Hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hydrogel as well as a preparation method and application thereof. The hydrogel contains a primer group for detecting a to-be-detected target, according to the hydrogel prepared by the invention, primers or probes are embedded in a hydrogel material, and the primers or probes are dispersed in different reaction areas, so that the mutual interference between the primers or probes is reduced, the detection sensitivity and accuracy can be effectively improved, and the experiment difficulty is reduced; the curve base line obtained through melting curve analysis is clean, the background is low, and the melting peak pattern is clear.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a molecular biology technique that utilizes the principle of DNA double-strand replication to replicate specific DNA fragments outside the organism, and has been widely used in genetics and disease diagnosis. Multiplex PCR, which simultaneously amplifies multiple target-specific sequences in a single-tube single-PCR reaction using multiple pairs of primers, is a more efficient PCR method than standard PCR using a single pair of primers. Simultaneous amplification of different target nucleic acids reduces the cost and time consumption of PCR, and multiplex PCR has been used in many fields of DNA detection, including microbial identification, mutation detection and polymorphism analysis, genotyping, and gene expression.

[0003] Common methods for monitoring the accumulation of amplicons in real time or at the end of amplification include: 1. Probe-based real-time fluorescent PCR: Taqman probes bind in the middle of the amplification region before the extension of the amplification primers, and the 5' exonuclease activity of the polymerase enzyme cleaves the probe, releasing the fluorescent group on the probe. For multiplex real-time fluorescent PCR based on Taqman or molecular beacon binding, different fluorescent groups are used to label the target-specific probes, and then the accumulation of different fluorescent signals during amplification is detected to identify the target. However, due to the limitation of the number of fluorescent labels and the number of fluorescence channels of the equipment, the number of targets detected in a single tube by this multiplex detection method is generally not more than 6. 2. Capillary electrophoresis: Capillary electrophoresis analysis can distinguish different lengths of fragment products to identify different target amplicons. This method involves product analysis, and the opening operation of the product is prone to contamination, and the cost of the equipment required for capillary electrophoresis analysis is also relatively high. 3. Melting curve method: Using probes or intercalating dyes such as SYBR Green, different target products can be distinguished by melting curve analysis due to the different melting temperatures or Tm of amplicons with different sequences.

[0004] The melting curve analysis technology combined with the use of probes can realize the detection and identification of more than twenty targets, and different probes or molecular beacons labeled with different fluorescent markers are used to distinguish different products according to the melting curve of the probe. In this method, the probe or molecular beacon is in a free state, and the quenching group it carries will close the fluorescent group it carries when the molecular beacon is in a curled state, but under the PCR reaction condition, the non-specific binding with part of the sequence will cause the fluorescence closure to be incomplete, thereby appearing a non-specific peak unrelated to the target sequence, affecting the accuracy and precision of the analysis result. The free probe or molecular beacon may combine with the primer end of the uncut probe primer to form a double strand, thereby affecting the cutting position of the enzyme when the probe end binds to the target sequence, resulting in a difference between the length of the cut product, i.e. the free probe primer, and the preset length. The length difference appears randomly and can affect the extension temperature of the probe primer after binding to the probe beacon, thereby causing a double peak / broad peak phenomenon in the final melting curve, affecting the accuracy and precision of the analysis result. In addition, the number of primers in multiplex amplification is large, and the mutual interference between primers also increases the design and experimental difficulty. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a hydrogel.

[0006] The present application also proposes a preparation method of the above-mentioned hydrogel.

[0007] The present application also proposes an application of the above-mentioned hydrogel.

[0008] The present application also proposes a method for detecting nucleic acid molecules.

[0009] The present application also proposes a primer combination for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes.

[0010] The present application also proposes a kit.

[0011] The present application also provides an application of the above-mentioned primer combination or kit.

[0012] The present application also provides a method for detecting novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene for non-diagnostic purposes.

[0013] The present application also provides a system for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes.

[0014] According to the first aspect of the present application, a hydrogel is provided, wherein the hydrogel contains a forward primer or a reverse primer in a primer set for detecting a target to be detected; and the 5' end of the forward primer or the reverse primer is modified by Acrydite.

[0015] The hydrogel according to the embodiments of the present application has at least the following beneficial effects:

[0016] Since multiple primers or probes are contained in the multiplex PCR system, the reaction system is complex, and multiple target points may not be compatible with each other in the amplification conditions, the amplification target points may inhibit each other, and the primers or probes may interfere with each other, which may result in a decrease in detection sensitivity, poor specificity, low accuracy, and difficulty in adjusting the system, and the experimental difficulty is increased sharply. In order to eliminate the influence of the mutual interference between the primers or probes in the multiplex PCR system on the PCR amplification, the hydrogel prepared in the present application embeds the primers or probes in the hydrogel material, disperses the primers or probes in different reaction zones, reduces the mutual interference between the primers or probes, and can effectively improve the detection sensitivity and accuracy, reduce the experimental difficulty, and obtain a clear curve baseline, low background, and clear melting peak type through melting curve analysis.

[0017] According to some embodiments of the present application, the target to be detected includes at least one of a novel coronavirus, an influenza A virus, an influenza B virus, a Mycoplasma pneumoniae, a Staphylococcus aureus, a Klebsiella pneumoniae, a Pseudomonas aeruginosa, a MecA gene, an OXA23 gene, an AmpC gene, a VIM gene, and a SHV gene.

[0018] According to some embodiments of the present application, the at least one can be one, two,..., eleven, or twelve.

[0019] According to some embodiments of the present application, when the target to be detected is a novel coronavirus, an influenza A virus, an influenza B virus, a Mycoplasma pneumoniae, a Staphylococcus aureus, a Klebsiella pneumoniae, a Pseudomonas aeruginosa, a MecA gene, an OXA23 gene, an AmpC gene, a VIM gene, and a SHV gene, the addition ratio of the forward primers or the reverse primers for detecting each target in the hydrogel is (1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2):(1-2).

[0020] According to some embodiments of the present application, the concentration of the forward primer or the reverse primer in the hydrogel is 80-120 µM.

[0021] According to some embodiments of the present application, the concentration of the forward primer or the reverse primer in the hydrogel is 90-110 µM.

[0022] According to some embodiments of the application, the concentration of the forward primer or the reverse primer in the hydrogel is 95-105 μΜ.

[0023] According to some embodiments of the application, the sequence of the forward primer of the specific primer set for detecting the novel coronavirus is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO: 2.

[0024] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting the influenza A virus is shown in SEQ ID NO: 3, and the sequence of the reverse primer is shown in SEQ ID NO: 4.

[0025] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting the influenza B virus is shown in SEQ ID NO: 5, and the sequence of the reverse primer is shown in SEQ ID NO: 6.

[0026] According to some embodiments of the application, the sequence of the forward primer of the specific primer set for detecting Mycoplasma pneumoniae is shown in SEQ ID NO: 7, and the sequence of the reverse primer is shown in SEQ ID NO: 8.

[0027] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting Staphylococcus aureus is shown in SEQ ID NO: 11, and the sequence of the reverse primer is shown in SEQ ID NO: 12.

[0028] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting Klebsiella pneumoniae is shown in SEQ ID NO: 13, and the sequence of the reverse primer is shown in SEQ ID NO: 14.

[0029] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting Pseudomonas aeruginosa is shown in SEQ ID NO: 15, and the sequence of the reverse primer is shown in SEQ ID NO: 16.

[0030] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting the MecA gene is shown in SEQ ID NO: 17, and the sequence of the reverse primer is shown in SEQ ID NO: 18.

[0031] According to some embodiments of the application, the sequence of the forward primer of the primer set for detecting the OXA23 gene is shown in SEQ ID NO: 19, and the sequence of the reverse primer is shown in SEQ ID NO: 20.

[0032] According to some embodiments of the present application, the forward primer sequence of the primer set for detecting AmpC gene is shown as SEQ ID NO: 21, and the reverse primer sequence is shown as SEQ ID NO: 22.

[0033] According to some embodiments of the present application, the forward primer sequence of the primer set for detecting VIM gene is shown as SEQ ID NO: 23, and the reverse primer sequence is shown as SEQ ID NO: 24.

[0034] According to some embodiments of the present application, the forward primer sequence of the primer set for detecting SHV gene is shown as SEQ ID NO: 25, and the reverse primer sequence is shown as SEQ ID NO: 26.

[0035] According to some embodiments of the present application, the 1-3 bp of the forward primer sequence or the reverse primer sequence is labeled with a fluorescent reporter group, and the 3' end of the forward primer sequence or the reverse primer sequence is labeled with a fluorescent quencher group.

[0036] According to some embodiments of the present application, the fluorescent reporter group comprises at least one of FAM, JOE, VIC, HEX, ROX, exasRed, Cy5.5, CY3 and CY5; and the fluorescent quencher group comprises Super Quencher.

[0037] According to some embodiments of the present application, the hydrogel further comprises a forward primer or a reverse primer in a primer set for detecting an internal reference gene, the forward primer sequence of the primer set for detecting the internal reference gene is shown as SEQ ID NO: 9, and the reverse primer sequence is shown as SEQ ID NO: 10.

[0038] According to a second aspect of the present application, a preparation method of the above hydrogel is provided, comprising the following steps: mixing a carboxymethyl cellulose solution with polyethylene glycol to obtain a mixed solution; adding a photoinitiator and an acrylate in the mixed solution to obtain a hydrogel solution; mixing the hydrogel solution with primers for detecting a target to be detected, dispensing, and obtaining a glue particle; and sequentially solidifying, washing and drying the glue particle to obtain a hydrogel containing a primer set for detecting the target to be detected.

[0039] According to some embodiments of the present application, the primer for detecting the target to be detected comprises a forward primer or a reverse primer for detecting the target to be detected.

[0040] According to some embodiments of the present application, the mass fraction of the carboxymethyl cellulose solution is 8%-12%.

[0041] According to some embodiments of the present application, the volume addition ratio of the carboxymethyl cellulose solution to polyethylene glycol is (8-12):(3-6).

[0042] According to some embodiments of the present application, the polyethylene glycol includes polyethylene glycol PEG-600, PEG-400, PEG-800.

[0043] According to some embodiments of the present application, the photoinitiator includes 2-hydroxy-2-methylprophenone, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0044] According to some embodiments of the present application, the addition amount of the photoinitiator is 4%-6%(wt) of the mixed solution.

[0045] According to some embodiments of the present application, the acrylate includes polyethylene glycol diacrylate PEG-DA400.

[0046] According to some embodiments of the present application, in the hydrogel containing primers for detecting the target to be detected, the addition volume ratio of the primers for detecting the target to be detected to the hydrogel solution is (1-3):9.

[0047] According to some embodiments of the present application, the addition volume ratio of the primers for detecting the target to be detected to the hydrogel solution is 2:9.

[0048] According to some embodiments of the present application, the concentration of the primer group in the primer solution for detecting the target to be detected is 80-120μM.

[0049] According to some embodiments of the present application, the curing method is ultraviolet curing.

[0050] According to some embodiments of the present application, the ultraviolet light used in the ultraviolet curing has a light intensity of 0.5-100mJ / cm 2 , and the irradiation time is 8-12min.

[0051] According to some embodiments of the present application, the ultraviolet light used in the ultraviolet curing has a wavelength of 206-247nm.

[0052] According to some embodiments of the present application, the reagent used in the washing includes a TE solution.

[0053] According to some embodiments of the present application, the washing time is 12-17min.

[0054] According to some embodiments of the present application, the drying time is 3-5h, and the temperature is 32-39℃.

[0055] According to a third aspect of the present application, an application of the hydrogel is provided, and the application is an application in any one of the following:

[0056] (1) detecting nucleic acid molecules;

[0057] (2) preparing products for detecting nucleic acid molecules;

[0058] (3) preparing products for PCR, melting curve analysis or real-time quantitative PCR detection.

[0059] According to some embodiments of the present application, the products are selected from reagents, kits or chips.

[0060] According to some embodiments of the present application, the detecting nucleic acid molecules comprises detecting nucleic acid molecules by using a melting curve method.

[0061] According to some embodiments of the present application, the nucleic acid molecules comprise nucleic acid molecules of novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene-containing nucleic acid molecules, OXA23 gene-containing nucleic acid molecules, AmpC gene-containing nucleic acid molecules, VIM gene-containing nucleic acid molecules and SHV gene-containing nucleic acid molecules.

[0062] According to a fourth aspect of the present application, a method for detecting nucleic acid molecules for non-disease diagnosis purposes is provided, and the method comprises the following steps: detecting a sample to be tested by using the hydrogel.

[0063] According to some embodiments of the present application, the method for detecting comprises a multi-color melting curve analysis technique.

[0064] The method for detecting nucleic acid molecules for non-disease diagnosis purposes according to the embodiments of the present application has at least the following beneficial effects:

[0065] The method of the present application combines the hydrogel with the multi-color melting curve analysis technique, can realize the detection of super-multiplex targets in a single tube, can improve the accuracy of detecting target sequences, reduce the difficulty of primer design and experiments, has no opening operation, effectively reduces environmental pollution, is easy to operate, has high detection sensitivity and accuracy, has low cost and is suitable for conventional real-time fluorescent quantitative PCR instruments.

[0066] According to a fifth aspect of the present application, a primer combination for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes is provided, which comprises a specific primer pair for detecting the novel coronavirus, a specific primer pair for detecting the influenza A virus, a specific primer pair for detecting the influenza B virus, a specific primer pair for detecting Mycoplasma pneumoniae, a specific primer pair for detecting Staphylococcus aureus, a specific primer pair for detecting Klebsiella pneumoniae, a specific primer pair for detecting Pseudomonas aeruginosa, a specific primer pair for detecting the MecA gene, a specific primer pair for detecting the OXA23 gene, a specific primer pair for detecting the AmpC gene, a specific primer pair for detecting the VIM gene, and a specific primer pair for detecting the SHV gene.

[0067] The forward primer sequence of the specific primer pair for detecting the novel coronavirus is shown as SEQ ID NO: 1, and the reverse primer sequence is shown as SEQ ID NO: 2.

[0068] The forward primer sequence of the specific primer pair for detecting the influenza A virus is shown as SEQ ID NO: 3, and the reverse primer sequence is shown as SEQ ID NO: 4.

[0069] The forward primer sequence of the specific primer pair for detecting the influenza B virus is shown as SEQ ID NO: 5, and the reverse primer sequence is shown as SEQ ID NO: 6.

[0070] The forward primer sequence of the specific primer pair for detecting Mycoplasma pneumoniae is shown as SEQ ID NO: 7, and the reverse primer sequence is shown as SEQ ID NO: 8.

[0071] The forward primer sequence of the specific primer pair for detecting Staphylococcus aureus is shown as SEQ ID NO: 11, and the reverse primer sequence is shown as SEQ ID NO: 12.

[0072] The forward primer sequence of the specific primer pair for detecting Klebsiella pneumoniae is shown as SEQ ID NO: 13, and the reverse primer sequence is shown as SEQ ID NO: 14.

[0073] The forward primer sequence of the specific primer pair for detecting Pseudomonas aeruginosa is shown as SEQ ID NO: 15, and the reverse primer sequence is shown as SEQ ID NO: 16.

[0074] The forward primer sequence of the specific primer pair for detecting the MecA gene is shown as SEQ ID NO: 17, and the reverse primer sequence is shown as SEQ ID NO: 18.

[0075] The sequence of the forward primer of the specific primer pair for detecting the OXA23 gene is shown as SEQ ID NO: 19, and the sequence of the reverse primer is shown as SEQ ID NO: 20;

[0076] The sequence of the forward primer of the specific primer pair for detecting the AmpC gene is shown as SEQ ID NO: 21, and the sequence of the reverse primer is shown as SEQ ID NO: 22;

[0077] The sequence of the forward primer of the specific primer pair for detecting the VIM gene is shown as SEQ ID NO: 23, and the sequence of the reverse primer is shown as SEQ ID NO: 24;

[0078] The sequence of the forward primer of the specific primer pair for detecting the SHV gene is shown as SEQ ID NO: 25, and the sequence of the reverse primer is shown as SEQ ID NO: 26.

[0079] According to an embodiment of the present application, a primer combination for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes has at least the following beneficial effects:

[0080] The primer combination for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes provided by the present application has the characteristics of high sensitivity, good repeatability and strong specificity. By cross-linking and fixing different primers with hydrogel, PCR reaction is carried out in the hydrogel, the space effect and charge repulsion in solid-phase PCR are eliminated, the amplification efficiency and accuracy are improved, and the primer combination can be used for specific detection of novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene.

[0081] According to some embodiments of the present application, the 5' end of the forward primer or the reverse primer has Acrydite modification.

[0082] According to some embodiments of the present application, 1-3 bp of the sequence of the forward primer or the reverse primer is labeled with a fluorescent reporter group, and the 3' end of the sequence of the forward primer or the reverse primer is labeled with a fluorescent quencher group.

[0083] According to some embodiments of the present application, the fluorescent reporter group comprises at least one of FAM, JOE, VIC, HEX, ROX, exasRed, Cy5.5, CY3 and CY5; and the fluorescent quencher group comprises Super Quencher. The present application designs specific amplification primers for targets, the length of the primers is 20-26 bp, Acrydite modification is performed at the 5' end of the forward or reverse primer, fluorescent group modification is performed at the 5' end with an interval of 1-3 bp, and quencher group Super Quencher modification is performed near the 3' end. Super Quencher is a self-quenching group, which almost does not emit fluorescence when it is in single-stranded state (primer), and emits fluorescence when it is in double-stranded state (product) after the primer binds to the target nucleic acid and the chain is extended under the action of polymerase. After the amplification reaction, melting curve analysis is performed to obtain the characteristic peak of the target amplification product and the corresponding Tm value. According to the length and GC content difference of the amplicon, primers for different targets can be designed so that the melting curve characteristic peaks of different targets can be distinguished. Then, different fluorescent reporter groups are used to label to distinguish targets with similar melting curve characteristic peak Tm values.

[0084] According to a sixth aspect of the present application, a kit is provided, which comprises the primer combination and / or the hydrogel described above.

[0085] According to some embodiments of the present application, the kit further comprises at least one of a positive control, a negative control and a PCR premix.

[0086] According to some embodiments of the present application, the PCR premix comprises at least one of a PCR Buffer, a DNA polymerase and a reverse transcriptase.

[0087] According to some embodiments of the present application, the positive control comprises a nucleic acid molecule of or containing a novel coronavirus, an influenza A virus, an influenza B virus, a Mycoplasma pneumoniae, a Staphylococcus aureus, a Klebsiella pneumoniae, a Pseudomonas aeruginosa, a MecA gene, an OXA23 gene, an AmpC gene, a VIM gene and / or a SHV gene.

[0088] According to some embodiments of the present application, the negative control comprises water.

[0089] According to some embodiments of the present application, the kit further comprises a primer set for detecting an internal reference gene, the sequence of the primer for detecting the internal reference gene is shown as SEQ ID NO: 9, SEQ ID NO: 10.

[0090] According to a seventh aspect of the present application, the use of the primer combination or kit described above is provided, the use is the use in any one of the following:

[0091] 1) detecting or assisting in detecting nucleic acid molecules of or containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene for non-diagnostic purposes;

[0092] 2) preparing products for detecting or assisting in detecting nucleic acid molecules of or containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene.

[0093] According to some embodiments of the present application, the product is selected from a reagent, a kit or a chip.

[0094] According to an eighth aspect of the present application, a method for detecting novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene for non-diagnostic purposes is provided, the method comprises the following steps: amplifying nucleic acid of a sample to be tested by using the primer combination or kit, obtaining an amplification product, obtaining a melting curve of the amplification product by using a multicolor melting curve analysis (MMCA) technology, and evaluating whether the sample to be tested contains novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene.

[0095] The method for detecting or assisting the novel coronavirus, the influenza A virus, the influenza B virus and / or the mycoplasma pneumoniae for non-diagnostic purposes according to the embodiments of the present application has at least the following beneficial effects:

[0096] The method of the present application adopts a single-tube multiplex detection, no opening operation, effectively reduces environmental pollution, convenient operation, high detection sensitivity and accuracy, low cost and adapts to conventional real-time fluorescent quantitative PCR instrument.

[0097] According to some embodiments of the present application, the amplification is a fluorescent quantitative PCR amplification.

[0098] According to some embodiments of the present application, the reaction conditions of the fluorescent quantitative PCR amplification are: 94-96℃ 2-4min; entering the pre-amplification stage: 93-96℃ 8-12s, 55-62℃ 40-50s, 12-18 cycles; entering the PCR cycle stage: 93-96℃ 8-12s, 55-62℃ 20-40s, 25-38 cycles.

[0099] According to some embodiments of the present application, the amplification procedure of the fluorescent quantitative PCR is: 95℃ 3min; entering the pre-amplification stage: 95℃ 10s, 60℃ 45s, 15 cycles; entering the PCR cycle stage: 95℃ 10s, 60℃ 30s, 30 cycles.

[0100] According to some embodiments of the present application, the melting procedure of the high-resolution melting curve method multi-color melting curve analysis technology is: setting the temperature from 58-62℃ gradually rising to 92-96℃, the rising rate is 0.01-0.03℃ / s, while continuously monitoring the fluorescence intensity, obtaining the fluorescence intensity change rate-temperature melting curve.

[0101] According to some embodiments of the present application, the melting procedure of the high-resolution melting curve method multi-color melting curve analysis technology is: setting the temperature from 60℃ gradually rising to 94℃, the rising rate is 0.02℃ / s, while continuously monitoring the fluorescence intensity, obtaining the fluorescence intensity change rate-temperature melting curve.

[0102] According to some embodiments of the present application, the evaluation of whether the sample contains the novel coronavirus, the influenza A virus, the influenza B virus, the Mycoplasma pneumoniae, the Staphylococcus aureus, the Klebsiella pneumoniae, the Pseudomonas aeruginosa, the MecA gene, the OXA23 gene, the AmpC gene, the VIM gene and / or the SHV gene includes: the TM value of the influenza B virus is 70.5℃, the TM value of the novel coronavirus is 74.4℃, the TM value of the influenza A virus is 77.6℃, the TM value of the Mycoplasma pneumoniae is 83.5℃, the TM value of the Staphylococcus aureus is 69.7℃, the TM value of the Pseudomonas aeruginosa is 76.3℃, the TM value of the Klebsiella pneumoniae is 81.1℃, the TM value of the MecA gene is 70.8℃, the TM value of the OXA23 gene is 75.1℃, the TM value of the AmpC gene is 78.9℃, the TM value of the VIM gene is 83.6℃, and the TM value of the SHV gene is 86.7℃.

[0103] According to the eleventh aspect of the present application, a system for detecting the novel coronavirus, the influenza A virus, the influenza B virus, the Mycoplasma pneumoniae, the Staphylococcus aureus, the Klebsiella pneumoniae, the Pseudomonas aeruginosa, the MecA gene, the OXA23 gene, the AmpC gene, the VIM gene and / or the SHV gene is provided, comprising:

[0104] The detection module is configured to perform fluorescent PCR amplification on the nucleic acid of the sample by using the primer combination or the kit, and obtain the melting curve of the fluorescent PCR amplification product.

[0105] The analysis module is configured to evaluate whether the sample contains the novel coronavirus, the influenza A virus, the influenza B virus, the Mycoplasma pneumoniae, the Staphylococcus aureus, the Klebsiella pneumoniae, the Pseudomonas aeruginosa, the MecA gene, the OXA23 gene, the AmpC gene, the VIM gene and / or the SHV gene.

[0106] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0107] Figure 1 The melting curve graph of the blank sample in the embodiment of the present application;

[0108] Figure 2 The melting curve graph of the negative sample in the embodiment of the present application;

[0109] Figure 3 The melting curve graph of the respiratory pathogen positive sample in the embodiment of the present application;

[0110] Figure 4The melting curve diagram of the FAM fluorescence channel of the pathogenic bacteria positive sample in the embodiment of the present application;

[0111] Figure 5 The melting curve diagram of the ROX fluorescence channel of the drug-resistant gene positive sample in the embodiment of the present application. DETAILED DESCRIPTION

[0112] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the following drawings, in which: the concept of the present application and the technical effects generated will be clearly and completely described below in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0113] The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be obtained by market purchase.

[0114] Unless otherwise specified, the nucleotide sequence is in the order of 5' to 3'.

[0115] Example 1 A primer combination for detecting respiratory pathogens, pathogenic bacteria and drug-resistant genes

[0116] This example provides a primer combination for detecting respiratory pathogens (containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae), pathogenic bacteria and drug-resistant genes. The design and screening steps of the primer combination are as follows:

[0117] (1) Design of primer combination for detecting respiratory pathogens

[0118] Through a large number of literature retrieval and analysis, at the same time through GenBank database sequence retrieval and alignment analysis, according to the conserved segment of nucleic acid sequence of new coronavirus (Severe Respiratory Syndrome Coronavirus 2, SARS-CoV-2), influenza A virus (Influence A, IVA), influenza B virus (Influence B, IVB), Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP)), a series of primers were designed, the length of the primers was 20-26 bp, Acrydite modification was carried out at the 5' end of the forward or reverse primer, and the fluorescent group modification was carried out at the 5' end with an interval of 1-3 bp, and the quencher group Super Quencher modification was carried out near the 3' end. After synthesis, the primers were screened and verified by test, and the primer group with the best effect was selected (the sequence is shown in Table 1). At the same time, human housekeeping gene GAPDH was selected as an internal reference. One of the primers for each target was modified as described above, and the fluorescent reporter group was selected as FAM.

[0119] Table 1

[0120]

[0121]

[0122] (2) Design of primer combination for detection of pathogenic bacteria and drug resistance genes

[0123] Through a large number of literature retrieval and analysis, at the same time through GenBank database sequence retrieval and alignment analysis, according to the conserved gene of pathogenic bacteria (Staphylococcus aureus (Staphylococcus aureus, Sau), Klebsiella pneumoniae (Klebsiella Pneumoniae, Kp), Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA)), specific primers were designed, and specific primers for drug resistance genes (MecA, OXA23, AmpC, VIM, SHV) were designed. Acrydite modification was carried out at the 5' end of the forward or reverse primer, and the fluorescent group modification was carried out at the 5' end with an interval of 1-3 bp, and the quencher group Super Quencher modification was carried out near the 3' end. After synthesis, the primers were screened and verified by test, and the primers with the best effect were selected (the sequence is shown in Table 2). The forward or reverse primers of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa were labeled with FAM, and the forward or reverse primers of MecA, OXA23, AmpC, VIM and SHV drug resistance genes were labeled with ROX.

[0124] Table 2

[0125]

[0126]

[0127] Example 2 A hydrogel particle for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes

[0128] The present embodiment provides a hydrogel particle for detecting respiratory pathogens, pathogenic bacteria and drug resistance genes, and the preparation method is as follows:

[0129] (1) Preparation of hydrogel

[0130] An appropriate amount of carboxymethyl cellulose is dissolved in water, stirred uniformly, and prepared into a carboxymethyl cellulose solution with a mass fraction of about 10%; then 40% (v / v) polyethylene glycol PEG-600 is added. Take 10 mL of the above mixture, add a photoinitiator 2-hydroxy-2-methylpropyl phenone with a mass fraction of 5% (4%-6% can be used), and finally add 0.5 mL of polyethylene glycol diacrylate PEG-DA400 dropwise, stir uniformly, and the glue solution needs to be prepared and used immediately.

[0131] (2) Preparation of hydrogel embedding different detection item (target) primers

[0132] The forward or reverse primer (concentration of 100 μM, specific primers are shown in Tables 1 and 2, and the primer sequence at the 5' end is modified by Acrydite) for detecting the target to be detected (including but not limited to respiratory pathogens, pathogenic bacteria and / or drug resistance genes) is mixed with the hydrogel at a volume ratio of 2:9 to prepare a hydrogel embedding different detection target primers; the hydrogel embedding different detection target primers is spotted (2-3 μL / grain) by a dispenser or a manual pipettor, sequentially subjected to ultraviolet (wavelength 206-247 nm, not less than 1 millijoule / square centimeter) curing for 10 minutes, immersed and washed with 1xTE solution for 15 min, and then placed in an oven for 4 h at 37°C for drying to obtain the hydrogel particle.

[0133] Example 3 A kit for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes

[0134] The embodiment provides a kit for detecting respiratory pathogens (novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae), pathogenic bacteria and / or drug resistance genes, which is prepared from the hydrogel particles for detecting respiratory pathogens, pathogenic bacteria and / or drug resistance genes, 2x PCR Buffer, primer Mixture (specific primers are shown in Tables 1 and 2, the 5' end of the primer sequence (forward or reverse primer) does not have Acrydite modification, the ratio of primers of each target (which can include novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA drug resistance gene, OXA23 drug resistance gene, AmpC drug resistance gene, VIM drug resistance gene, SHV drug resistance gene) is 1:1:1:1:1:1:1:1:1:1:1:1), positive control, negative control.

[0135] The negative control is sterile nucleic acid-free water.

[0136] The positive control is a mixed sample of novel coronavirus, influenza A virus, influenza B virus and Mycoplasma pneumoniae quality control.

[0137] The recombinant vector contains Staphylococcus aureus, Klebsiella pneumoniae and / or Pseudomonas aeruginosa nucleic acid molecules.

[0138] The recombinant vector contains MecA, OXA23, AmpC, VIM and / or SHV nucleic acid molecules.

[0139] Example 4: A method for detecting respiratory pathogens (novel coronavirus, influenza A virus, influenza B virus and / or Mycoplasma pneumoniae)

[0140] The embodiment provides a method for detecting respiratory pathogens (novel coronavirus, influenza A virus, influenza B virus and / or Mycoplasma pneumoniae), which comprises the following steps:

[0141] (1) Extracting nucleic acid from the sample to be tested.

[0142] (2) Prepare the PCR amplification reaction solution according to Table 3 below.

[0143] Table 3

[0144]

[0145] The water gel particles embedded with primers and 2 μL nucleic acid of the biological sample to be detected are added to the above PCR amplification reaction solution, and the PCR amplification is carried out according to the procedure in Table 4. After the amplification product is obtained, the following procedure is used to detect the multi-color melting curve analysis technology: the instrument melting curve parameter setting temperature is gradually increased from 60 ℃ to 94 ℃ at a rate of 0.02 ℃ / s, and the fluorescence signal is collected every 10 seconds. Then the results are analyzed by the gene scanning software module, and the results are determined by the following steps: signal normalization processing, temperature coordinate translation and differentiation. Figure Four

[0146] Table 4

[0147]

[0148] In the above result determination, the TM value of the influenza B virus is 70.5 ℃, the TM value of the novel coronavirus is 74.4 ℃, the TM value of the influenza A virus is 77.6 ℃, and the TM value of the Mycoplasma pneumoniae is 83.5 ℃. The TM value of the internal reference characteristic peak is 80.5 ℃.

[0149] Performance evaluation of detecting respiratory pathogens in Example 5

[0150] 1. Respiratory tract joint detection sample test

[0151] (1) Blank sample

[0152] The blank sample is a sample extracted with sterile nucleic acid-free water.

[0153] (2) Negative sample

[0154] The negative sample is a healthy human throat swab sample.

[0155] (3) Positive sample

[0156] The positive sample is a mixed sample of novel coronavirus, influenza A virus, influenza B virus and Mycoplasma pneumoniae quality control.

[0157] The kit prepared in Example 3 and the detection method provided in Example 4 are used for detection.

[0158] The detection results of the blank sample are shown in Figure 1. Figure 1 As can be seen from the figure, there is no melting peak in the melting curve. It can be seen that the reagent for detecting multiple nucleic acids in the present application does not produce non-specific melting peaks, and does not cause false positives, and the results are accurate and reliable.

[0159] The detection results of the negative sample are shown in Figure 2. Figure 2 As can be seen from the figure, only the internal reference characteristic peak with a TM value of 80.5 appears. ​

[0160] The detection results of the positive samples, as shown in the figure, five characteristic peaks appeared in the FAM channel, the TM value of 70.5℃ corresponds to the influenza B virus, the TM value of 74.4℃ corresponds to the novel coronavirus, the TM value of 77.6℃ corresponds to the influenza A virus, and the TM value of 83.5℃ corresponds to the Mycoplasma pneumoniae. The TM value of 80.5℃ is the internal reference characteristic peak, and the detection result is accurate. Figure 3

[0161] 2. Specificity analysis

[0162] 31 kinds of culture representing common respiratory tract pathogens or pathogens possibly existing in nasopharynx were used to evaluate the analysis specificity of the method for detecting respiratory tract pathogens (novel coronavirus, influenza A virus, influenza B virus and / or Mycoplasma pneumoniae) of the application.

[0163] Positive samples or nucleic acid of pathogens including adenovirus, human coronavirus OC43, human coronavirus 229E, cytomegalovirus, enterovirus, Epstein-Barr virus, herpes simplex virus, measles virus, human metapneumovirus, mumps virus, human parainfluenza virus type 1, rhinovirus, Burkholderia cenocepacia, Candida albicans, Bordetella pertussis, Chlamydophila pneumoniae, Escherichia coli, Enterococcus faecalis, Haemophilus influenzae, Lactobacillus reuteri, Legionella, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria meningitidis, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, as well as novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae quality control sample mixed sample were used as detection samples, the concentration of bacteria in the detection sample was 10 6 CFU / mL, and the concentration of virus was 10 5 PFU / mL. Sterile nucleic acid-free water was used as a blank detection sample.

[0164] The kit prepared in Example 3 and the detection method provided in Example 4 were used for detection.

[0165] The detection results showed that only the novel coronavirus, influenza A virus, influenza B virus and Mycoplasma pneumoniae presented positive amplification and specific peaks, and other results were negative. The analysis specificity was 100%.

[0166] 3. Sensitivity analysis

[0167] ​The positive reference sample after concentration calibration is used for gradient dilution to determine the sensitivity of the application. The positive reference samples of new coronavirus, influenza A virus, influenza B virus and Mycoplasma pneumoniae are diluted according to the requirements to obtain concentration gradients of 1000 copies / mL, 500 copies / mL, 200 copies / mL, 100 copies / mL and 50 copies / mL, and the sensitivity of the application is determined by repeating the detection 20 times for each concentration gradient of each sample.

[0168] The kit prepared in Example 3 and the detection method provided in Example 4 are used for detection.

[0169] The data is subjected to Probit regression analysis by using the SSPS software, and the concentration when the positive detection rate is ≥95% is the minimum detection limit of the application.

[0170] The detection results show that the sensitivity of the new coronavirus of the application is 100 copies / mL, the sensitivity of the influenza A virus and the influenza B virus is 500 copies / mL, and the sensitivity of the Mycoplasma pneumoniae is 200 copies / mL.

[0171] Example 6 A method for detecting pathogenic bacteria and drug resistance genes

[0172] The present embodiment provides a method for detecting pathogenic bacteria (Staphylococcus aureus, Klebsiella pneumoniae and / or Pseudomonas aeruginosa) and / or drug resistance genes (MecA, OXA23, AmpC, VIM and / or SHV), comprising the following steps:

[0173] (1) Extracting nucleic acid of the sample to be tested.

[0174] (2) Preparing the PCR amplification reaction solution according to Table 5.

[0175] Table 5

[0176] Component Add volume 2x PCR Buffer 8 μL DNA Polymerase (1.25 U / reaction) 0.5 μL Primer Mixture (10 μM each) 0.32 μL Enzyme-free water Add water to make reaction 14 μL

[0177] In the above PCR amplification reaction solution, add water gel particles embedded with primers and 2 μL of nucleic acid of the biological sample to be tested, and perform PCR amplification according to the program in Table 6 to obtain the amplification product. Then, the amplification product is subjected to multi-color melting curve analysis technology detection by using the following program: setting the temperature of the instrument melting curve parameters from 60℃ to gradually rise to 94℃, the rising rate is 0.02℃ / s, collecting the fluorescence signal every 10 seconds. Then, the results are analyzed by using the gene scanning software module, and the results are determined by the following steps: marking negative, signal normalization processing, temperature coordinate translation and differentiation Figure Four , obtaining the result image, and determining the result.

[0178] Table 6

[0179]

[0180] The TM value of Staphylococcus aureus is 69.7℃, the TM value of Pseudomonas aeruginosa is 76.3℃, and the TM value of Klebsiella pneumoniae is 81.1℃.

[0181] The TM value of MecA gene is 70.8℃, the TM value of OXA23 gene is 75.1℃, the TM value of AmpC gene is 78.9℃, the TM value of VIM gene is 83.6℃, and the TM value of SHV gene is 86.7℃.

[0182] Example 7 Performance evaluation of detection of pathogenic bacteria and drug resistance genes

[0183] 1. Nucleic acid detection of positive samples

[0184] The positive sample is a mixture of nucleic acid extracted from Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa bacterial liquid and MecA, OXA23, AmpC, VIM, and SHV gene synthetic plasmids.

[0185] The kit prepared in Example 3 and the detection method provided in Example 6 are used for detection.

[0186] The detection results of the positive sample are shown in FIG. 1. Figures 4-5 As can be seen from the figure, three characteristic peaks appear in the FAM channel, the TM value of Staphylococcus aureus is 69.7℃, the TM value of Pseudomonas aeruginosa is 76.3℃, and the TM value of Klebsiella pneumoniae is 81.1℃.

[0187] Five characteristic peaks appear in the ROX channel, the TM value of MecA gene is 70.8℃, the TM value of OXA23 gene is 75.1℃, the TM value of AmpC gene is 78.9℃, the TM value of VIM gene is 83.6℃, and the TM value of SHV gene is 86.7℃, and the detection result is accurate.

[0188] 2. Specificity analysis

[0189] The close relatives of pathogenic bacteria (Staphylococcus aureus, Klebsiella pneumoniae, and / or Pseudomonas aeruginosa) are used to evaluate the analysis specificity of the application.

[0190] The positive sample or nucleic acid of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa is detected by using the kit prepared in Example 3 and the detection method provided in Example 4. The concentration of the bacteria used for detection is 10 6 CFU / mL.

[0191] The kit prepared in Example 3 and the detection method provided in Example 4 are used for detection.

[0192] The results show that the method of the present application does not produce cross-reaction with Enterococcus faecalis, Enterococcus faecium, Corynebacterium diphtheria, Haemophilus influenzae, Proteus vulgaris, Proteus mirabilis, Acinetobacter baumannii, Neisseria meningitidis, Streptococcus sanguis, Escherichia coli, Klebsiella oxytoca, Burkholderia cepacia, Candida albicans, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Moraxella catarrhalis, Streptococcus pyogenes, Enterobacter cloacae, Serratia marcescens and Enterobacter aerogenes, and the specificity of analysis is 100%.

[0193] 3. Sensitivity analysis

[0194] The positive reference product after concentration calibration is used for gradient dilution to determine the sensitivity of the present application. The positive reference product of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa is diluted by gradient according to the requirements, and the concentration gradient of 2000 CFU / mL, 1000 CFU / mL, 500 CFU / mL, 200 CFU / mL, 100 CFU / mL and 50 CFU / mL is used to determine the sensitivity of the present application, and each concentration gradient is repeated for 20 times.

[0195] The kit prepared in Example 3 and the detection method provided in Example 6 are used for detection.

[0196] The data is analyzed by Probit regression analysis by using the SSPS software, and the concentration when the positive detection rate is ≥95% is the minimum detection limit of the present application. The sensitivity of Staphylococcus aureus of the present application is 200 CFU / mL, and the sensitivity of Klebsiella pneumoniae and Pseudomonas aeruginosa is 100 CFU / mL.

[0197] The above examples of the present application are described in detail, but the present application is not limited to the above examples, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the present application.

Claims

1. A hydrogel, characterized in that The hydrogel contains a forward primer or a reverse primer in a primer set for detecting a target to be detected; the 5' end of the forward primer or the reverse primer is modified with Acrydite.

2. The hydrogel according to claim 1, wherein The target to be tested includes at least one of a novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and SHV gene.

3. A method for preparing the hydrogel according to any one of claims 1 to 2, characterized in that: The following steps are involved: mixing the carboxymethyl cellulose solution and polyethylene glycol to obtain a mixed solution; adding a photoinitiator and acrylate to the mixed solution to prepare a hydrogel solution; The hydrogel solution is mixed with primers for detecting the target to be detected, and then dispensed to obtain gel particles; the gel particles are sequentially solidified, washed, and dried to prepare a hydrogel containing a primer set for detecting the target to be detected; Preferably, the mass fraction of the carboxymethyl cellulose solution is 8%-12%; Preferably, the volume addition ratio of the carboxymethyl cellulose solution to polyethylene glycol is (8-12): (3-6); Preferably, the polyethylene glycol comprises polyethylene glycol PEG-600; Preferably, the photoinitiator comprises 2-hydroxy-2-methylacetophenone; Preferably, in the hydrogel containing the primers for detecting the target to be detected, the added volume ratio of the solution of the primers for detecting the target to be detected to the hydrogel solution is (1-3):

9.

4. Use of the hydrogel according to any one of claims 1 to 2 in any of the following: (1) Detection of nucleic acid molecules; (2) preparing products for detecting nucleic acid molecules; (3) Prepare products for PCR, melting curve analysis, or real-time quantitative PCR detection.

5. A method for detecting nucleic acid molecules for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: using the hydrogel according to any one of claims 1 to 2 to detect a sample to be tested; Preferably, the detection method includes multi-color melting curve analysis technology.

6. A primer combination for detecting respiratory pathogens, pathogenic bacteria and / or drug-resistant genes, characterized in that: The primer combination includes a specific primer pair for detecting a novel coronavirus, a specific primer pair for detecting influenza A virus, a specific primer pair for detecting influenza B virus, a specific primer pair for detecting Mycoplasma pneumoniae, a specific primer pair for detecting Staphylococcus aureus, a specific primer pair for detecting Klebsiella pneumoniae, a specific primer pair for detecting Pseudomonas aeruginosa, a specific primer pair for detecting the MecA gene, a specific primer pair for detecting the OXA23 gene, a specific primer pair for detecting the AmpC gene, a specific primer pair for detecting the VIM gene, and a specific primer pair for detecting the SHV gene; The forward primer sequence of the specific primer pair for detecting the novel coronavirus is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 2; The forward primer sequence of the specific primer pair for detecting influenza A virus is shown in SEQ ID NO: 3, and the reverse primer sequence is shown in SEQ ID NO: 4; The forward primer sequence of the specific primer pair for detecting influenza B virus is shown in SEQ ID NO: 5, and the reverse primer sequence is shown in SEQ ID NO: 6; The sequence of the specific primer pair for detecting Mycoplasma pneumoniae is shown in SEQ ID NO:7 for the forward primer and in SEQ ID NO:8 for the reverse primer. The forward primer sequence of the specific primer pair for detecting Staphylococcus aureus is shown in SEQ ID NO: 11, and the reverse primer sequence is shown in SEQ ID NO: 12; The forward primer sequence of the specific primer pair for detecting Klebsiella pneumoniae is shown in SEQ ID NO: 13, and the reverse primer sequence is shown in SEQ ID NO: 14; The forward primer sequence of the specific primer pair for detecting Pseudomonas aeruginosa is shown in SEQ ID NO: 15, and the reverse primer sequence is shown in SEQ ID NO: 16; The forward primer sequence of the specific primer pair for detecting the MecA gene is shown in SEQ ID NO: 17, and the reverse primer sequence is shown in SEQ ID NO: 18; The forward primer sequence of the specific primer pair for detecting the OXA23 gene is shown in SEQ ID NO: 19, and the reverse primer sequence is shown in SEQ ID NO: 20; The forward primer sequence of the specific primer pair for detecting the AmpC gene is shown in SEQ ID NO: 21, and the reverse primer sequence is shown in SEQ ID NO: 22; The forward primer sequence of the specific primer pair for detecting the VIM gene is shown in SEQ ID NO: 23, and the reverse primer sequence is shown in SEQ ID NO: 24; The forward primer sequence of the specific primer pair for detecting the SHV gene is shown in SEQ ID NO: 25, and the reverse primer sequence is shown in SEQ ID NO: 26; Preferably, the 5' end of the forward primer or the reverse primer has an Acrydite modification; Preferably, the 1-3 bp position of the forward primer or the reverse primer sequence is marked with a fluorescent reporter group, and the 3' end of the forward primer or the reverse primer sequence is marked with a fluorescent quencher group; More preferably, the fluorescent reporter group includes at least one of FAM, JOE, VIC, HEX, ROX, exas Red, Cy5.5, CY3 and CY5; More preferably, the fluorescence quenching group includes Super Quencher.

7. A kit, characterized in that The kit comprises the primer combination of claim 6 and / or the hydrogel of any one of claims 1-2; Preferably, the kit further comprises at least one of a positive control, a negative control, and a PCR premix; More preferably, the PCR premix comprises at least one of PCR Buffer, DNA polymerase and reverse transcriptase; More preferably, the positive control comprises a nucleic acid molecule of a novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene, or a recombinant vector containing a novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene; More preferably, the negative control comprises water.

8. Use of the primer combination according to claim 6 or the kit according to claim 7 in any of the following: 1) Non-diagnostic detection or auxiliary detection of nucleic acid molecules containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene, or nucleic acid molecules containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene; 2) Preparation of nucleic acid molecules for detecting or assisting in the detection of novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene, or products containing novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene; Preferably, the product is selected from a reagent, a kit or a chip.

9. A method for detecting novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: using the primer combination of claim 6 or the kit of claim 7 to amplify the nucleic acid of the sample to be tested to obtain an amplified product, using a multicolor melting curve analysis technique to obtain a melting curve of the amplified product, and evaluating whether the sample to be tested contains a novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene; Preferably, the amplification is fluorescent quantitative PCR amplification; More preferably, the reaction conditions for the fluorescent quantitative PCR amplification are: 94-96°C for 2-4 min; entering the pre-amplification stage: 93-96°C for 8-12 s, 55-62°C for 40-50 s, for 12-18 cycles; entering the PCR cycling stage: 93-96°C for 8-12 s, 55-62°C for 20-40 s, for 25-38 cycles; Preferably, the melting procedure of the multicolor melting curve analysis technique is as follows: setting the temperature to gradually rise from 58-62°C to 92-96°C at a rate of 0.01-0.03°C / s, while continuously monitoring the fluorescence intensity to obtain a fluorescence intensity change rate-temperature melting curve; Preferably, the assessment of whether the sample to be tested contains a new coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumonia, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene includes: the TM value of influenza B virus is 70.5°C, the TM value of the new coronavirus is 74.4°C, the TM value of influenza A virus is 77.6°C, the TM value of Mycoplasma pneumonia is 83.5°C, the TM value of Staphylococcus aureus is 69.7°C, the TM value of Pseudomonas aeruginosa is 76.3°C, the TM value of Klebsiella pneumonia is 81.1°C, the TM value of MecA gene is 70.8°C, the TM value of OXA23 gene is 75.1°C, the TM value of AmpC gene is 78.9°C, the TM value of VIM gene is 83.6°C, and the TM value of SHV gene is 86.7°C.

10. A system for detecting novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene, characterized in that: include: Detection module: used to perform PCR amplification on the nucleic acid of the sample to be tested using the primer combination according to claim 6 or the kit according to claim 7, and obtain the melting curve of the PCR amplification product; Analysis module: used to evaluate whether the sample to be tested contains novel coronavirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, MecA gene, OXA23 gene, AmpC gene, VIM gene and / or SHV gene.

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