in one embodiment of this application;

[0041] Figure 7 This is a schematic diagram illustrating an adenovirus positive result in one embodiment of this application;

[0042] Figure 8 This is a schematic diagram illustrating a positive result of combined target detection in one embodiment of this application;

[0043] Figure 9 This is the result of novel coronavirus sensitivity detection in one embodiment of this application;

[0044] Figure 10 This is the result of the sensitivity test for influenza A virus in one embodiment of this application;

[0045] Figure 11 This is the result of human rhinovirus sensitivity detection in one embodiment of this application;

[0046] Figure 12 This is the result of the influenza B virus sensitivity test in one embodiment of this application;

[0047] Figure 13 This is the result of respiratory syncytial virus sensitivity detection in one embodiment of this application;

[0048] Figure 14 This is the result of Mycoplasma pneumoniae sensitivity detection in one embodiment of this application;

[0049] Figure 15 This is the result of adenovirus sensitivity detection in one embodiment of this application;

[0050] Figure 16 This is the result of joint target sensitivity detection in one embodiment of this application;

[0051] Figure 17 This is a specific detection result in one embodiment of this application;

[0052] Figure 18 This is the result of anti-interference detection of the novel coronavirus in one embodiment of this application;

[0053] Figure 19 This is the result of the anti-interference detection of influenza A virus in one embodiment of this application;

[0054] Figure 20 This is the result of the human rhinovirus anti-interference detection in one embodiment of this application;

[0055] Figure 21 This is the result of the anti-interference detection of influenza B virus in one embodiment of this application;

[0056] Figure 22 This is the result of the respiratory syncytial virus anti-interference detection in one embodiment of this application;

[0057] Figure 23 This is the result of the anti-interference detection of Mycoplasma pneumoniae in one embodiment of this application;

[0058] Figure 24 This is the result of adenovirus anti-interference detection in one embodiment of this application;

[0059] Figure 25 The test results are for other primers and probes used in one embodiment of this application;

[0060] Figure 26 The morphology of each lyophilized reagent in Comparative Example 2;

[0061] Figure 27The results are for the detection of the lyophilized reagent prepared using raffinose in Comparative Example 2;

[0062] Figure 28 The results are for the lyophilized reagent prepared using sucrose in Comparative Example 2.

[0063] Figure 29 The results are for the lyophilized reagent prepared using dextran in Comparative Example 2.

[0064] Figure 30 The results are for the lyophilized reagent prepared using maltose in Comparative Example 2. Detailed Implementation

[0065] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0067] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0068] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0069] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0070] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0071] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0072] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0073] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0074] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0075] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0076] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0077] In multi-pathogen nucleic acid co-detection technologies, the reaction system contains a large number of primers and probes, which are prone to dimerization or non-specific binding, leading to non-specific amplification. Although this can be predicted and mitigated to some extent during the design phase, the design difficulty increases significantly under the practical conditions of multiplex detection. The core challenge lies in ensuring the conservation of primer and probe sequences to guarantee detection accuracy while minimizing their mutual interference. This design difficulty often manifests in practical applications as incomplete amplification curves, low amplification efficiency, poor repeatability, and missed detection of some targets, severely impacting the overall detection effect and reliability.

[0078] Currently, multiplex nucleic acid detection technologies for respiratory pathogens have made some progress. For example, some reports utilize multiple fluorescent channels to label corresponding detection targets, and monitor the amplification of different targets by monitoring changes in fluorescence signals within different channels, thus enabling the detection of multiple pathogenic microorganisms. However, its limitations lie in the fact that a single system can only detect a maximum of five respiratory pathogens, and it cannot detect combinations of more than seven pathogens in one tube. In addition, it has poor resistance to interference from some interfering substances.

[0079] Based on this, the embodiments of this application provide at least nucleic acid combination products for detecting multiple respiratory pathogens, kits containing such products, and methods.

[0080] In a first aspect of this application, a nucleic acid combination product for detecting multiple respiratory pathogens is provided, comprising primer pairs and probes with nucleotide sequences as shown in SEQ ID NO: 1-21, respectively.

[0081] In some implementations, the following primer-probe set is included:

[0082] Primer and probe set 1: The sequences are shown in SEQ ID NO: 1~3, which are primer pairs and probes used to detect the novel coronavirus;

[0083] Primer and probe set 2: The sequences are shown in SEQ ID NO: 4~6, which are primer pairs and probes used to detect influenza A virus;

[0084] Primer and probe set 3: The sequences are shown in SEQ ID NO: 7~9, and the primer pairs and probes are used to detect human rhinovirus.

[0085] Primer and probe set 4: The sequences are shown in SEQ ID NO: 10~12, which are primer pairs and probes used to detect influenza B virus;

[0086] Primer and probe set 5: The sequences are shown in SEQ ID NO: 13~15, which are primer pairs and probes used to detect respiratory syncytial virus;

[0087] Primer-probe set 6: The sequences are shown in SEQ ID NO: 16~18, representing primer pairs and probes for detecting Mycoplasma pneumoniae; and,

[0088] Primer and probe set 7: The sequences are shown in SEQ ID NO: 19~21, which are primer pairs and probes used to detect adenovirus.

[0089] In some implementations, the nucleic acid combo product further includes primer pairs and probes for detecting the internal standard, with sequences shown in SEQ ID NO: 22-24, respectively.

[0090] In some embodiments, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. Exemplarily, all of the above probes use dual quenching groups to enhance the quenching effect and improve detection sensitivity and signal-to-noise ratio.

[0091] In some embodiments, the fluorescent group includes one or more of FAM, HEX, ROX, CY5, QUSAR705, ATTO425, CY7 and AF405.

[0092] In some embodiments, the quenching group includes one or more of BHQ1, BHQ2, and TAMRA.

[0093] In a second aspect of this application, a kit for detecting multiple respiratory pathogens is provided, comprising the above-described nucleic acid combination product.

[0094] In some embodiments, the kit further includes one or more of nucleic acid extraction reagents and PCR amplification reagents. Further, the PCR amplification reagents include PCR buffer, DNA polymerase, reverse transcriptase, dNTPs, dUTPs, and Mg. 2+ One or more of them.

[0095] In some embodiments, the nucleic acid combination product and the PCR amplification reagent each independently include a lyophilization protectant and / or a lyophilization excipient, and the nucleic acid combination product and the PCR amplification reagent are packaged in the form of lyophilized reagents. Further, the lyophilization protectant includes trehalose, and the lyophilization excipient includes polyethylene glycol.

[0096] In some implementations, the kit also includes one or more of a positive control and a negative control.

[0097] In a third aspect of this application, a lyophilized PCR premix is ​​provided, comprising the aforementioned nucleic acid product, lyophilization protectant, lyophilization excipient, and PCR amplification reagent, wherein the PCR amplification reagent comprises PCR buffer, DNA polymerase, UDG enzyme, dNTPs, dUTPs, and Mg. 2+ One or more of the following, the freeze-drying protectant includes trehalose, and the freeze-drying excipient includes polyethylene glycol.

[0098] In a third aspect of this application, a method for detecting multiple respiratory pathogens is provided, comprising the following operations in a closed reaction tube: placing the sample to be tested in a nucleic acid release reagent for pretreatment, and quantitatively mixing the resulting pretreatment product with a lyophilized PCR premix as defined above to achieve detection of nucleic acids in the sample to be tested.

[0099] In some implementations, the methods described above for detecting multiple pathogens are for non-diagnostic purposes. It is understood that they can be used for public health and food safety monitoring, as well as animal and plant quarantine and healthy aquaculture.

[0100] In some implementations, the above-described methods for detecting multiple pathogens are for diagnostic purposes.

[0101] In some embodiments, in the mixed reaction system, the working concentration of each primer is 200 nM to 400 nM, and the working concentration of each probe is 100 nM to 200 nM. Non-limitingly, the working concentration of each primer can be, but is not limited to, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, or any value or range between two of the above values; the working concentration of each probe can be, but is not limited to, 100 nM, 150 nM, 200 nM, or any value or range between two of the above values.

[0102] In this application, "working concentration" refers to the actual effective concentration of the indicator or probe in the total reaction system of quantitative real-time PCR.

[0103] In some implementations, the sample is a swab sample; further, the swab sample is a throat swab or a nasal swab.

[0104] The following are some examples.

[0105] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0106] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0107] Unless otherwise specified, all test materials used in the following examples are commercially available products.

[0108] In the following examples, extraction solution 1 was used to release nucleic acids, and its main components include surfactants, etc.; washing solution 1 and washing solution 2 were used for washing and purification, and their main components include anhydrous ethanol, etc. All the above reagents are conventional reagents in the art.

[0109] Example 1: Primer and Probe Design

[0110] The primers and probes used in this application are shown in Table 1 below:

[0111] Table 1

[0112]

[0113] The fluorescent reporter groups for probes SEQ ID NO: 3 are FAM; for probe SEQ ID NO: 6, HEX; for probe SEQ ID NO: 9, ROX; for probe SEQ ID NO: 12, CY5; for probe SEQ ID NO: 15, QUASAR 705; for probe SEQ ID NO: 18, ATTO 425; for probe SEQ ID NO: 21, CY7; and for probe SEQ ID NO: 24, AF 405. All probes use dual-quencher groups.

[0114] Example 2: Method for detecting pathogens

[0115] 1. Reagent preparation

[0116] 1.1 Remove the reagent from the packaging box and place it at room temperature until it reaches room temperature before use.

[0117] 1.2 Add 1000 μL of reconstitution solvent (TE buffer) to the positive control (lyophilized), shake well to mix, centrifuge at 2000 rpm for 10 seconds, and set aside.

[0118] 1.3 Based on the number of samples to be tested, negative controls (physiological saline), and positive controls (pseudovirus solution), take the corresponding amount of reagents for later use.

[0119] 1.4 Transfer the prepared reagents to the sample processing area for later use.

[0120] 2. Sample processing and loading

[0121] The sample used in this application was a pharyngeal swab. Viral nucleic acid was extracted using the magnetic bead method, and the following procedures were performed in the sample processing room:

[0122] 2.1 Add glass beads to a 1.5 mL cryovial. Add 300 μL of the processed sample and 300 μL of LTE buffer to the 1.5 mL cryovial containing the mixed glass beads, and grind using a grinder. After grinding, remove the cryovial and centrifuge briefly. Collect the supernatant liquid as the sample to be tested for later use.

[0123] 2.2 Take several 1.5 mL centrifuge tubes according to the number of samples to be tested, and add 300 μL of the sample to be tested to each tube;

[0124] 2.3 Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cap the tube, shake to mix for 30 s, and heat at 60 °C for 10 min.

[0125] 2.4 Let stand at room temperature for 1 minute, then centrifuge briefly at low speed. Place the centrifuge tube on a magnetic separator and slowly aspirate the waste liquid after 5 minutes (*Be careful not to touch the magnetic beads adsorbed on the inner wall of the tube).

[0126] 2.5 Add 200 μL of washing solution 1 and 600 μL of washing solution 2, vortex to mix for 30 seconds, centrifuge briefly at low speed, and then place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, then completely aspirate and discard the liquid.

[0127] 2.6 Place the centrifuge tubes in the centrifuge and centrifuge briefly at low speed. Then place the centrifuge tubes back into the magnetic separator. Magnetize for 3 minutes to completely remove the liquid from the bottom of the tubes.

[0128] 2.7 Add 30~100μL of elution buffer S (ultrapure water, use 80μL for elution), vortex and mix for 30s, wash the magnetic beads on the centrifuge tube wall to the bottom of the tube, let stand at room temperature for 3min; centrifuge briefly at low speed, place the centrifuge tube on the magnetic separator again for 3min, and then transfer the eluted nucleic acid to a clean 1.5mL centrifuge tube.

[0129] 2.8 Add the above-treated test sample (mixed by pipetting and aspirating) and 25 μL each of negative control and positive control to the PCR reaction tubes containing lyophilized reagents (Table 2), respectively, cap the tubes (if there are air bubbles, tap them with your finger to remove them), and centrifuge at 2000 rpm for 10 seconds until there are no air bubbles or obvious liquid droplets on the tube wall.

[0130] Table 2

[0131]

[0132] PCR amplification

[0133] PCR amplification was performed on PCR instruments such as the SLAN-48S fully automated medical PCR analysis system, following a specific temperature and time setting program. The amplification program of this application is shown in Table 3 below.

[0134] Table 3

[0135]

[0136] The interpretation of the test results is shown in Tables 4 and 5 below.

[0137] Samples with a typical S-shaped amplification curve detected in the FAM channel and Ct≤40 are reported as positive for novel coronavirus; samples without a typical S-shaped amplification curve detected in the FAM channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for novel coronavirus.

[0138] Samples with a typical S-type amplification curve detected in the HEX channel and Ct≤40 are reported as positive for influenza A virus; samples without a typical S-type amplification curve detected in the HEX channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for influenza A virus.

[0139] Samples with a typical S-type amplification curve detected in the ROX channel and Ct≤40 are reported as positive for human rhinovirus; samples without a typical S-type amplification curve detected in the ROX channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for human rhinovirus.

[0140] Samples with a typical S-type amplification curve detected in the CY5 channel and Ct≤40 are reported as positive for influenza B virus; samples without a typical S-type amplification curve detected in the CY5 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for influenza B virus.

[0141] Samples with a typical S-type amplification curve detected in the QUASAR 705 channel and Ct≤40 are reported as positive for respiratory syncytial virus (RSV); samples without a typical S-type amplification curve detected in the QUASAR 705 channel, or with Ct>40 and an amplification curve in the AF 405 channel and Ct≤40, are reported as negative for RSV.

[0142] For samples where a typical S-type amplification curve is detected in the ATTO 425 channel and Ct≤40, the report is positive for Mycoplasma pneumoniae; for samples where a typical S-type amplification curve is not detected in the ATTO 425 channel, or Ct>40, and an amplification curve is present in the AF 405 channel and Ct≤40, the report is negative for Mycoplasma pneumoniae.

[0143] Samples with a typical S-type amplification curve detected in the CY7 channel and Ct≤40 are reported as adenovirus positive; samples without a typical S-type amplification curve detected in the CY7 channel, or Ct>40, but with an amplification curve in the AF 405 channel and Ct≤40, are reported as adenovirus negative.

[0144] Table 4

[0145]

[0146] Table 5

[0147]

[0148] Example 3: Detection results of the test sample in this application

[0149] Using the method described in Example 2, one sample each of novel coronavirus, influenza A virus, human rhinovirus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, and adenovirus (all real human pharyngeal swab samples, directly spotted after release with nucleic acid release agent) were used for PCR detection on the Hongshi Real-Time PCR instrument. The results are as follows. Figures 1-8 As shown.

[0150] Example 4: Sensitivity of this application

[0151] Serially diluted plasmids were taken at concentrations of 5000, 2500, 1000, 500, and 50 copies / mL, and detection was performed at each of the five concentrations. 25 µL of each concentration was used as a template. The results show that the method of this application has high sensitivity, and the detection concentration can reach 200 copies / mL. Figures 9-16 These are the results of tests for novel coronavirus, influenza A virus, human rhinovirus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, adenovirus, and combined target tests.

[0152] Example 5, Specificity of this application

[0153] Using the method described in Example 2, other pathogen standards were added to the pharyngeal swab sample and mixed thoroughly for nucleic acid extraction. The system in this application showed no non-specific amplification in Escherichia coli, Streptococcus pyogenes, Acinetobacter baumannii, Pneumocystis jirovecii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Epstein-Barr virus, human metapneumovirus, measles virus, mumps virus, norovirus, rotavirus, Candida albicans, and Mycobacterium tuberculosis, demonstrating high specificity. Detection results are shown below. Figure 17 This indicates that there is no cross-reaction with other pathogens.

[0154] Example 6: Anti-interference capability of this application

[0155] Using the method described in Example 2, the sample was a spurious virus simulation sample. The spurious virus simulation sample was diluted with a solution containing interfering substances. The system described in this application exhibited strong anti-interference ability in 50 μg / mL dexamethasone, 50 μg / mL cefotaxime hydrochloride, 100 μg / mL ribavirin, 100 μg / mL azithromycin, 320 μg / mL budesonide, 125 μg / mL phenformin, 50 μg / mL beclomethasone, 100 μg / mL mometasone, 200 μg / mL fluticasone, 200 μg / mL histamine hydrochloride, 100 μg / mL lopinavir, 100 μg / mL triamcinolone, 100 μg / mL arbidol, and 10 μg / mL heme. (See [link to relevant documentation]). Figures 18-24 .

[0156] Comparative Example 1: Using other primers and probes

[0157] Based on the principle of complementary base pairing, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful design and validation of primers and probes are necessary.

[0158] The inventors also designed other primers and probes (see Table 6 below) to form different detection systems, using the method of Example 2, for the joint detection of targets including novel coronavirus, influenza A virus, human rhinovirus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, and adenovirus. The joint detection sample was an artificially simulated low-concentration sample, prepared by mixing novel coronavirus, influenza A virus, human rhinovirus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, and adenovirus, with a concentration of approximately 500 copies / mL. The results showed that only partial amplification curves appeared, and the amplification curves had low amplification and poor repeatability, and false negatives occurred, such as... Figure 25 As shown, the overall detection effect is poor.

[0159] Table 6

[0160]

[0161] Comparative Example 2 used other lyophilization auxiliary reagents

[0162] In the preparation of lyophilized reagents for nucleic acid detection, lyophilization auxiliary reagents (protectants and excipients) are usually added. Protectants protect active ingredients such as nucleases, primers, and probes from damage during lyophilization and ensure reagent stability during long-term storage. Excipients maintain the physical structure of the reagent, prevent disintegration after lyophilization, and rapidly restore activity upon reconstitution, thereby improving batch consistency and detection sensitivity. However, improper use of auxiliary reagents can cause the lyophilized reagent to collapse, or even lead to a decline in detection performance. To ensure optimal stability, reconstitution efficiency, and detection performance after lyophilization, it is necessary to repeatedly optimize the ratio and amount of auxiliary reagents, gradually adjust the concentration of each component, and compare the protective effects of different formulations to select the optimal one.

[0163] The inventors also added other lyophilized auxiliary reagents to form different detection systems, which are also used for the joint detection of targets such as novel coronavirus, influenza A virus, human rhinovirus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, and adenovirus. Specifically:

[0164] (1) Lyophilized reagent form

[0165] Referring to Table 2 in Example 2, the inventors replaced trehalose with dextran, sucrose, and maltose, and polyethylene glycol with raffinose, respectively, to prepare PCR reaction lyophilized reagents. The results showed that, compared to the PCR reaction lyophilized reagents in Example 2 of this application, the above-mentioned PCR reaction lyophilized reagents exhibited shrunken morphology, rough and non-rounded surfaces, and poor overall appearance. Figure 26 As shown.

[0166] (2) Detection performance of lyophilized reagents

[0167] The artificially simulated low-concentration sample (same as Comparative Example 1) was tested using the above-described lyophilized reagent, following the same procedure as in Example 2. The results are as follows: Figures 27-30 As shown, some curves fluctuate greatly in the early stage and are low, with the highest point of the curve being below the threshold line, indicating that the target point corresponding to this curve was missed and the detection performance is poor.

[0168] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0169] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A nucleic acid combination product for detecting multiple respiratory pathogens, characterized in that, It includes the following primer and probe set: Primer and probe set 1: The sequences are shown in SEQ ID NO: 1~3, which are primer pairs and probes used to detect the novel coronavirus; Primer and probe set 2: The sequences are shown in SEQ ID NO: 4~6, which are primer pairs and probes used to detect influenza A virus; Primer and probe set 3: The sequences are shown in SEQ ID NO: 7~9, and the primer pairs and probes are used to detect human rhinovirus. Primer and probe set 4: The sequences are shown in SEQ ID NO: 10~12, which are primer pairs and probes used to detect influenza B virus; Primer and probe set 5: The sequences are shown in SEQ ID NO: 13~15, which are primer pairs and probes used to detect respiratory syncytial virus; Primer-probe set 6: The sequences are shown in SEQ ID NO: 16~18, representing primer pairs and probes for detecting Mycoplasma pneumoniae; and, Primer and probe set 7: The sequences are shown in SEQ ID NO: 19~21, which are primer pairs and probes used to detect adenovirus.

2. The nucleic acid combination product for detecting multiple respiratory pathogens as described in claim 1, characterized in that, It also includes primer pairs and probes with sequences such as SEQ ID NO: 22~24.

3. The nucleic acid combination product for detecting multiple respiratory pathogens as described in claim 1 or 2, characterized in that, The probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.

4. The nucleic acid combination product for detecting multiple respiratory pathogens as described in claim 3, characterized in that, One or more of the following conditions must be met: The fluorescent group includes one or more of FAM, HEX, ROX, CY5, QUSAR705, ATTO425, CY7 and AF405; The quenching group includes one or more of BHQ1, BHQ2 and TAMRA.

5. A kit for detecting multiple respiratory pathogens, characterized in that, The kit includes the nucleic acid combination product as described in any one of claims 1 to 4, and further includes one or more of nucleic acid extraction reagents and PCR amplification reagents.

6. The kit for detecting multiple respiratory pathogens as described in claim 5, characterized in that, One or more of the following conditions must be met: (1) The PCR amplification reagents include PCR buffer, DNA polymerase, reverse transcriptase, dNTPs, dUTPs and Mg 2+ One or more of the following; (2) The nucleic acid combination product and the PCR amplification reagent each independently include a lyophilization protectant and / or a lyophilization excipient, and the nucleic acid combination product and the PCR amplification reagent are packaged in the form of lyophilized reagents; (3) The kit also includes one or more of positive control and negative control.

7. The kit for detecting multiple respiratory pathogens as described in claim 6, characterized in that, The freeze-drying protectant includes trehalose and / or the freeze-drying excipient includes polyethylene glycol.

8. A method for detecting multiple respiratory pathogens, characterized in that, The method includes performing the following operations in a closed reaction tube: placing the sample to be tested in a nucleic acid release reagent for pretreatment, and quantitatively mixing the resulting pretreatment product with the nucleic acid combination product according to any one of claims 1 to 4 to achieve nucleic acid detection of the sample to be tested.

9. The method for detecting multiple respiratory pathogens as described in claim 8, characterized in that, In the mixed reaction system, the working concentration of each primer is 200nM~400nM, and the working concentration of each probe is 100nM~200nM.

10. The method for detecting multiple respiratory pathogens as described in claim 8 or 9, characterized in that, The samples to be tested include throat swabs or nasal swabs.

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