Combination for detecting target genes and use thereof

Abstract

Provided is a combination for specifically detecting P target genes. The combination comprises P pairs of primers used for amplifying P genes and P probes used for hybridizing the P genes. The P probes are respectively labelled with M different fluorescent groups having N concentrations; the types or concentrations of fluorescent groups carried by each probe among the P probes are different; and the P target genes and the fluorescent groups meet the following formula: 2≤P≤(MN+M)-X, M being a positive integer greater than or equal to 2, N ranging from 1 to 3, and X ranging from 0 to 19.

Description

A combination for detecting target genes and its application Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a combination for detecting target genes and its application. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, triggering an inflammatory response syndrome at the primary site of infection and throughout the body, and in severe cases, even septic shock. Infections in any part of the human body can develop into sepsis, with common sites including the digestive system (such as the gastrointestinal tract and gallbladder), lungs, kidneys, and other parts of the urinary system, the central nervous system, the bloodstream, catheter-related sites, and severe trauma and burns. Timely and accurate detection of the infecting pathogen is essential for the early identification, diagnosis, and prompt initiation of effective treatment for sepsis. It is also crucial for the accurate use of antibiotics, improving treatment efficacy, and saving patients' lives.

[0003] Traditional bacterial and fungal detection methods, such as culture, smear microscopy, and antigen testing, generally suffer from low sensitivity and high rates of missed detection. Furthermore, culture methods are time-consuming. For example, using traditional culture plus drug sensitivity testing takes an average of 57.4 hours to diagnose, far from meeting the needs of early diagnosis and treatment for sepsis patients. Furthermore, culture methods have a detection rate of only approximately 10%, and are unable to accurately detect pathogens that are difficult or impossible to culture.

[0004] Methods based on blood culture followed by PCR (e.g. BioFire Blood culture test and Luminex Blood culture tests have shown good performance and have been shown to shorten the time it takes to optimize antibiotic regimens. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) can also be used for bacterial identification after blood culture (such as Biomerieux Bruker's Vitekms Biotyper), with good consistency (over 90%) for Gram-negative bacteria but only 80% consistency for Gram-positive bacteria. These methods still require a period of culturing the pathogen (over 4 hours) and are unable to provide test results in a shorter time. Furthermore, these methods rely on bacterial identification after a positive blood culture, leaving gaps for detecting some unculturable pathogens.

[0005] Methods for directly detecting pathogens and drug resistance markers in blood samples by fluorescent quantitative PCR technology without prior blood culture (such as Roche SeptiFast, SeeGene Sepsis Test, Abbott Iridica) has been launched, but because their sensitivity and specificity do not meet clinical level requirements, they cannot be widely promoted and used.

[0006] While smear microscopy is time-efficient, pathogen identification relies on the physician's experience and judgment, and can be difficult to distinguish between morphologically similar microorganisms. Antigen testing is convenient and rapid, but it can be challenging due to numerous interference factors, making it difficult to distinguish between false positives and false negatives.

[0007] Digital PCR (dPCR) is considered the third generation of PCR technology, following conventional PCR and fluorescent PCR. Its principle is to divide a PCR system into multiple smaller units using limiting dilution, ensuring that each unit contains at most one target template. The presence or absence of endpoint fluorescence in each partition after PCR is used as the criterion. Data analysis is then performed using a Poisson distribution, enabling absolute quantification of the target gene in the specimen. With its advantages of ease of operation, absolute quantification, high sensitivity, and high specificity, digital PCR is widely used in the quantitative detection of rare tumor mutations in in vitro diagnosis, the quantitative detection of infectious pathogens, the quantitative detection of foodborne pathogens in food safety control, and the determination of GMO abundance in imported agricultural products by customs inspection and quarantine.

[0008] Digital PCR takes only 3-4 hours from sample nucleic acid extraction to test result reading. Its high sensitivity, high specificity, and absolute quantification enable accurate identification of infectious pathogens, helping clinicians make timely early diagnoses. Furthermore, its absolute quantification facilitates precise efficacy monitoring during treatment, offering significant application value in optimizing medication types and dosages. It can be used for both the prevention and diagnosis of sepsis. Test targets include individuals at high risk for sepsis requiring preventive measures, patients with focal infections, those with a high suspicion of sepsis, and patients at all stages of confirmed sepsis. Summary of the Invention

[0009] The purpose of the present application is to provide PCR primer pairs, probes, microfluidic cartridges, test kits, detection systems and detection methods for specific detection of target genes, and the PCR primer pairs, probes, microfluidic cartridges, test kits, detection systems and detection methods can be used for any PCR detection system, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc., preferably for digital PCR. For example, the purpose of the present application is to provide PCR primer pairs, probes, microfluidic cartridges, test kits, detection systems and detection methods for specific detection of multiple pathogens in bloodstream infections. The purpose of the present application is to provide PCR primer pairs, probes, microfluidic cartridges, test kits, detection systems and detection methods for specific detection of multiple different drug resistance genes.

[0010] To achieve the above objectives, this application adopts the following technical solutions:

[0011] The present application relates to a combination for specifically detecting P target genes, wherein the combination includes P pairs of primers for amplifying the P genes and P probes for hybridizing the P genes. The P probes are labeled with N concentrations of M different fluorescent groups, and each of the P probes carries a different type or concentration of the fluorescent group. The P target genes and the fluorescent groups satisfy the following formula: 2≤P≤(M N +M)-X, M is a positive integer greater than or equal to 2; N ranges from 1 to 3; X ranges from 0 to 19.

[0012] In a specific embodiment, wherein M is 2, 3, 4, 5, 6, 7 or 8, N is 1, 2 or 3, and X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0013] In a specific embodiment, the N concentrations are achieved by utilizing the ratio of any two different fluorescent groups, wherein the ratio of any two different fluorescent groups A and B is in the range of A:B being 1:(0.20-10).

[0014] In a specific embodiment, the target gene is a pathogen gene or a drug resistance gene that causes bloodstream infection.

[0015] In a specific embodiment, the pathogen is one or more pathogens selected from the group consisting of Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Haemophilus influenzae, Serratia marcescens, Candida tropicalis, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, and / or Candida albicans, human herpesvirus type 1, human alphaherpesvirus type 1, varicella zoster virus, Listeria monocytogenes, Mycobacterium tuberculosis, human cytomegalovirus, human herpesvirus type 5, human herpesvirus type 2, human herpesvirus type 6, Epstein-Barr virus, human herpesvirus type 4, parvovirus B19, primate erythrocytic parvovirus type 1, human Herpesvirus-like 7, JC virus, JC polyoma, human polyomavirus type 2, pseudorabies virus, porcine herpesvirus type 1, porcine herpesvirus 1, Neisseria meningitidis, Streptococcus agalactiae, Brucella, Brucella melitensis, Brucella melitensis, Cryptococcus gattii, Cryptococcus gothi, Aspergillus fumigatus, Angiostrongylus cantonensis, Taenia solium, Toxoplasma gondii, Balamuthiae baboon, adenovirus C subgroup 1, adenovirus B subgroup 4, human Mammary adenovirus B, human polyomavirus type 1, Merkel multicellular oncovirus, human polyomavirus 5, parvovirus, Streptococcus intermedius, Fusobacterium spp., Porphyromonas endodontis, Klebsiella oxytoca, Streptococcus suis, Campylobacter coli, Streptococcus constellatus, Proteus, Salmonella, Prevotella, Rhizopus oryzae, Mucor, Histoplasma capsulatum, Cryptococcus laurentii, Cryptococcus albidus, Aspergillus flavus, Treponema pallidum, and Orientia tsutsugamushi; or

[0016] The drug-resistant gene is one or more drug-resistant genes selected from the following: mecA, mecC, vanA, vanB, MCR-1, blaKPC, blaCTX-M, blaOXA-23, blaNDM-1, blaTEM, blaSHV, blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-4, SmeDEF, Sul1, QnrA, QnrB, gyrA, gyrB, Aac(6')-Ib-cr, ermB, MefA, aac(3')-VI, aph(3')-VI, ant(2")-Ia, DfrA1, catA1, tetA, tetB, and cfr.

[0017] In a specific embodiment, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quencher group. When the probes are in the same reaction system, the spectral ranges of the fluorescent reporter groups for screening different types of pathogens and drug-resistant genes are different.

[0018] In a specific embodiment, the fluorescent reporter group is selected from FAM, VIC, ROX, Cy5, and / or HEX; the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0019] The combination for specifically detecting P target genes of the present application is a combination used in digital PCR (dPCR) reaction.

[0020] The present application also relates to a digital PCR reaction premix for specifically detecting target genes, wherein the digital PCR reaction premix comprises the above-mentioned combination of the present application for specifically detecting P target genes.

[0021] In a specific embodiment, the digital PCR reaction premix further comprises one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ ion.

[0022] The present application also relates to a digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge includes the above-mentioned digital PCR reaction premix for specifically detecting a target gene of the present application.

[0023] In a specific embodiment, the microfluidic cartridge comprises at least one hole or at least two holes.

[0024] The present application also relates to a kit for detecting a target gene, wherein the kit includes the digital PCR reaction premix for specifically detecting the target gene described above.

[0025] In a specific embodiment, the kit further comprises a positive control, a negative control, and / or droplet-generating oil.

[0026] In a specific embodiment, the kit further comprises the digital PCR microfluidic cartridge of the present application.

[0027] The present application also relates to a method for detecting a target gene for a non-disease diagnosis purpose, the method comprising the following steps:

[0028] (1) Release nucleic acid from the sample to be tested;

[0029] (2) preparing a digital PCR amplification mixture containing the nucleic acid of the sample to be tested provided in step (1), the combination for specifically detecting P target genes of the present application and / or the digital PCR reaction premix for specifically detecting target genes of the present application, and distilled water;

[0030] (3) preparing droplets in a digital PCR microfluidic cartridge and performing PCR amplification reactions;

[0031] (4) Use a digital PCR microfluidic cartridge reader to read the fluorescence signal after the droplet PCR amplification reaction.

[0032] In one embodiment, the sample types involved in the detection include whole blood, serum, plasma, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid or ascites.

[0033] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here.

[0034] The beneficial effect achieved by the present application is to provide a primer probe set for detecting multiple target genes, wherein the 5' end of the probe sequence of the present application is labeled with a fluorescent group of different colors and different concentrations, and the target sequence of each target gene is efficiently amplified by digital PCR, so as to achieve absolute quantification of each target gene to be detected, and directly give the number of copies per microliter (CFU / microliter) of each target gene in the reaction system. In addition, the present application also provides a method for detecting target genes for the purpose of non-disease diagnosis using the primer and probe set of the method of the present application. For example, the combination of the present application can be used to detect multiple pathogens in bloodstream infections. The sensitivity and specificity of the method of the present application are 92.88% and 100%, respectively, which exceed the existing technology; and the minimum detection limit is 500cps / ml, which is much lower than the minimum detection limit of the existing qPCR technology for directly detecting bloodstream infection samples.

[0035] Specifically, 1) a digital PCR detection kit is used to directly detect pathogenic microbial nucleic acids in 2-10 mL of human peripheral whole blood, eliminating the need for a 7-8 hour blood culture enrichment step; 2) a digital PCR primer-probe set fluorescence color coding technology is used to enable single-well detection of 11-300 pathogens; 3) detection targets include, but are not limited to, Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, yeast, and drug-resistant genes; and 4) the detection kit contains an internal reference gene to prevent false negatives caused by insufficient sample extraction quality or failed PCR amplification reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0037] Figures 1A-1B illustrate the results of a 10-plex digital PCR amplification test performed simultaneously in a single well. Numbers 1-10 represent the 10 pathogens being tested. When multiple infections are present in a bloodstream infection sample, corresponding positive droplets will appear at the double, triple, and quadruple positions.

[0038] Figures 2A-2F show the results of simultaneous detection of 21 pathogens plus one internal reference gene in two wells using the primer probe set of the present application in Example 1. Figure 2A is a two-dimensional image of the positive control FAM&HEX in well 1; Figure 2B is a two-dimensional image of the positive control ROX&Cy5 in well 1; Figure 2C is a one-dimensional image of the positive control Cy5.5 in well 1; Figure 2D is a two-dimensional image of the positive control FAM&HEX in well 2; Figure 2E is a two-dimensional image of the positive control ROX&Cy5 in well 2; and Figure 2F is a one-dimensional image of the positive control Cy5.5 in well 1. Note: In this design, since the Cy5.5 fluorescence channel does not form a two-dimensional image with other channels, only its one-dimensional image is displayed. The horizontal axis is the serial number of the droplets that are not displayed, and the vertical axis is the fluorescence intensity.

[0039] Figures 3A-3F show examples of shifting the position of a two-dimensional image of a positive droplet cluster by adjusting the ratio of different color probes, illustrating the principle of multi-color fluorescence color coding for ultra-multiplexed target detection. Figure 3A shows a boxed droplet cluster with a FAM:HEX ratio of 1:3, Figure 3B shows a boxed droplet cluster with a FAM:HEX ratio of 1.8:3, and Figure 3C shows an overlay of Figures 3A and 3B, allowing comparison of the cluster movement. Figure 3D shows a boxed droplet cluster with a ROX:Cy5 ratio of 4:1, Figure 3E shows a boxed droplet cluster with a ROX:Cy5 ratio of 5.6:1, and Figure 3F shows an overlay of Figures 3D and 3E, allowing comparison of the cluster movement.

[0040] FIG4 shows the detection limit range test results of 21 bacterial liquid samples using the kit of the present application (Staphylococcus aureus is shown as an example).

[0041] Figure 5 shows the specificity test results. The 21-pathogen detection kit reacted with 115 non-target bacteria in triplicate, and no positive droplet clusters were produced in any of the three wells. This demonstrates that the 21-pathogen detection kit has no nonspecific cross-reactivity with the 115 non-target bacteria.

[0042] Figures 6A-6C show the test results of a real pathogen-positive sample using the kit of the present application.

[0043] FIG7 shows the results of detecting Staphylococcus epidermidis in patient sputum samples using the kit of the present application.

[0044] FIG8 shows the results of detecting cerebrospinal fluid samples of patients using the kit of the present application.

[0045] 9A-9D are the results of detecting Acinetobacter baumannii, Pseudomonas maltophilia, Enterobacter cloacae, and Candida albicans in patient sputum samples using the kit of the present application.

[0046] Figures 10A-10D are the results of detecting the patient's throat swab using the kit of the present application; Figures 10E-10H are the results of detecting the patient's anal swab samples using the kit of the present application.

[0047] FIG11 shows the results of detecting a patient's urine sample using the kit of the present application.

[0048] 12A-12I show the results of simultaneously detecting 32 drug-resistant genes plus one internal reference gene in three wells using the primer-probe set of the present application in Example 1.

[0049] Figures 13A-13F show examples of shifting the position of a positive droplet cluster in a two-dimensional image by adjusting the ratio of different color probes, illustrating the principle of achieving ultra-multiplexed target detection using mixed-color fluorescence coding. Figure 13A shows the arrow pointing to a droplet cluster with a FAM:HEX ratio of 1:2.5, Figure 13B shows the arrow pointing to a droplet cluster with a FAM:HEX ratio of 1:3.2, and Figure 13C shows the superposition of a mecA resistance gene-positive droplet cluster with a FAM:HEX probe concentration of 1:2.5 and a mecA resistance gene-positive droplet cluster with a FAM:HEX probe concentration of 1:3.2. It is clearly shown that adjusting the FAM and HEX probe concentration ratios can shift the position of the positive droplet cluster in the two-dimensional image of the digital PCR assay. Figure 13D shows the droplet cluster indicated by the arrow with ROX:Cy5=4:1, Figure 13E shows the droplet cluster indicated by the arrow with ROX:Cy5=5.5:1, and Figure 13F superimposes the positive droplet clusters with two different ROX:Cy5 ratios, which shows that by adjusting the ROX:Cy5 ratio, the position of the mecC resistance gene positive droplet cluster in the two-dimensional image of the digital PCR detection can be moved.

[0050] Figure 14 shows the specificity test results. The 32-drug resistance gene detection kit was tested against 115 strains that did not carry resistance genes. Three replicates were performed, and no positive droplet clusters were produced in any of the three wells. This demonstrates that the 32-drug resistance gene detection kit has no nonspecific cross-reactivity with the 115 strains that do not carry resistance genes.

[0051] Figures 15A-15E show the detection results of samples of strains carrying drug-resistant genes using the kit of the present application. Figure 15A shows the detection of the blaOXA-48 resistance gene from Escherichia coli strains carrying drug-resistant genes; Figure 15B shows the detection of the smeDEF resistance gene from five strains of Stenotrophomonas maltophilia carrying drug-resistant genes; Figure 15C shows the detection of the smeDEF resistance gene from Klebsiella pneumoniae carrying drug-resistant genes; Figure 15D shows the detection of the mecA resistance gene from two strains of Staphylococcus aureus carrying drug-resistant genes; and Figure 15E shows the detection of the vanA or vanB resistance gene from three strains carrying the VanA gene and the blaCTX-M gene.

[0052] FIG16 shows the results of detecting patient sputum samples using the kit of the present application. DETAILED DESCRIPTION

[0053] The following describes the specific embodiments of the present invention in detail, but it should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific experimental conditions are not specified are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0054] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0055] Digital PCR (dPCR) is a new absolute quantitative PCR technology that primarily involves limiting dilution of PCR reactants, followed by PCR amplification in separate reaction chambers. Finally, the starting copy number or concentration of the target molecule is determined based on the Poisson distribution principle and the number and ratio of positive droplets. Because multiplex digital PCR reaction systems involve multiple primers and probes, the requirements for the primer-probe combination in the detection system are very high.

[0056] So far, there has been no report on multiplex PCR technology for efficiently detecting multiple mixed nucleic acid samples using digital PCR methods.

[0057] In some embodiments, P pairs of primers are used to amplify P genes, P probes are used to hybridize the P genes, and the P probes are labeled with N concentrations of M different fluorescent groups, each of the P probes carries a different type or concentration of the fluorescent group, wherein the P target genes and the fluorescent groups satisfy the following formula: 2≤P≤(M N +M)-X, M is a positive integer greater than or equal to 2; N ranges from 1 to 3; X ranges from 0 to 19.

[0058] Preferably, M is any number among 2, 3, 4, 5, 6, 7, 8; more preferably, M is 2 or 3.

[0059] N is 1, 2 or 3, for example.

[0060] X is, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. Preferably, the range of X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. More preferably, the range of X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0061] The ratio of any two fluorescent groups A and B is in the range of 1:(0.20-10), for example, 1:0.2, 1:0.25, 1:0.3, 1:0.33, 1:0.4, 1:0.75, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.7, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:5. :4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.5, 1:5.6, 1:5.8, 1:6, 1:6.2, 1:6.4, 1:6.6, 1:6.8, 1:7, 1:7.2, 1:7.4, 1:7.6, 1:7.8, 1:8, 1:8.2, 1:8.4, 1:8.6, 1:8.8, 1:9, 1:9.2, 1:9.4, 1:9.6, 1:9.8, 1:10, etc.

[0062] Preferably, the ratio of any two fluorescent groups A and B is in the range of 1:0.25, 1:0.33, 1:0.75, 1:1, 1:2, 1:1.4, 1:1.6, 1:1.7, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.6, 1:5.8, 1:6.

[0063] In the present application, A and B represent any fluorescent groups, for example, A can represent any one of FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX, and B represents a fluorescent group different from A, and can also be any one of FAM, VIC, ROX, Cy5, Cy5.5 and / or HEX.

[0064] More preferably, the ratio of the two fluorescent groups ranges from 1:0.2, 1:0.25, 1:0.3, 1:0.33, 1:0.4, 1:0.75, 1:1, 1:1.7, 1:2, 1:2.5, 1:3, 1:3.2, 1:4, and 1:5.5.

[0065] In one embodiment of the present application, when M is 4, N is 2, and X is 8, the identified pathogen P is 12.

[0066] In one embodiment of the present application, when M is 5, N is 2, and X is 8, the identified pathogen P is 22.

[0067] In another embodiment of the present application, when M is 6, N is 2, and X is 8, the identified pathogen P is 34.

[0068] In a specific embodiment, P probes are labeled with N concentrations of M different fluorescent groups, and the difference in the type or concentration of the fluorescent group carried by each of the P probes means that the type of fluorescent group carried by each of the P probes is different, or the concentration of the fluorescent group carried by each of the P probes is different, or the type or concentration of the fluorescent group carried by each of the P probes is different. It will be understood by those skilled in the art that the core of this application is to use different types or fluorescence intensities of different fluorescent groups to distinguish each probe, as long as they can be distinguished and can be detected by detection means.

[0069] In a specific embodiment, the first probe is labeled with fluorescent groups A and B at a ratio of 1:0.33, the second probe is labeled with fluorescent groups A and B at a ratio of 1:0.75, the third probe is labeled with fluorescent groups A and B at a ratio of 1:1, the fourth probe is labeled with fluorescent groups A and B at a ratio of 1:2, and the fifth probe is labeled with fluorescent groups A and B at a ratio of 1:4. By adjusting the ratio of the two fluorescent groups A and B, different pathogens can be detected.

[0070] For example, as shown in Example 2 of this application, by adjusting the ratios of the fluorescent probes FAM and HEX, and the fluorescent probes ROX and Cy5, it is possible to detect positive droplet clusters at different locations. Adjusting the ratio of the FAM probe to the HEX probe from 1:3 to 1.8:3, and the ratio of the ROX and Cy5 probes from 4:1 to 5.6:1, both enabled the identification of positive droplet clusters at different locations.

[0071] In some embodiments, the target gene can be any type of gene, including but not limited to animal genes, plant genes, and / or microbial genes.

[0072] Preferably, the microbial genes include pathogenic microbial genes, including bacteria, fungi, viruses, spirochetes, mycoplasmas, rickettsiae, chlamydiae, parasites (protozoa, worms, medical insects), and the like.

[0073] In some embodiments, the target genes are multiple different drug-resistance genes.

[0074] Preferably, the drug-resistant genes include 32 types of pathogenic microorganism drug-resistant genes.

[0075] The combination of the present application can effectively control the number of multiple probes and utilize multiple different concentrations of multiple probes to specifically and efficiently detect multiple pathogen resistance genes.

[0076] In one embodiment of the present application, the aforementioned primer-probe set was used to enable simultaneous detection of 11 pathogens using five fluorescent colors in a single well, and 21 pathogens and an internal control using five fluorescent colors in two wells, covering the most common pathogens in clinical bloodstream infections. Simultaneous detection of 16 targets using seven colors in a single well, coupled with three fluorescent color coding, enabled detection of over 30 targets in a single well.

[0077] In one embodiment of the present application, the above-mentioned primer probe set is used to achieve simultaneous detection of 11 drug-resistant genes by 5-color fluorescence in 1 well through digital PCR, and simultaneous detection of 32 drug-resistant genes and internal reference by 5-color fluorescence in 3 wells. The detection range covers the most common drug-resistant genes in clinical bacterial infections.

[0078] In some embodiments, the present application identifies multiple target genes by the relative positional relationship of negative clusters, single-positive clusters, double-positive clusters, and diversity clusters. The definitions and divisions of negative clusters, single-positive clusters, double-positive clusters, and diversity clusters are determined as follows: the position of each droplet cluster in the two-dimensional map is determined by two factors: ① the type and combination of fluorescence in the droplet cluster; ② the intensity of fluorescence in the droplet cluster. (a) Negative cluster: A droplet group (cluster) composed of droplets that do not contain any target DNA detectable by primer probes, that is, a negative droplet cluster. In this droplet cluster, the primer probe does not undergo an amplification reaction, does not produce a positive strong fluorescence signal, and only emits a background signal intensity (the intensity detected by each fluorescence channel is not 0). Therefore, this cluster is usually the cluster with the weakest fluorescence intensity. In the two-dimensional map generated by the multiplex PCR positive control (as shown in Figures 1A-1B), the position of the negative cluster in the two-dimensional map is usually in the lower left corner. In addition, another method for determining the location of the negative cluster is to use the single droplet cluster (i.e., the negative cluster) produced by the negative control reaction to calibrate its position. (b) Single-positive cluster: This droplet cluster contains only a single target DNA and therefore can only trigger the reaction of its corresponding primer probe. By controlling the type of fluorescein labeled on the probe and the probe concentration, the positive cluster can be positioned at the expected position in the two-dimensional map, that is, consistent with the expected (X, Y) coordinate value. In Figures 1A-1B, the distance between the five single-positive clusters and the negative cluster is the shortest compared to other clusters (referring to double-positive clusters and multi-positive clusters). Each single-positive cluster has a different (X, Y) value due to the different types (and combinations) and concentrations of fluorescein labeled on its corresponding probe, but the position of the single-positive cluster triggered by a specific DNA type is fixed. (c) Double-positive cluster: A cluster composed of droplets containing two specific target DNA reactions. In these droplets, the two target DNAs each trigger a reaction with their corresponding primer probes, activating the corresponding fluorophores to emit the expected intensity. Therefore, the fluorescence of the droplet cluster is actually the superposition of the fluorescence produced by the two target DNAs under the background fluorescence (i.e., the intensity of the negative cluster). If you draw a vector pointing to the single positive cluster of target 1 from the negative cluster as the starting point and the single positive cluster of target 1 as the end point, and similarly draw a vector arrow from the negative cluster to target 2, and apply the "parallelogram rule" or "triangle rule" of vector synthesis, summing vectors 1 and 2, you can know the coordinates of the double positive cluster. If the coordinates of the single-positive cluster of target 1 are (X1, Y1), the coordinates of the single-positive cluster of target 2 are (X2, Y2), and the negative cluster is (X0, Y0), then the coordinates of the double-positive cluster containing targets 1 and 2 are ((X1-X0)+(X2-X0)+X0), ((Y1-Y0)+(Y2-Y0)+Y0)), that is, (X1+X2-X0, Y1+Y2-Y0). Therefore, in a two-dimensional graph, for any two target DNAs, their negative cluster, two single-positive clusters, and corresponding double-positive clusters must be located at the four vertices of a parallelogram. (d) Triple-positive and multi-positive clusters: droplet clusters composed of droplets containing three or more specified target DNA reactions.The coordinates of these clusters also conform to the principle of vector synthesis. For example, the coordinates of a triple positive cluster containing targets 1, 2, and 3 are (X1+X2+X3-2X0, Y1+Y2+Y3-2Y0). The coordinates of a multi-(n) positive cluster are (X1+X2+…Xn-(n-1)X0, Y1+Y2+…Yn-(n-1)Y0).

[0079] As described above, the present application controls the concentration ratio of different fluorescent reporter genes when they are labeled by designing the number of different fluorescent reporter genes, that is, the distribution method of N concentrations can achieve a reasonable configuration of negative clusters, single positive clusters, double positive clusters, and diversity clusters, thereby effectively realizing rapid detection of multiple target genes at the same time.

[0080] The primers and probes of this application can be applied to any PCR detection system for detecting target genes, including but not limited to conventional PCR, RL-PCR, RNA-PCR, fluorescent quantitative PCR, digital PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELISA), nested PCR-high resolution melting analysis (nPCR-HRM), etc. Preferably, the primers and probes involved in this application are used for fluorescent quantitative PCR or digital PCR. Most preferably, the primers and probes determined in this application are used for digital PCR.

[0081] The digital PCR reaction premix in this application contains primers, probes, enzymes (hot start Taq enzyme and / or UDG enzyme), dNTPs, glycerol, betaine, NH4 + Mg 2+ , bovine serum albumin, etc.

[0082] Compared with the digital PCR reaction premix, the digital PCR amplification mixture in the present application contains an amplification template (such as a sample template, a positive control or a negative control) and distilled water.

[0083] In some embodiments, the present application provides a digital PCR reaction premix for detecting a target gene, wherein the PCR reaction premix comprises the above-mentioned primer pair, a combination of any of the above-mentioned primer pairs and a probe, or a combination of the above-mentioned primer pair and any probe.

[0084] In a specific embodiment, the present application targets the specific nucleotide sequences of 21 pathogens including Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Serratia marcescens, Haemophilus influenzae, Candida tropicalis, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida albicans. Through multiple sequence comparison and analysis, multiplex digital PCR primers and probes that can be used to detect the above pathogens are designed. By using different concentrations of the probes, up to 11-12 multiplex digital PCRs can be detected in one well. At the same time, internal standard primers and internal standard probes are also designed to monitor the sample collection and extraction process to avoid false negative results.

[0085] In a specific embodiment, the present application is based on mecA, mecC, vanA, vanB, MCR-1, blaKPC, blaCTX-M, blaOXA-23, blaNDM-1, blaTEM, blaSHV, blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-4, SmeDEF, Sul1, QnrA, QnrB, gyrA, gyrB, Aac(6')-Ib-cr, ermB, MefA, aac The specific nucleotide sequences of 32 drug-resistant genes, including (3')-VI, aph(3')-VI, ant(2")-Ia, DfrA1, catA1, tetA, tetB, and cfr, were targeted. Through multiple sequence comparison and analysis, multiplex digital PCR primers and probes that can be used to detect the above-mentioned drug-resistant genes were designed. By using different probe concentrations, up to 11-12 multiplex digital PCR tests can be achieved in one well. At the same time, internal standard primers and internal standard probes were also designed to monitor the sample collection and extraction process to avoid false negative results.

[0086] To improve the efficiency of screening for bloodstream infection pathogens, on the one hand, the present application provides a method for efficiently screening for bloodstream infection pathogens using a PCR method, wherein an example of an upstream primer for Bacteroides fragilis is shown in SEQ ID NO: 1, and an example of a downstream primer is shown in SEQ ID NO: 2; an example of an upstream primer for Staphylococcus epidermidis is shown in SEQ ID NO: 4, and an example of a downstream primer is shown in SEQ ID NO: 5; an example of an upstream primer for Enterococcus faecalis is shown in SEQ ID NO: 7, and an example of a downstream primer is shown in SEQ ID NO: 8; an example of an upstream primer for Streptococcus pneumoniae is shown in SEQ ID NO: 10, and an example of a downstream primer is shown in SEQ ID NO: 11; an example of an upstream primer for Acinetobacter baumannii is shown in SEQ ID NO: 13, and an example of a downstream primer is shown in SEQ ID NO: 14; an example of an upstream primer for Enterobacter cloacae is shown in SEQ ID NO: 16, and an example of a downstream primer is shown in SEQ ID NO: 17; an example of an upstream primer for Enterococcus faecium is shown in SEQ ID NO: an example of the upstream primer for Staphylococcus aureus is shown in SEQ ID NO:22, and an example of the downstream primer is shown in SEQ ID NO:23; an example of the upstream primer for Klebsiella pneumoniae is shown in SEQ ID NO:25, and an example of the downstream primer is shown in SEQ ID NO:26; an example of the upstream primer for Pseudomonas aeruginosa is shown in SEQ ID NO:28, and an example of the downstream primer is shown in SEQ ID NO:29; an example of the upstream primer for Escherichia coli is shown in SEQ ID NO:31, and an example of the downstream primer is shown in SEQ ID NO:32; an example of the upstream primer for Staphylococcus capitis is shown in SEQ ID NO:34, and an example of the downstream primer is shown in SEQ ID NO:35; an example of the upstream primer for Stenotrophomonas maltophilia is shown in SEQ ID NO:37, and an example of the downstream primer is shown in SEQ ID NO:38; an example of the upstream primer for Haemophilus influenzae is shown in SEQ ID NO: An example of the upstream primer for Serratia marcescens is shown in SEQ ID NO:43, and an example of the downstream primer is shown in SEQ ID NO:44; an example of the upstream primer for Candida tropicalis is shown in SEQ ID NO:46, and an example of the downstream primer is shown in SEQ ID NO:47;An example of an upstream primer for Candida krusei is shown in SEQ ID NO:49, and an example of a downstream primer is shown in SEQ ID NO:50; an example of an upstream primer for Candida parapsilosis is shown in SEQ ID NO:52, and an example of a downstream primer is shown in SEQ ID NO:53; an example of an upstream primer for Candida glabrata is shown in SEQ ID NO:55, and an example of a downstream primer is shown in SEQ ID NO:56; an example of an upstream primer for Candida albicans is shown in SEQ ID NO:58, and an example of a downstream primer is shown in SEQ ID NO:59; an example of an upstream primer for Cryptococcus neoformans is shown in SEQ ID NO:61, and an example of a downstream primer is shown in SEQ ID NO:62; an example of an upstream primer for an internal reference is shown in SEQ ID NO:64, and an example of a downstream primer is shown in SEQ ID NO:65.

[0087] On the other hand, the present application provides a probe that can screen Bacteroides fragilis, an example of which is shown in SEQ ID NO: 3: 5'-CAGTTGTCCAAGTGGCGACG-3'; a probe that can screen Staphylococcus epidermidis, an example of which is shown in SEQ ID NO: 6: 5'-TGTGTATCATTATGCCATGAGCTTG-3'; a probe that can screen Enterococcus faecalis, an example of which is shown in SEQ ID NO: 9: 5'-GGCTTCATCCTAATCTTCAAGACAA-3'; a probe that can screen Streptococcus pneumoniae, an example of which is shown in SEQ ID NO: 12: 5'-TTAGAAAACGTGGGCAGGGAA-3'; a probe that can screen Acinetobacter baumannii, an example of which is shown in SEQ ID NO: 15: 5'-TCACAACCCGACAACGGTGAGCAA-3'; a probe that can screen Enterobacter cloacae, an example of which is shown in SEQ ID NO: NO:18: 5'-AACCTTTGCCGACGTTGTCGGTCA-3'; a probe that can screen Enterococcus faecium, an example of which is shown in SEQ ID NO:21: 5'-TTCATCCATTTTGGACTGATGCA-3'; a probe that can screen Staphylococcus aureus, an example of which is shown in SEQ ID NO:24: 5'-TGGCTGAGATGAACTGTTCAGACCC-3'; a probe that can screen Klebsiella pneumoniae, an example of which is shown in SEQ ID NO:27: 5'-CCGATTGAAAAACGCTCCGGGC-3'; a probe that can screen Pseudomonas aeruginosa, an example of which is shown in SEQ ID NO:30: 5'-AACACAAACGCACTCGGAAAAATCG-3'; a probe that can screen Escherichia coli, an example of which is shown in SEQ ID NO:33: 5'-GCCTGCAGCTTCCATACGCT-3'; a probe that can screen Staphylococcus capitis, an example of which is shown in SEQ ID NO: NO:36: 5'-ACCTCTTGCGAATAGTTCAGTACTTTC-3'; a probe that can screen for Stenotrophomonas maltophilia, an example of which is shown in SEQ ID NO:39: 5'-TCTTGCCTTCGTTCATCAGCTCGT-3'; a probe that can screen for Haemophilus influenzae, an example of which is shown in SEQ ID NO:42: 5'-CAAATATTGAAATTGGGCTTTGACG-3';A probe that can screen for Serratia marcescens, an example of which is shown in SEQ ID NO:45: 5'-GCGCGCTGAACTACACCACT-3'; a probe that can screen for Candida tropicalis, an example of which is shown in SEQ ID NO:48: 5'-GGATCATACGTTCCATTTGCTTTA-3'; a probe that can screen for Candida krusei, an example of which is shown in SEQ ID NO:51: 5'-AGCATCTGGCCCTGGCTATAACAC-3'; a probe that can screen for Candida parapsilosis, an example of which is shown in SEQ ID NO:54: 5'-TAACGTATCTGCAGACGTGGCTGC-3'; a probe that can screen for Candida glabrata, an example of which is shown in SEQ ID NO:57: 5'-CTGCCGCAAGTCATGGGTTCTTG-3'; a probe that can screen for Candida albicans, an example of which is shown in SEQ ID NO:60: 5'-AACAAACTTGCTTTGGCGGTGGG-3'; a probe capable of screening Cryptococcus neoformans, an example of which is shown in SEQ ID NO:63: 5'-CCTGTCAGCCCGGCGTAATAAGTT-3'; an internal control (human) probe, an example of which is shown in SEQ ID NO:66: 5'-TGCCTGCCGTGTGAACCATGTGACT-3'.

[0088] In one aspect, the present application provides a combination of primers and probes that can simultaneously screen 21 pathogens, the combination of primers and probes comprising any one or more of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0089] To improve the efficiency of screening for drug-resistant genes of bloodstream infection pathogens, on the one hand, the present application provides a method for efficiently screening for drug-resistant genes of bloodstream infection pathogens using a PCR method, wherein an example of an upstream primer for mecA is shown in SEQ ID NO: 67, and an example of a downstream primer is shown in SEQ ID NO: 68; an example of an upstream primer for mecC is shown in SEQ ID NO: 70, and an example of a downstream primer is shown in SEQ ID NO: 71; an example of an upstream primer for vanA is shown in SEQ ID NO: 73, and an example of a downstream primer is shown in SEQ ID NO: 74; an example of an upstream primer for vanB is shown in SEQ ID NO: 76, and an example of a downstream primer is shown in SEQ ID NO: 77; an example of an upstream primer for MCR-1 is shown in SEQ ID NO: 79, and an example of a downstream primer is shown in SEQ ID NO: 80; an example of an upstream primer for blaKPC is shown in SEQ ID NO: 82, and an example of a downstream primer is shown in SEQ ID NO: 83; an example of an upstream primer for blaCTX-M is shown in SEQ ID NO: an example of an upstream primer for blaOXA-23 is shown in SEQ ID NO:88, and an example of a downstream primer is shown in SEQ ID NO:89; an example of an upstream primer for blaNDM-1 is shown in SEQ ID NO:91, and an example of a downstream primer is shown in SEQ ID NO:92; an example of an upstream primer for blaTEM is shown in SEQ ID NO:94, and an example of a downstream primer is shown in SEQ ID NO:95; an example of an upstream primer for blaSHV is shown in SEQ ID NO:97, and an example of a downstream primer is shown in SEQ ID NO:98; an example of an upstream primer for blaVIM-1 is shown in SEQ ID NO:100, and an example of a downstream primer is shown in SEQ ID NO:101; an example of an upstream primer for blaVIM-2 is shown in SEQ ID NO:103, and an example of a downstream primer is shown in SEQ ID NO:104; an example of an upstream primer for blaIMP-1 is shown in SEQ ID NO: An example of an upstream primer for blaIMP-4 is shown in SEQ ID NO: 109, and an example of a downstream primer is shown in SEQ ID NO: 110;An example of an upstream primer for SmeDEF is shown in SEQ ID NO: 112, and an example of a downstream primer is shown in SEQ ID NO: 113; an example of an upstream primer for Sul1 is shown in SEQ ID NO: 115, and an example of a downstream primer is shown in SEQ ID NO: 116; an example of an upstream primer for QnrA is shown in SEQ ID NO: 118, and an example of a downstream primer is shown in SEQ ID NO: 119; an example of an upstream primer for QnrB is shown in SEQ ID NO: 121, and an example of a downstream primer is shown in SEQ ID NO: 122; an example of an upstream primer for gyrB is shown in SEQ ID NO: 124, and an example of a downstream primer is shown in SEQ ID NO: 125; an example of an upstream primer for gyrA is shown in SEQ ID NO: 127, and an example of a downstream primer is shown in SEQ ID NO: 128; an example of an upstream primer for Aac(6')-Ib-cr is shown in SEQ ID NO: an example of an upstream primer for ant(2')-Ia is shown in SEQ ID NO: 148, and an example of a downstream primer is shown in SEQ ID NO: 149; an example of an upstream primer for cfr is shown in SEQ ID NO: 150, and an example of a downstream primer is shown in SEQ ID NO: 151; an example of an upstream primer for ermB is shown in SEQ ID NO: 151, and an example of a downstream primer is shown in SEQ ID NO: 152; an example of an upstream primer for aac(3')-VI is shown in SEQ ID NO: 153, and an example of a downstream primer is shown in SEQ ID NO: 154; an example of an upstream primer for ant(2')-Ia is shown in SEQ ID NO: 155, and an example of a downstream primer is shown in SEQ ID NO: 156. An example of an upstream primer for DfrA1 is shown in SEQ ID NO:151, and an example of a downstream primer is shown in SEQ ID NO:152; an example of an upstream primer for catA1 is shown in SEQ ID NO:154, and an example of a downstream primer is shown in SEQ ID NO:155; an example of an upstream primer for tetA is shown in SEQ ID NO:157, and an example of a downstream primer is shown in SEQ ID NO:158;An example of an upstream primer for tetB is shown in SEQ ID NO: 160, and an example of a downstream primer is shown in SEQ ID NO: 161; an example of an upstream primer for the internal reference B2M gene is shown in SEQ ID NO: 163, and an example of a downstream primer is shown in SEQ ID NO: 164.

[0090] On the other hand, the present application provides a probe that can screen the mecA gene, an example of which is shown in SEQ ID NO: 69: 5'-AGCACTTGTAAGCACACCTTCATATGACG-3'; a probe that can screen the mecC gene, an example of which is shown in SEQ ID NO: 72: 5'-TCACTACATCACCAGGTTCAACCCA-3'; a probe that can screen the VanA gene, an example of which is shown in SEQ ID NO: 75: 5'-TCAGGCTCATCCTTCGGTGTGAAA-3'; a probe that can screen the VanB gene, an example of which is shown in SEQ ID NO: 78: 5'-TTACGCCAAAGGACGAACCTGACC-3'; a probe that can screen the MCR-1 gene, an example of which is shown in SEQ ID NO: 81: 5'-AGTTTCTTTCGCGTGCATAAGCCG-3'; a probe that can screen the blaKPC gene, an example of which is shown in SEQ ID A probe that can screen for the blaCTX-M gene, an example of which is shown in SEQ ID NO:87: 5'-TTCATCCATTTTGGACTGATGCA-3'; a probe that can screen for the blaOXA-23 gene, an example of which is shown in SEQ ID NO:90: ​​5'-TGGCTGAGATGAACTGTTCAGACCC-3'; a probe that can screen for the blaNDM-1 gene, an example of which is shown in SEQ ID NO:93: 5'-CAGGACAAGATGGGCGGTATGGAC-3'; a probe that can screen for the blaTEM gene, an example of which is shown in SEQ ID NO:96: 5'-TCACCCAGAAACGCTGGTGAAAGT-3'; a probe that can screen for the blaSHV gene, an example of which is shown in SEQ ID NO: NO:99: 5'-AGGTGCTCATCATGGGAAAGCGTT-3'; a probe that can screen the blaVIM-1 gene, an example of which is shown in SEQ ID NO:102: 5'-CAAACGACTGCGTTGCGATATGCG-3'; a probe that can screen the blaVIM-2 gene, an example of which is shown in SEQ ID NO:105: 5'-CTGCGAGTGTGCTCTATGGTGGTT-3';A probe that can screen the blaIMP-1 gene, an example of which is shown in SEQ ID NO: 108: 5'-AGTGGTTTGGTTGCCTGAAAGGAA-3'; a probe that can screen the blaIMP-4 gene, an example of which is shown in SEQ ID NO: 111: 5'-AGAAGCTTGGCCAAAGTCCGCTAA-3'; a probe that can screen the SmeDEF gene, an example of which is shown in SEQ ID NO: 114: 5'-TCAGCGTGGCGGAAGAAGAGTTT-3'; a probe that can screen the Sul1 gene, an example of which is shown in SEQ ID NO: 117: 5'-TCAATCGACAGCTTCCAACCGGAA-3'; a probe that can screen the QnrA gene, an example of which is shown in SEQ ID NO: 120: 5'-TTTCAAGGCCTGCCGTCTGTCTTT-3'; a probe that can screen the QnrB gene, an example of which is shown in SEQ ID NO: A probe that can screen for the Aac(6')-Ib-cr gene, an example of which is shown in SEQ ID NO: 132: 5'-CATCACAACTGGGCAAAGGCTTGG-3'; a probe that can screen for the ermB gene, an example of which is shown in SEQ ID NO: 135: 5'-TGCCAGCGGAATGCTTTCATCCTA-3'; a probe that can screen for the MefA gene, an example of which is shown in SEQ ID NO: 136: 5'-TGCCAGCGGAATGCTTTCATCCTA-3'; a probe that can screen for the MefA gene, an example of which is shown in SEQ ID NO: 137: 5'-CGCATCGCGCTTCACTTCAATCAC-3'; a probe that can screen for the Aac(6')-Ib-cr gene, an example of which is shown in SEQ ID NO: 138: 5'-CATCACAACTGGGCAAAGGCTTGG-3'; a probe that can screen for the ermB gene, an example of which is shown in SEQ ID NO: 139: 5'-TGCCAGCGGAATGCTTTCATCCTA-3'; a probe that can screen for the MefA gene, an example of which is shown in SEQ ID NO: 140: 5'-CGCATCGCGCTTCACTTCAATCAC-3'; a probe that can screen for the NO:138: 5'-CCCAGCACTCAATTCGGTTACACCA-3'; a probe capable of screening the cfr gene, an example of which is shown in SEQ ID NO:141: 5'-TACCTGCCCTTCGTTTGCTTCTCC-3'; a probe capable of screening the aac(3')-VI gene, an example of which is shown in SEQ ID NO:144: 5'-TTCTGGCGTCTGGAGAATGTGACG-3';A probe that can screen the aph(3')-VI gene, an example of which is shown in SEQ ID NO: 147: 5'-TTTAGATCTTGGCCGTGCTGGGTT-3'; a probe that can screen the ant(2")-Ia gene, an example of which is shown in SEQ ID NO: 150: 5'-TACTTGACTGCGAACCTGCTTGGT-3'; a probe that can screen the DfrA1 gene, an example of which is shown in SEQ ID NO: 153: 5'-AATGGCTGTTGGTTGGACGCAAG-3'; a probe that can screen the catA1 gene, an example of which is shown in SEQ ID NO: 156: 5'-ACGGTGTAACAAGGGTGAACACTATCC-3'; a probe that can screen the tetA gene, an example of which is shown in SEQ ID NO: 159: 5'-ATATCACTGATGGCGATGAGCGCG-3'; a probe that can screen the tetB gene, an example of which is shown in SEQ ID NO: NO: 162: 5'-TGGATGGAATAGCATGATGGTTGGCT-3'; internal reference (human B2M gene) probe, an example of which is shown in SEQ ID NO: 165: 5'-TGCCTGCCGTGTGAACCATGTGACT-3'.

[0091] In one aspect, the present application provides a combination of primers and probes that can simultaneously screen for 32 types of pathogenic microbial resistance genes, the combination of primers and probes comprising any one or more of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96.

[0092] In some embodiments, the 5' end of the probe sequence used in the present application is labeled with a fluorescent group, and the 3' end of the probe sequence used in the present application is labeled with a fluorescence quenching group. When the above probes are located in the same reaction system, the spectral ranges of the fluorescent groups for screening different types of bloodstream infection pathogens and microbial resistance genes are different. The fluorescent group of the probe sequence used in the present application can be any fluorescent group commonly used in the art, including but not limited to FAM, VIC, ROX, Cy5, and / or HEX; the fluorescence quenching group of the probe sequence used in the present application can be any fluorescent quenching group commonly used in the art, including but not limited to BHQ1, BHQ2, BHQ3, and / or Eclipse.

[0093] On the one hand, the probes used in the present application and the fluorescent reporter group labeled at the 5' end and the fluorescent quencher group labeled at the 3' end are as follows: the probe for screening Bacteroides fragilis is shown in SEQ ID NO: 3, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-CAGTTGTCCAAGTGGCGACG-3'-BHQ1; the probe for screening Staphylococcus epidermidis is shown in SEQ ID NO: 6, whose 5' end is fluorescently labeled with HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-TGTGTATCATTATGCCATGAGCTTG-3'-BHQ1; the probe for screening Enterococcus faecalis is shown in SEQ ID NO: 9, whose 5' end is fluorescently labeled with ROX and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-GGCTTCATCCTAATCTTCAAGACAA-3'-BHQ2; the probe for screening Streptococcus pneumoniae is shown in SEQ ID NO:12, whose 5' end is fluorescently labeled with Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-Cy5-TTAGAAAACGTGGGCAGGGAA-3'-BHQ2; the probe for screening Acinetobacter baumannii is shown in SEQ ID NO:15, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1 / BHQ2, i.e., 5'-FAM-TCACAACCCGACAACGGTGAGCAA-3'-BHQ1 and 5'-HEX-TCACAACCCGACAACGGTGAGCAA-3'-BHQ2; the probe for screening Enterobacter cloacae is shown in SEQ ID NO:18, whose 5' end is fluorescently labeled with HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-AACCTTTGCCGACGTTGTCGGTCA-3'-BHQ1; the probe for screening Enterococcus faecium is shown in SEQ ID NO: NO:21, whose 5' end is fluorescently labeled with ROX and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TTCATCCATTTTGGACTGATGCA-3'-BHQ2; the probe for screening Staphylococcus aureus is shown in SEQ ID NO:24, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TGGCTGAGATGAACTGTTCAGACCC-3'-BHQ2 and 5'-Cy5-TGGCTGAGATGAACTGTTCAGACCC-3'-BHQ2;The probe for screening Klebsiella pneumoniae is shown in SEQ ID NO: 27, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CCGATTGAAAAACGCTCCGGGC-3'-BHQ2, 5'-Cy5-CCGATTGAAAAACGCTCCGGGC-3'-BHQ2; the probe for screening Pseudomonas aeruginosa is shown in SEQ ID NO: 30, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-AACACAAACGCACTCGGAAAAATCG-3'-BHQ2, 5'-Cy5-AACACAAACGCACTCGGAAAAATCG-3'-BHQ2; the probe for screening Escherichia coli is shown in SEQ ID NO: NO:33, the 5' end of which is fluorescently labeled with FAM / HEX and the 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-GCCTGCAGCTTCCATACGCT-3'-BHQ1, 5'-HEX-GCCTGCAGCTTCCATACGCT-3'-BHQ1; the probe for screening Staphylococcus capitis is shown in SEQ ID NO:36, the 5' end of which is fluorescently labeled with FAM and the 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-ACCTCTTGCGAATAGTTCAGTACTTTC-3'-BHQ1; the probe for screening Stenotrophomonas maltophilia is shown in SEQ ID NO: NO:39, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TCTTGCCTTCGTTCATCAGCTCGT-3'-BHQ2, 5'-Cy5-TCTTGCCTTCGTTCATCAGCTCGT-3'-BHQ2; the probe for screening Haemophilus influenzae is shown in SEQ ID NO:42, whose 5' end is fluorescently labeled with ROX / Cy5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CAAATATTGAAATTGGGCTTTGACG-3'-BHQ2, 5'-Cy5-CAAATATTGAAATTGGGCTTTGACG-3'-BHQ2; the probe for screening Serratia marcescens is shown in SEQ ID NO: As shown in NO:45, its 5' end is fluorescently labeled with Cy5.5, and its 3' end is fluorescently labeled with BHQ3, i.e. 5'-Cy5.5-GCGCGCTGAACTACACCACT-3'-BHQ3;The probe for screening Candida tropicalis is shown in SEQ ID NO: 48, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-GGATCATACGTTCCATTTGCTTTA-3'-BHQ1, 5'-HEX-GGATCATACGTTCCATTTGCTTTA-3'-BHQ1; the probe for screening Candida krusei is shown in SEQ ID NO: 51, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AGCATCTGGCCCTGGCTATAACAC-3'-BHQ1, 5'-HEX-AGCATCTGGCCCTGGCTATAACAC-3'-BHQ1; the probe for screening Candida parapsilosis is shown in SEQ ID NO:54, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TAACGTATCTGCAGACGTGGCTGC-3'-BHQ1, 5'-HEX-TAACGTATCTGCAGACGTGGCTGC-3'-BHQ1; the probe for screening Candida glabrata is shown in SEQ ID NO:57, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-CTGCCGCAAGTCATGGGTTCTTG-3'-BHQ1, 5'-HEX-CTGCCGCAAGTCATGGGTTCTTG-3'-BHQ1; the probe for screening Candida albicans is shown in SEQ ID NO:60, whose 5' end is fluorescently labeled with ROX / Cy5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-AACAAACTTGCTTTGGCGGTGGG-3'-BHQ2, 5'-Cy5-AACAAACTTGCTTTGGCGGTGGG-3'-BHQ2; the probe for screening Cryptococcus neoformans is shown in SEQ ID NO:63, whose 5' end is fluorescently labeled with ROX / Cy5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CCTGTCAGCCCGGCGTAATAAGTT-3'-BHQ2, 5'-Cy5-CCTGTCAGCCCGGCGTAATAAGTT-3'-BHQ2; the probe for the internal reference (human) is shown in SEQ ID As shown in NO:66, its 5' end is fluorescently labeled with Cy5.5, and its 3' end is fluorescently labeled with BHQ3, i.e. 5'-Cy5.5-TGCCTGCCGTGTGAACCATGTGACT-3'-BHQ3.

[0094] On the other hand, the probes used in the present application and the fluorescent reporter group labeled at the 5' end and the fluorescent quencher group labeled at the 3' end are as follows: the probe for screening the mecA gene is shown in SEQ ID NO: 69, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AGCACTTGTAAGCACACCTTCATATGACG-3'-BHQ1; the probe for screening the mecC gene is shown in SEQ ID NO: 72, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TCACTACATCACCAGGTTCAACCCA-3'-BHQ1; the probe for screening the VanA gene is shown in SEQ ID NO: NO:75, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TCAGGCTCATCCTTCGGTGTGAAA-3'-BHQ1, 5'-HEX-TCAGGCTCATCCTTCGGTGTGAAA-3'-BHQ1; the probe for screening VanB gene is shown in SEQ ID NO:78, whose 5' end is fluorescently labeled with FAM and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TTACGCCAAAGGACGAACCTGACC-3'-BHQ1; the probe for screening MCR-1 gene is shown in SEQ ID NO: NO:81, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AGTTTCTTTCGCGTGCATAAGCCG-3'-BHQ1, 5'-HEX-AGTTTCTTTCGCGTGCATAAGCCG-3'-BHQ1; the probe for screening the blaKPC gene is shown in SEQ ID NO:84, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AAACCGGAACCTGCGGAGTGTATG-3'-BHQ1, 5'-HEX-AAACCGGAACCTGCGGAGTGTATG-3'-BHQ1; the probe for screening the blaCTX-M gene is shown in SEQ ID NO:85, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AAACCGGAACCTGCGGAGTGTATG-3'-BHQ1, 5'-HEX-AAACCGGAACCTGCGGAGTGTATG-3'-BHQ1; As shown in NO:87, its 5' end is fluorescently labeled with FAM / HEX, and its 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TTCATCCATTTTGGACTGATGCA-3'-BHQ1, 5'-HEX-TTCATCCATTTTGGACTGATGCA-3'-BHQ1;The probe for screening the blaOXA-23 gene is shown in SEQ ID NO: 90, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TGGCTGAGATGAACTGTTCAGACCC-3'-BHQ1, 5'-HEX-TGGCTGAGATGAACTGTTCAGACCC-3'-BHQ1; the probe for screening the blaNDM-1 gene is shown in SEQ ID NO: 93, whose 5' end is fluorescently labeled with HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-CAGGACAAGATGGGCGGTATGGAC-3'-BHQ1; the probe for screening the blaTEM gene is shown in SEQ ID NO: NO:96, whose 5' end is fluorescently labeled with FAM / HEX, and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-TCACCCAGAAACGCTGGTGAAAGT-3'-BHQ1, 5'-HEX-TCACCCAGAAACGCTGGTGAAAGT-3'-BHQ1; the probe for screening the blaSHV gene is shown in SEQ ID NO:99, whose 5' end is fluorescently labeled with FAM / HEX, and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AGGTGCTCATCATGGGAAAGCGTT-3'-BHQ1, 5'-HEX-AGGTGCTCATCATGGGAAAGCGTT-3'-BHQ1; the probe for screening the blaVIM-1 gene is shown in SEQ ID SEQ ID NO: 102, whose 5'-end is fluorescently labeled with HEX and whose 3'-end is fluorescently labeled with BHQ1, i.e., 5'-HEX-CAAACGACTGCGTTGCGATATGCG-3'-BHQ1; the probe for screening the blaVIM-2 gene is shown in SEQ ID NO: 105, whose 5'-end is fluorescently labeled with FAM / HEX and whose 3'-end is fluorescently labeled with BHQ1, i.e., 5'-FAM-CTGCGAGTGTGCTCTATGGTGGTT-3'-BHQ1 and 5'-HEX-CTGCGAGTGTGCTCTATGGTGGTT-3'-BHQ1; the probe for screening the blaIMP-1 gene is shown in SEQ ID NO: 108, whose 5'-end is fluorescently labeled with ROX and whose 3'-end is fluorescently labeled with BHQ2, i.e., 5'-ROX-AGTGGTTTGGTTGCCTGAAAGGAA-3'-BHQ2;The probe for screening the blaIMP-4 gene is shown in SEQ ID NO: 111, whose 5' end is fluorescently labeled with FAM / HEX and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-FAM-AGAAGCTTGGCCAAAGTCCGCTAA-3'-BHQ1, 5'-HEX-AGAAGCTTGGCCAAAGTCCGCTAA-3'-BHQ1; the probe for screening the SmeDEF gene is shown in SEQ ID NO: 114, whose 5' end is fluorescently labeled with ROX / CY5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TCAGCGTGGCGGAAGAAGAGTTT-3'-BHQ2, 5'-CY5-TCAGCGTGGCGGAAGAAGAGTTT-3'-BHQ2; the probe for screening the Sul1 gene is shown in SEQ ID NO: NO:117, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TCAATCGACAGCTTCCAACCGGAA-3'-BHQ2, 5'-CY5-TCAATCGACAGCTTCCAACCGGAA-3'-BHQ2; the probe for screening QnrA gene is shown in SEQ ID NO:120, whose 5' end is fluorescently labeled with HEX, and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-TTTCAAGGCCTGCCGTCTGTCTTT-3'-BHQ1; the probe for screening QnrB gene is shown in SEQ ID NO:131, whose 5' end is fluorescently labeled with HEX, and whose 3' end is fluorescently labeled with BHQ1, i.e., 5'-HEX-TTTCAAGGCCTGCCGTCTGTCTTT-3'-BHQ1. NO:123, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-ATTGCACCCTTTCTGGCTTTCACG-3'-BHQ2, 5'-CY5-ATTGCACCCTTTCTGGCTTTCACG-3'-BHQ2; the probe for screening the gyrB gene is shown in SEQ ID NO:126, whose 5' end is fluorescently labeled with Cy5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-Cy5-ATCATGACCGATGCTGACGTCGAC-3'-BHQ2; the probe for screening the gyrA gene is shown in SEQ ID NO:129, whose 5' end is fluorescently labeled with ROX, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-CGCATCGCGCTTCACTTCAATCAC-3'-BHQ2; the probe for screening the Aac(6')-Ib-cr gene is shown in SEQ ID As shown in NO:132, its 5' end is fluorescently labeled with CY5.5, and its 3' end is fluorescently labeled with BHQ3, i.e., 5'-CY5.5-CATCACAACTGGGCAAAGGCTTGG-3'-BHQ3;The probe for screening the ermB gene is shown in SEQ ID NO: 135, whose 5' end is fluorescently labeled with ROX / CY5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TGCCAGCGGAATGCTTTCATCCTA-3'-BHQ2, 5'-CY5-TGCCAGCGGAATGCTTTCATCCTA-3'-BHQ2; the probe for screening the MefA gene is shown in SEQ ID NO: 138, whose 5' end is fluorescently labeled with CY5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-CY5-CCCAGCACTCAATTCGGTTACACCA-3'-BHQ2; the probe for screening the cfr gene is shown in SEQ ID NO: NO:141, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TACCTGCCCTTCGTTTGCTTCTCC-3'-BHQ2, 5'-CY5-TACCTGCCCTTCGTTTGCTTCTCC-3'-BHQ2; the probe for screening aac(3')-VI gene is shown in SEQ ID NO:144, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TTCTGGCGTCTGGAGAATGTGACG-3'-BHQ2, 5'-CY5-TTCTGGCGTCTGGAGAATGTGACG-3'-BHQ2; the probe for screening aph(3')-VI gene is shown in SEQ ID NO: NO:147, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TTTAGATCTTGGCCGTGCTGGGTT-3'-BHQ2, 5'-CY5-TTTAGATCTTGGCCGTGCTGGGTT-3'-BHQ2; the probe for screening ant(2")-Ia gene is shown in SEQ ID NO:150, whose 5' end is fluorescently labeled with ROX / CY5, and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-TACTTGACTGCGAACCTGCTTGGT-3'-BHQ2, 5'-CY5-TACTTGACTGCGAACCTGCTTGGT-3'-BHQ2; the probe for screening DfrA1 gene is shown in SEQ ID NO: As shown in NO:153, its 5' end is fluorescently labeled with ROX / CY5, and its 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-AATGGCTGTTGGTTGGACGCAAG-3'-BHQ2, 5'-CY5-AATGGCTGTTGGTTGGACGCAAG-3'-BHQ2;The probe for screening the catA1 gene is shown in SEQ ID NO: 156, whose 5' end is fluorescently labeled with CY5 and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-CY5-ACGGTGTAACAAGGGTGAACACTATCC-3'-BHQ2; the probe for screening the tetA gene is shown in SEQ ID NO: 159, whose 5' end is fluorescently labeled with ROX and whose 3' end is fluorescently labeled with BHQ2, i.e., 5'-ROX-ATATCACTGATGGCGATGAGCGCG-3'-BHQ2; the probe for screening the tetB gene is shown in SEQ ID NO: 162, whose 5' end is fluorescently labeled with CY5.5 and whose 3' end is fluorescently labeled with BHQ3, i.e., 5'-CY5.5-TGGATGGAATAGCATGATGGTTGGCT-3'-BHQ3; the probe for the internal reference (human) is shown in SEQ ID As shown in NO:99, its 5' end is fluorescently labeled with Cy5.5, and its 3' end is fluorescently labeled with BHQ3, i.e. 5'-Cy5.5-TGCCTGCCGTGTGAACCATGTGACT-3'-BHQ3.

[0095] In some embodiments, the digital PCR reaction premix provided herein, in addition to the above primer pairs, or the combination of any of the above primer pairs and probes, may further include any one, two, three, four, five, six, seven, or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + and / or Mg 2+ ion.

[0096] The present application provides a kit for detecting a target gene, which includes the primer pair mentioned above, or a combination of any primer pair and a probe mentioned above, or a combination of the primer pair and any probe mentioned above. The kit of the present application may also include any one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ The kit of the present application may also include a positive control and / or a negative control.

[0097] In some embodiments, the present application provides a method for detecting a target gene for a purpose other than disease diagnosis, the method comprising the steps of:

[0098] (1) Processing the sample to be tested;

[0099] The sample volume should be 2-10 mL, preferably 5-10 ml, 6-10 ml, 7-10 ml, 8-10 ml, or 9-10 ml, with 10 ml being the most preferred volume to increase the detection rate. Centrifuge at 800 rpm for 10 minutes and collect the supernatant. The separated sample can be stored at -20°C ± 5°C for 3 years. It is recommended to use a commercial kit to extract DNA from the sample. After DNA extraction, it is recommended to perform testing immediately; otherwise, it is best to store it below -20°C for no more than 3 months.

[0100] (2) Prepare a digital PCR amplification mixture according to Table 1, which contains the sample to be detected provided in step (1) and the following digital PCR reaction premix; the final concentrations of the forward and reverse primers in the digital PCR reaction premix are 50-900 nM, preferably 50-500 nM, 200-500 nM, and most preferably 450 nM; the final concentrations of each probe are 50-800 nM, preferably 50-500 nM, 100-400 nM, and most preferably 250 nM; Mg 2+ The final concentration of ions is 0.5-5mM, preferably 1-4.5mM, 1.5-4mM, 2-3.5mM, and most preferably 3mM; the final concentration of bovine serum albumin is 0.01-1wt%, preferably 0.02-0.5wt%, 0.03-0.1wt%, 0.04-0.08wt%, and most preferably 0.05%; the final concentration of hot-start Taq enzyme (which can be an antibody-modified hot-start Taq enzyme, a chemically modified hot-start Taq enzyme, or an aptamer-modified hot-start Taq enzyme) is 0.5-10U / reaction, preferably 1-5U / reaction, 1.5-4U / reaction, 2-3.5U / reaction, and optimally 2.5U / reaction; the final concentration of dNTP is 20-300μM, preferably 50-250μM, 100-230μM, 150-220μM, and most preferably 200μM. The digital PCR reaction premix may also contain UNG enzyme at a final concentration of 0.1-10 U / reaction, 0.5-5 U / reaction, 1-2.5 U / reaction, 1-2 U / reaction, and most preferably 1.5 U / reaction. The pH of the digital PCR reaction premix is ​​7-9, preferably 7.4-8.5, preferably 7.8-8.2, and most preferably 8.0.

[0101] Those skilled in the art will understand that the substances and concentrations listed in the above-mentioned PCR amplification mixture are schematic and can be selected by those skilled in the art based on the actual system.

[0102] Table 1 Digital PCR amplification mixture

[0103] (3) preparing microdroplets and performing PCR amplification reaction;

[0104] Step 1: Prepare droplets: First, add 50-100 μl of droplet generation oil into the square filling hole on the top of the microfluidic cartridge, preferably 70-100 μl, and most preferably 75 μl.

[0105] Step 2: Add 13-25 μl of digital PCR amplification mixture (containing amplified nucleic acid template, primers, probes, etc.) into the circular sample well on the top of the microfluidic cartridge, preferably 15-25 μl, most preferably 20 μl;

[0106] Step 3: Use a pipette to add 5-10 μl of sealing oil on top of the digital PCR amplification mixture, preferably 10 μl.

[0107] Step 4: Cover the sample and oil wells on the top of the microfluidic cartridge and the waste well on the bottom with two plastic sealing caps. Place the microfluidic cartridge containing the PCR amplification mixture on a PCR amplifier, preferably an SG-2000 PCR amplifier. Perform the amplification reaction according to the recommended amplification protocol in Table 2:

[0108] Table 2 An example of a PCR amplification reaction program

[0109] (4) Signal collection

[0110] Select the MUT mode on the biochip reader (e.g., Dscanner4-1000, registration number: Su Xie Zhu Zhun 20202220818), set the fluorescence channels to FAM, HEX, ROX, and Cy5, remove the high-throughput digital PCR cartridge (e.g., microfluidic cartridge) from the PCR amplifier, preferably the SG-2000 PCR amplifier (registration number: Su Xie Zhu Zhun 20202220887), and transfer it smoothly to the biochip reader for signal collection.

[0111] (5) Result analysis

[0112] Click "Analyze" on the biochip reader to analyze the experimental data. Check that "Accepted Droplets" is ≥ 10,000 before proceeding to further analysis. Click "2D Amplitude" to view the 2D scatter plots for Channels 1, 2, 3, and 4. Click on the 2D plot to divide the regions based on the four sets of positive controls. The darkest black dots at the bottom of each figure represent droplets without amplification template, green dots represent droplets containing FAM-labeled probes, blue dots represent droplets containing HEX-labeled probes, orange dots represent droplets containing ROX-labeled probes, purple dots represent droplets labeled with CY5 probes, and red dots represent droplets labeled with both sets of probes.

[0113] The primers, probes, digital PCR reaction premix containing primer probes, kits, and microfluidic cartridges provided in this application can be used to prepare quantitative detection kits for detecting multiple pathogens in bloodstream infections, and can also be used alone or together with other reagents for the detection of multiple pathogens in bloodstream infections. The processing steps for plasma samples are: take 2-10mL of peripheral blood sample, preferably use 5-10ml, 6-10ml, 7-10ml, 8-10ml, 9-10ml, and most preferably use 10ml to improve the detection rate. It is preferred to use EDTA anticoagulant, and heparin anticoagulant cannot be used. Separate plasma within 2 hours after the blood is removed from the body, generally not more than 4 hours; the plasma volume is 0.5-4mL, preferably 1-4ml, 2-4ml, 3-4ml, and most preferably 4ml. Centrifuge at 800 rpm for 10 minutes to obtain the supernatant. If plasma cannot be separated in time, it can be stored at room temperature in a vacuum blood collection tube, or in a dedicated room temperature blood collection tube (Streck tube) containing a free DNA protectant and an anti-cell lysis protectant. The storage time should not exceed 3 days. The separated plasma can be stored at -20℃±5℃ for 3 years. It is recommended to use a commercial kit to extract microbial DNA from plasma samples. After DNA extraction, it is recommended to test immediately, otherwise it is best to store it below -20℃ for no more than 3 months.

[0114] In some embodiments, the sample types involved in the test also include whole blood (also called blood), plasma, serum, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid or ascites.

[0115] A whole blood test involves drawing peripheral blood from the patient and examining the various blood cells and plasma components within the whole blood. Alternatively, plasma or serum samples can be tested as needed.

[0116] A throat swab test is a medical testing method that uses a medical cotton swab to collect a small amount of secretions from the human throat, inoculates it into a special culture dish, and then places it in a temperature-controlled device for incubation. The throat swab sample in this application can be a sample directly obtained by a skilled practitioner from the clinic or a throat swab sample that has been incubated for a period of time, depending on the item being tested.

[0117] Sputum is the secretion of alveoli, bronchi and trachea. Healthy people have very little sputum. Under normal circumstances, bronchial mucosal glands and goblet cells can secrete a small amount of mucus to keep the respiratory mucosa moist. Under pathological conditions, the respiratory mucosa and alveoli are stimulated and the mucosa becomes congested and edematous, serous fluid exudates, and mucus secretion increases. Various cells (red blood cells, white blood cells, macrophages, etc.), exudates such as fibrin and mucus, inhaled dust, certain tissue necrosis, etc. are mixed to form sputum. The sputum samples used in this application are samples that can be commonly obtained by those skilled in the art.

[0118] Urine and feces samples are also samples that can be obtained by those skilled in the art through routine means.

[0119] Cerebrospinal fluid (CSF) is a colorless, transparent liquid that fills the ventricles, subarachnoid space, and central canal of the spinal cord. Produced by the choroid plexus within the ventricles, CSF has properties similar to those of plasma and lymph and is slightly viscous. CSF is considered extracellular fluid. The CSF samples used in this application are those commonly available to those skilled in the art.

[0120] In this application, body fluid refers to various body fluids derived from blood, urine, feces, serous cavity fluid, cerebrospinal fluid, ascites, etc.

[0121] The ascites involved in this application refers to the fluid in the human abdominal cavity, which can be obtained by conventional means.

[0122] For convenience, the sequences of the primers and probes used in this application are summarized in the following table:

[0123] Table 3 Primer and probe sequences for 21 pathogens in this application

[0124] Table 4 An example of fluorescent labeling of probes used in this application

[0125] Table 5 Primer and probe sequences for 32 microbial resistance genes in this application

[0126] Table 6 An example of fluorescent labeling of probes used in this application

[0127] Example

[0128] Example 1 Multiple pathogens and drug resistance gene detection kit

[0129] 1.1 Detection kits for multiple pathogens in bloodstream infections

[0130] This application develops a digital PCR detection kit for detecting 21 pathogens and one internal reference gene in bloodstream infections. The kit contains primer and probe mix 1, primer and probe mix 2, digital PCR premix (digital PCR buffer, dNTPs, MgCl2, Taq hot start enzyme mix, glycerol, betaine), negative quality control, and positive quality control.

[0131] Each sample requires two sets of testing. Digital PCR droplet generation and PCR amplification are performed on the sample using the primer-probe mixture and digital PCR premix in the kit. The volume of the primer-probe mixture is 2 μL, the volume of the digital PCR premix is ​​10 μL, the DNA sample volume is 8 μL, and the total reaction volume is 20 μL. The reaction system for digital PCR amplification is shown in Table 7-8:

[0132] Table 7 Group 1 Multiple Digital Reaction System

[0133] Table 8 Group 2 Multiple Digital Reaction System

[0134] The components of the kit of this application are shown in Table 9.

[0135] Table 9 Composition of the kit

[0136] The kit of this application is equipped with an internal quality control system to monitor the sample collection and extraction process to avoid false negatives. The kit of this application is equipped with negative and positive quality controls to monitor the digital PCR amplification and chip reading of the sample and assist in the interpretation of the final results. The specific steps are as follows:

[0137] 1) Release nucleic acid from the sample to be tested;

[0138] 2) In one well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, a mixture of 11 pathogen primers and fluorescently labeled probes (corresponding to group 1) and a digital PCR buffer were added;

[0139] 3) In another well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, add another 10 pathogen primers and fluorescent-labeled probe mixtures (corresponding to Group 2), an internal reference primer and fluorescent-labeled probe mixture, and digital PCR buffer;

[0140] 4) performing digital PCR droplet generation and PCR amplification in a digital PCR amplifier;

[0141] Add 75 μl of oil-phase encapsulated reagent to the oil wells in the high-throughput digital PCR chip, add 20 μl of amplification mixture to the reagent wells, cover with a sealing cap, and place on a PCR amplifier for droplet generation and PCR amplification.

[0142] Perform the amplification reaction according to the following recommended amplification protocol:

[0143] 5) Signal collection

[0144] After amplification is completed using the nucleic acid obtained in step 4) as a template, the chip is used as a test object, and fluorescence is detected by a biochip reader. The results are interpreted based on the presence and intensity of fluorescence in each droplet.

[0145] Interpretation of results:

[0146] The results are shown in FIG2A-FIG2F , wherein in FIG2A-FIG2F , the labels of the 11 bacterial species detected in the first well and the 10 bacterial species detected in the second well plus 1 internal reference are shown in Table 10 .

[0147] Table 10

[0148] If, in addition to negative droplets, there are positive droplets in the positive droplet area in the 2D graph, and the number of droplets is greater than the threshold, the strain test result corresponding to the position is determined to be positive. The test results are shown in Table 11 below:

[0149] Table 11

[0150] 1.2 Kits for detecting multiple drug-resistant genes in bacterial pathogen infections

[0151] This application develops a digital PCR detection kit for detecting 32 pathogen resistance genes in bacterial infections plus one internal reference gene. The kit contains primer and probe mixture 1, primer and probe mixture 2, primer and probe mixture 3, digital PCR premix (digital PCR buffer, dNTPs, MgCl2, Taq hot start enzyme mixture, glycerol, betaine), negative quality control, and positive quality control.

[0152] Each sample requires three sets of testing. Digital PCR droplet generation and PCR amplification are performed on the test sample using the primer-probe mix and digital PCR premix provided in the kit. The volume of the primer-probe mix is ​​2 μL, the volume of the digital PCR premix is ​​10 μL, the DNA sample volume is 8 μL, and the total reaction volume is 20 μL. The reaction system for digital PCR amplification is shown in Tables 7-8 above.

[0153] The components of the kit of this application are shown in Table 12.

[0154] Table 12 Composition of the kit

[0155] The kit of this application is equipped with an internal quality control system to monitor the sample collection and extraction process to avoid false negatives. The kit of this application is equipped with negative and positive quality controls to monitor the digital PCR amplification and chip reading of the sample and assist in the interpretation of the final results. The specific steps are as follows:

[0156] 1) Release nucleic acid from the sample to be tested;

[0157] 2) In one well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, a mixture of 11 pathogen resistance gene primers and fluorescently labeled probes (corresponding to group 1) and a digital PCR buffer were added;

[0158] 3) In another well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, a mixture of primers and fluorescently labeled probes for another 11 pathogen resistance genes (corresponding to group 2) and digital PCR buffer were added;

[0159] 4) In the third well of the microfluidic chip, using the nucleic acid obtained in step 1) as a template, add another mixture of 10 pathogen resistance gene primers and fluorescently labeled probes (corresponding to Group 3), a mixture of an internal reference primer and fluorescently labeled probe, and digital PCR buffer;

[0160] 5) performing digital PCR droplet generation and PCR amplification in a digital PCR amplifier;

[0161] Add 75 μl of oil-phase encapsulated reagent to the oil wells in the high-throughput digital PCR chip, add 20 μl of amplification mixture to the reagent wells, cover with a sealing cap, and place on a PCR amplifier for droplet generation and PCR amplification.

[0162] Perform the amplification reaction according to the following recommended amplification protocol:

[0163] 5) Signal collection

[0164] After amplification is completed using the nucleic acid obtained in step 4) as a template, the chip is used as a test object, and fluorescence is detected by a biochip reader. The results are interpreted based on the presence and intensity of fluorescence in each droplet.

[0165] Interpretation of results:

[0166] The results are shown in FIG12 . The markers of the 11 drug-resistant genes detected in the first well, the 11 drug-resistant genes detected in the second well, and the 10 drug-resistant genes plus 1 internal reference detected in the third well are shown in Table 13 .

[0167] Table 13

[0168] If, in addition to negative droplets, there are positive droplets in the positive droplet area in the 2D graph, and the number of droplets is greater than the threshold, the strain test result corresponding to the position is determined to be positive. The test results are shown in Table 14 below:

[0169] Table 14 Note: Schematic diagram of 32 drug-resistance genes and 1 internal reference detected in 3 wells. The first well detects 11 drug-resistance genes, the second well detects 11 drug-resistance genes, and the third well detects 10 drug-resistance genes plus 1 internal reference.

[0170] Example 2 Adjusting the Probe Ratio for Detection of Different Pathogens and Drug Resistance Genes

[0171] 2.1 Adjusting the ratio of probes for detection of different pathogens

[0172] Figures 3A-3C illustrate an example of how the ratio of FAM and HEX probes can be adjusted to shift the position of a Candida tropicalis-positive droplet cluster. In Figure 3A, the FAM:HEX probe ratio for the Candida tropicalis-positive droplet cluster is 1:3, and the position indicated by the box is shown. Adjusting the FAM:HEX probe ratio to 1.8:3, i.e., increasing the FAM probe concentration, shows that Candida tropicalis has moved to the position indicated by the box in Figure 3B. Figure 3C shows an overlay of a Candida tropicalis-positive cluster with a FAM:HEX probe concentration of 1:3 and a Candida tropicalis-positive cluster with a FAM:HEX probe concentration of 1.8:3. It is clearly shown that adjusting the FAM:HEX probe ratio can shift the position of the positive droplet cluster in the 2D image of the digital PCR assay. Figures 3D-3F illustrate an example of how the ratio of ROX and Cy5 probes can be adjusted to shift the position of a Haemophilus influenzae-positive droplet cluster. Figure 3D shows an image of H. influenzae-positive droplets with a ROX:Cy5 probe ratio of 4:1, with the positions indicated by the boxes. Adjusting the ROX:Cy5 probe ratio to 5.6:1, i.e., increasing the ROX probe concentration, reveals that the H. influenzae-positive droplet cluster has shifted to a new position in Figure 3E, as indicated by the boxes. Figure 3F overlays the positive droplet clusters with two different ROX:Cy5 ratios, demonstrating that adjusting the ROX:Cy5 ratio can shift the position of the H. influenzae-positive droplet cluster in the 2D image of the digital PCR assay.

[0173] 2.2 Adjusting the ratio of probes for detection of different drug-resistant genes

[0174] Figures 13A-13C show an example of how to adjust the position of the mecA resistance gene positive droplet cluster by adjusting the ratio of FAM and HEX probes. In Figure 13A, the position of the mecA resistance gene positive droplet cluster with a ratio of FAM and HEX probes of 1:2.5 is indicated by the arrow. When the ratio of FAM and HEX probes is adjusted to 1:3.2, that is, the concentration of the FAM probe is increased, it can be seen that the mecA resistance gene positive droplet cluster has moved to the position indicated by the arrow in 13B. Figure 13C shows the superposition of the mecA resistance gene positive droplet cluster with a FAM:HEX probe concentration of 1:2.5 and the mecA resistance gene positive droplet cluster with a FAM:HEX probe concentration of 1:3.2. It can be clearly seen that by adjusting the concentration ratio of the two probes FAM and HEX, the position of the mobile positive droplet cluster in the digital PCR detection 2D map (as shown by the arrow) can be achieved. Figures 13D-13F illustrate an example of how the position of the mecC resistance gene-positive droplet cluster can be adjusted by adjusting the ratio of ROX and Cy5 probes. Figure 13D shows a mecC resistance gene-positive droplet cluster with a ROX:Cy5 probe ratio of 4:1, with the position indicated by the arrow. Adjusting the ROX:Cy5 probe ratio to 5.5:1, i.e., increasing the ROX probe concentration, shows that the mecC resistance gene-positive droplet cluster has moved to a new position in Figure 13E, as indicated by the arrow. Figure 13F superimposes the positive droplet clusters with two different ROX:Cy5 ratios, demonstrating that adjusting the ROX:Cy5 ratio can achieve the effect of shifting the position of the mecC resistance gene-positive droplet cluster in the 2D image of the digital PCR assay.

[0175] Example 3 Detection limit range test

[0176] The present application uses Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Serratia marcescens, Haemophilus influenzae, Candida tropicalis, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida albicans liquid solutions for dilution to test the detection limit range of the primer and probe combination of the present application.

[0177] (1) Bacterial liquid dilution

[0178] The above bacterial suspension was diluted in series to form 5 concentrations of samples (S9: 5×10 6 、S10:5×10 5 、S11:5×10 4 、S12:5×10 3 、S13 5×10 2, S14: 5.0, S15: 0.5, S16: 0 copies / ml), so that the detection concentrations were 4×10 4 , 4×10 3 , 4×10 2 , 4×10 1 , 4, 0.4, and 0 copies / reaction. The formula for calculating copies / reaction is: copies / mL x 8 μL. The loading volume for each digital PCR reaction was 8 μL.

[0179] (2) Digital PCR droplet formation and amplification

[0180] Digital PCR droplet formation and digital PCR amplification were performed with reference to the reaction system and reaction procedure described in Example 1.

[0181] (3) Signal collection

[0182] Remove the digital PCR chip from the digital PCR amplification instrument, transfer it smoothly and place it on the biochip reader for signal collection.

[0183] (4) Interpretation and analysis of results:

[0184] A threshold line is drawn midway between the negative and positive droplets of each strain's positive control sample. If the number of positive droplets exceeds the threshold, the strain corresponding to that positive droplet is considered positive. The software reports the copy number corresponding to the positive droplet. A linear regression graph is constructed with the dilution as the horizontal axis and the logarithm of the measured value for each dilution as the vertical axis.

[0185] Figure 4 shows the linear range results for 21 bacterial sample dilution series. As shown in Figure 4, the detection kit of this application achieved a minimum detection limit of 4 copies / reaction, calculated from the dilution results. No positive droplets were detected in the 0 copy / mL test wells. This demonstrates that the blank detection limit of the kit is 0 copies / mL.

[0186] Example 4 Precision Test

[0187] The present application uses plasmids containing target fragments of Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus haemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Staphylococcus hominis, Candida tropicalis, Pneumocystis jiroveci, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida albicans to test the precision of the primer and probe combination of the present application.

[0188] Detection method: A high concentration of plasmid mixture (a synthetic plasmid mixture of 21 target pathogens, with a concentration of approximately 5×106 copies / ml), diluted 10,000 times to about 5×10 2 A low-concentration sample was prepared using 100 copies / ml. Following the procedure in Example 3, two concentration samples were measured continuously for 5 days, with the test repeated 4 times each day. The mean, standard deviation, and coefficient of variation (CV) of the log values ​​were calculated. The test results are as follows:

[0189] Table 15 Bacterial group CV%

[0190] Table 16 Fungal Group CV%

[0191] High concentration (5×10 6 The CV values ​​(copies / ml level) ranged from 0.15% to 0.43%, with an average CV of 0.275%.

[0192] Low concentration (5×10 2 The CV values ​​of the two groups (copies / ml level) ranged from 5.74% to 10.89%, with an average CV of 8.87%.

[0193] Example 5 Correctness Test

[0194] The present application uses Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Serratia marcescens, Haemophilus influenzae, Candida tropicalis, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, Candida albicans, and plasmids of the target fragment of the internal reference gene to test the accuracy of the primer and probe combination of the present application.

[0195] (1) Plasmid dilution

[0196] The above plasmid 5×10 6 The high concentration standard of 10 copies / ml was diluted into 12 concentrations (2 / 5 / 10 / 20 / 50 / 100 / 200 / 500 / 1000 / 2000 / 5000 / 10000 times) so that the detection concentrations were 2.5×10 6 , 1×10 6 , 5×10 5 , 2.5×10 5 , 1×10 5 , 5×10 4 , 2.5×10 4 , 1×10 4 , 5×10 3 , 2.5×10 3 , 1×10 3 , 5×102 Copy / React.

[0197] (2) Digital PCR droplet formation and amplification

[0198] Digital PCR droplet formation and digital PCR amplification were performed with reference to the reaction system and reaction procedure described in Example 1.

[0199] (3) Signal collection

[0200] Remove the digital PCR chip from the digital PCR amplification instrument, transfer it smoothly and place it on the biochip reader for signal collection.

[0201] Result interpretation and analysis:

[0202] Table 17 Bacteria detection results

[0203] Table 18 Fungal detection results

[0204] Example 6 Specificity Detection

[0205] The present application selects purple bacillus, intermedius streptococcus, bronchiseptica Bordetella, marcescens serratia, sobrinus streptococcus, listeria ei, cepacia, streptococcus anginosus, anaerobic peptostreptococcus, streptococcus sanguinis, constellation streptococcus constellation subspecies, small veillonella, streptococcus agalactiae, harmless listeria, banded bacillus, aeromonas temperate, streptococcus salivarius, aeromonas caviae, terrestrial raouli bacteria (terrestrial klebsiella), Staphylococcus capitis subspecies, dysgalactiae streptococcus, micrococcus luteus, sonnei shigella, staphylococcus squirrel, listeria monocytogenes, aquatic raenella, salmonella typhimurium, lactobacillus acidophilus, aeromonas hydrophila, foam agglutinating bacteria. Polybacterium, Legionella pneumophila subspecies, Staphylococcus stutzeri subspecies, Pseudomonas stutzeri, Enterobacter carcinogenes, Sphingomonas paucimobilis, Neisseria lactis, Staphylococcus carnosus, Japanese Gouweiduo bacteria, Streptococcus canis, Enterococcus lead yellow, Proteus mirabilis, Pseudomonas rice, Bacteroides vulgaris, Acinetobacter pitei, Ralstonia pitei, Streptococcus bovis, Eikenella corrodens, Streptococcus pyogenes, Neisseria meningitidis, Enterococcus durable, Staphylococcus xylosus, Moraxella morrhagica, Morganella morganii, Pseudomonas mendocina, Enterococcus caecum, Streptococcus equi subspecies zooepidemicus, Rhodococcus equi, Talaromyces marneffei, Gemini cocci, Bacteroides ovatus, Staphylococcus lugdunensis, Acinetobacter rhus Bacillus, Haemophilus influenzae, Neisseria gonorrhoeae, Shigella dysenteriae, Pseudomonas shigellae, Bacillus cereus, Bacillus subtilis, Streptococcus oralis, Acinetobacter radioresistance, Enterobacter cobequi, Kingella, Streptococcus gallolyticus, Staphylococcus pseudointermedius, Enterobacter hallii, Streptococcus mitis, Enterococcus hirae, Listeria monocytogenes, Streptococcus gordonii, Neisseria dry, Streptococcus parasanguinis, Clostridium difficile, Bordetella parapertussis, Staphylococcus saprophyticus, Shigella flexneri, Yersinia flexneri, Hafnia alvei, Photobacterium nonsymbioticum, Staphylococcus auris, Pseudomonas putida, Bacteroides thetaiotaomicron, Burkholderia multivora, Pasteurella multocida, Parasitoid The test cross-reacted with 115 strains, including Bacillus, Bacillus licheniformis, Bacteroides monocytogenes, Listeria monocytogenes, Propionibacterium acnes, Vibrio vulnificus, Pantoea agglomerans, Salmonella enteritidis, Yersinia enterocolitica, Klebsiella oxytoca, Pseudomonas alcaligenes, Streptococcus mutans, Activeinobacter bayenii, Acinetobacter berezzii, Shigella boydii, Actinobacillus actinomycetemcomitans, Cronobacter sakazakii, Bordetella pertussis, Enterococcus gallinarum, Clostridium perfringens, Staphylococcus warfarinii and Haemophilus parainfluenzae. These strains are pathogen strains that have homology with the nucleic acid sequences of the target pathogens and are likely to cause the same or similar clinical symptoms. It has been confirmed that all of the above strains do not carry drug-resistant genes.

[0206] (1) Release nucleic acid of the strain to be tested;

[0207] (2) mixing nucleic acids extracted from the above strains;

[0208] (3) Digital PCR droplet formation and amplification;

[0209] Digital PCR droplet formation and digital PCR amplification were performed according to the reaction system and reaction procedures described in Example 1;

[0210] (4) Signal collection:

[0211] Remove the digital PCR chip from the digital PCR amplification instrument, transfer it smoothly and place it on the biochip reader for signal collection;

[0212] (5) Interpretation and analysis of results:

[0213] A threshold line is drawn midway between the negative and positive droplets of each strain's positive control sample. If the number of positive droplets exceeds the threshold, the strain corresponding to that positive droplet is considered positive. The software reports the copy number corresponding to the positive droplet. A linear regression graph is constructed with the dilution as the horizontal axis and the logarithm of the measured value for each dilution as the vertical axis.

[0214] The results of cross-reactions with the 115 strains are shown in Figures 5 and 14, respectively. No cross-reactions were detected between these 115 strains and the primer probes for the 21 pathogens included in the kit. Figures 5 and 14 contain only negative droplet clusters; no positive droplet clusters were detected that cross-reacted with any of the 115 strains.

[0215] Example 7: Detection of a real pathogen-positive sample by the detection kit

[0216] Clinical samples were tested according to the method of Example 1. Figure 6A is a 2D graph of the clinical sample test results, and Figure 6B is a 2D graph of the positive quality control product. Figure 6C shows the results of superimposing and comparing the 2D graphs of the clinical sample and the positive quality control product. Through superimposition and comparison, it can be seen that the negative droplet cluster in the lower left corner of the clinical sample and the negative droplet cluster of the positive quality control product overlap. The droplet cluster in the upper right corner of the negative droplet cluster in the 2D graph of the clinical sample overlaps with the position of Klebsiella pneumoniae in the positive quality control product, that is, Klebsiella pneumoniae was detected in the clinical sample.

[0217] Example 8 Detection Kit for Whole Blood Samples from Patients with Hemorrhage

[0218] Blood samples were collected from 7 patients who tested positive for blood culture (confirmed bloodstream infection), with 1.8-2 mL of blood collected from each patient. After nucleic acid extraction, 21 pathogen detection kits were tested using a digital PCR method. The test results of blood culture were compared with the results of the digital PCR detection kit, and the results are shown in Table 19. Through the blind test comparison of the above two methods, it can be seen that the digital PCR bloodstream infection detection kit and blood culture detection have high consistency. Pathogens in patient blood can be quickly detected with 2 mL of whole blood within 5 hours, avoiding the tedious steps of blood culture requiring 40-60 ml (2-3 sets of blood culture bottles) and several days of culture time, providing a fast and effective method for detecting bloodstream infection pathogens. At the same time, blood culture only detected 1 pathogen infection due to sensitivity, culture medium selection, inhibition caused by competition between strains, and inhibition caused by patients taking antibiotics in advance. The digital PCR method detected more pathogens than blood culture, indicating that the digital PCR method has higher detection sensitivity.

[0219] Table 19 Comparison of blood culture test and digital PCR test reagents and kits test results

[0220] Example 9: Detection of other samples from infected patients using the detection kit

[0221] Samples were collected from 6 patients with clinical symptoms of infection or suspected infection. The sample types included urine, ascites, and cerebrospinal fluid, with a collection volume of 3-5 ml. The sputum was about 1 ml. Throat swabs and anal swabs were conventional swabs stored in 2 ml of preservative solution. After nucleic acid extraction, 21 pathogens were detected by digital PCR using the pathogen detection kit of Example 1. Comparing the test results of the smear microscopy method with the results of the digital PCR detection kit, it can be seen that the digital PCR bloodstream infection detection kit and the smear test have high consistency, and can accurately distinguish specific bacterial species that cannot be interpreted by the smear.

[0222] As shown in Figure 7, the digital PCR result was positive for Staphylococcus epidermidis. The patient's sputum smear was observed to have a coccal infection, but the specific type of coccus could not be identified. Digital PCR specifically identified the specific type of coccus as Staphylococcus epidermidis. As shown in Figure 8, the digital PCR results and the cerebrospinal fluid smear method results were both negative, and the results of the two detection methods were consistent. Figures 9A-9D show that the test kit of the present application can detect Acinetobacter baumannii, Pseudomonas maltophilia (a type of bacillus), Enterobacter cloacae, and Candida albicans in the patient's sputum samples; while the sputum smear results only detected Acinetobacter baumannii or bacilli. The above results show that the smear method only determines whether there is a bacillus infection based on the morphology under the microscope after bacterial staining, but it cannot accurately distinguish which type of bacillus infection it is, while digital PCR can be used to identify the type of bacillus infected. Figures 10A-10H show that the patient's throat swab and anal swab were both positive for Candida albicans, Acinetobacter baumannii, and Klebsiella pneumonia. The patient's anal swab was positive for Enterococcus faecium. As shown in Figure 11, urine culture showed that the patient had Candida tropicalis infection.

[0223] Example 10: Detection of a sample of a bacterial strain carrying a drug-resistant gene by a detection kit

[0224] The samples of bacterial strains carrying drug-resistant genes were detected according to the method of Example 1. All bacterial strain samples were obtained from the standard bacterial strain library. Figure 15A shows the detection results of Escherichia coli strains carrying drug-resistant genes, indicating that the blaOXA-48 resistance gene was detected in the strain sample; Figure 15B shows the detection results of Stenotrophomonas maltophilia carrying drug-resistant genes, indicating that the smeDEF resistance gene was detected in all 5 strain samples tested; Figure 15C shows the detection results of Klebsiella pneumoniae carrying drug-resistant genes, indicating that the smeDEF resistance gene was detected in the strain sample; Figure 15D shows the detection results of Staphylococcus aureus carrying drug-resistant genes, indicating that the mecA resistance gene was detected in both 2 strain samples tested; Figure 15E shows the detection results of VanA gene and blaCTX-M gene carrying drug-resistant genes, among which the vanA resistance gene (FAM fluorescent label) was detected in strain samples #130 and #148, and the vanB resistance gene (HEX fluorescent label) was detected in strain sample #135. All three strains could be detected by the vanA+vanB mixed primer probe (ROX fluorescent labeling).

[0225] Example 11: Detection of pathogen resistance genes in a real ICU patient's sputum sample using the detection kit

[0226] Four sputum samples obtained from patients at the First Affiliated Hospital of Soochow University were tested according to the method of Example 1. The results showed that blaNDM resistance gene was detected in three of the four samples ( FIG. 16 ).

[0227] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A combination for specifically detecting P target genes, wherein the combination includes P pairs of primers for amplifying the P genes and P probes for hybridizing the P genes, and N concentrations of M different fluorescent groups are used to label the P probes respectively, and there are differences in the types and / or concentrations of the fluorescent groups carried by each of the P probes; The P target genes and the fluorescent groups satisfy the following formula: 2 ≤ P ≤ (M N + M) - X, M is a positive integer greater than or equal to 2; The range of N is 1 - 3; The range of X is 0 - 19.

2. The combination according to claim 1, wherein M is 2, 3, 4, 5, 6, 7 or 8, N is 1, 2 or 3, and X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

3. The combination according to claim 1, wherein for the N concentrations, it is achieved by using the ratio of any two different fluorescent groups, wherein, The ratio range of any two different fluorescent groups A and B is: A:B is 1:(0.20 - 10).

4. The combination according to any one of claims 1 - 3, wherein the target gene is a pathogen gene or a drug - resistant gene.

5. The combination according to claim 4, wherein the pathogen is one or more pathogens selected from the following: Bacteroides fragilis, Staphylococcus epidermidis, Enterococcus faecium, Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacter cloacae, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis, Stenotrophomonas maltophilia, Serratia marcescens, Candida tropicalis, Cryptococcus neoformans, Candida krusei, Candida parapsilosis, Candida glabrata, Candida albicans, Human herpesvirus 1, Human alpha - herpesvirus 1, Varicella - zoster virus, Listeria monocytogenes, Mycobacterium tuberculosis, Human cytomegalovirus, Human herpesvirus 5, Human herpesvirus 2, Human herpesvirus 6, Epstein - Barr virus, Human herpesvirus 4, Parvovirus B19, Primate erythroparvovirus 1, Human herpesvirus 7, JC virus, JC polyomavirus, Human polyomavirus 2, Pseudorabies virus, Suid herpesvirus 1, Pseudorabies virus (Suid herpesvirus 1), Neisseria meningitidis, Streptococcus agalactiae, Brucella, Brucella melitensis, Brucella ovis, Haemophilus influenzae, Cryptococcus gattii, Cryptococcus gattii, Aspergillus fumigatus, Angiostrongylus cantonensis, Taenia solium, Toxoplasma gondii, Balamuthia mandrillaris, Adenovirus subgroup C type 1, Adenovirus subgroup B type 4, Human mastadenovirus B, Human polyomavirus 1, Merkel cell polyomavirus, Human polyomavirus 5, Torque teno virus, Streptococcus intermedius, Fusobacterium, Porphyromonas endodontalis, Neisseria meningitidis, Klebsiella oxytoca, Streptococcus suis, Campylobacter coli, Streptococcus constellatus, Proteus, Salmonella, Prevotella, Rhizopus oryzae, Mucor, Histoplasma capsulatum, Cryptococcus laurentii, Cryptococcus albidus, Aspergillus flavus, Treponema pallidum, and Orientia tsutsugamushi; or The drug-resistant gene is one or more drug-resistant genes selected from the following: mecA, mecC, vanA, vanB, MCR-1, blaKPC, blaCTX-M, blaOXA-23, blaNDM-1, blaTEM, blaSHV, blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-4, SmeDEF, Sul1, QnrA, QnrB, gyrA, gyrB, Aac(6’)-Ib-cr, ermB, MefA, aac(3’)-VI, aph(3’)-VI, ant(2”)-Ia, DfrA1, catA1, tetA, tetB, and cfr.

6. According to the combination described in claim 5, wherein the primers and probes for the pathogen consist of two sets of primers and probes. The first set of primers and probes is: a PCR primer and probe combination that specifically amplifies Staphylococcus capitis. The primers include a forward primer shown in SEQ ID NO: 34 and a reverse primer shown in SEQ ID NO: 35, and the probe includes a probe shown in SEQ ID NO: 36; A PCR primer and probe combination for specifically amplifying Acinetobacter baumannii, the primer comprising a forward primer as shown in SEQ ID NO:13 and a reverse primer as shown in SEQ ID NO:14, and the probe comprising a probe as shown in SEQ ID NO:15; A PCR primer and probe combination for specifically amplifying Candida tropicalis, the primer comprising a forward primer as shown in SEQ ID NO:46 and a reverse primer as shown in SEQ ID NO:47, and the probe comprising a probe as shown in SEQ ID NO:48; A PCR primer and probe combination for specifically amplifying Candida krusei, the primer comprising a forward primer as shown in SEQ ID NO:49 and a reverse primer as shown in SEQ ID NO:50, and the probe comprising a probe as shown in SEQ ID NO:51; A PCR primer and probe combination for specifically amplifying Staphylococcus epidermidis, the primer comprising a forward primer as shown in SEQ ID NO:4 and a reverse primer as shown in SEQ ID NO:5, and the probe comprising a probe as shown in SEQ ID NO:6; A PCR primer and probe combination for specifically amplifying Enterococcus faecium, the primer comprising a forward primer as shown in SEQ ID NO:19 and a reverse primer as shown in SEQ ID NO:20, and the probe comprising a probe as shown in SEQ ID NO:21; A PCR primer and probe combination for specifically amplifying Cryptococcus neoformans, the primer comprising a forward primer as shown in SEQ ID NO:61 and a reverse primer as shown in SEQ ID NO:62, and the probe comprising a probe as shown in SEQ ID NO:63; A PCR primer and probe combination for specifically amplifying Stenotrophomonas maltophilia, the primer comprising a forward primer as shown in SEQ ID NO:37 and a reverse primer as shown in SEQ ID NO:38, and the probe comprising a probe as shown in SEQ ID NO:39; A PCR primer and probe combination for specifically amplifying Klebsiella pneumoniae, wherein the primer comprises a forward primer as shown in SEQ ID NO: 25 and a reverse primer as shown in SEQ ID NO: 26, and the probe comprises a probe as shown in SEQ ID NO: 27; A PCR primer and probe combination for specifically amplifying Streptococcus pneumoniae, wherein the primer comprises a forward primer as shown in SEQ ID NO: 10 and a reverse primer as shown in SEQ ID NO: 11, and the probe comprises a probe as shown in SEQ ID NO: 12; A PCR primer and probe combination for specifically amplifying Serratia marcescens, wherein the primer comprises a forward primer as shown in SEQ ID NO: 43 and a reverse primer as shown in SEQ ID NO: 44, and the probe comprises a probe as shown in SEQ ID NO: 45; The second group of primers and probes are: A PCR primer and probe combination for specifically amplifying Bacteroides fragilis, wherein the primer comprises a forward primer as shown in SEQ ID NO: 1 and a reverse primer as shown in SEQ ID NO: 2, and the probe comprises a probe as shown in SEQ ID NO: 3; A PCR primer and probe combination for specifically amplifying Enterococcus faecalis, wherein the primer comprises a forward primer as shown in SEQ ID NO: 7 and a reverse primer as shown in SEQ ID NO: 8, and the probe comprises a probe as shown in SEQ ID NO: 9; A PCR primer and probe combination for specifically amplifying Enterobacter cloacae, wherein the primer comprises a forward primer as shown in SEQ ID NO: 16 and a reverse primer as shown in SEQ ID NO: 17, and the probe comprises a probe as shown in SEQ ID NO: 18; A PCR primer and probe combination for specifically amplifying Staphylococcus aureus, wherein the primer comprises a forward primer as shown in SEQ ID NO: 22 and a reverse primer as shown in SEQ ID NO: 23, and the probe comprises a probe as shown in SEQ ID NO: 24; A PCR primer and probe combination for specifically amplifying Pseudomonas aeruginosa, wherein the primer comprises a forward primer as shown in SEQ ID NO: 28 and a reverse primer as shown in SEQ ID NO: 29, and the probe comprises a probe as shown in SEQ ID NO: 30; A PCR primer and probe combination for specifically amplifying Escherichia coli, wherein the primer comprises a forward primer as shown in SEQ ID NO: 31 and a reverse primer as shown in SEQ ID NO: 32, and the probe comprises a probe as shown in SEQ ID NO: 33; A PCR primer and probe combination for specifically amplifying Haemophilus influenzae, wherein the primer comprises a forward primer as shown in SEQ ID NO: 40 and a reverse primer as shown in SEQ ID NO: 41, and the probe comprises a probe as shown in SEQ ID NO: 42; A PCR primer and probe combination for specifically amplifying Candida parapsilosis, wherein the primers comprise a forward primer as shown in SEQ ID NO:52 and a reverse primer as shown in SEQ ID NO:53, and the probe comprises a probe as shown in SEQ ID NO:54; A PCR primer and probe combination for specifically amplifying Candida glabrata, wherein the primers comprise a forward primer as shown in SEQ ID NO:55 and a reverse primer as shown in SEQ ID NO:56, and the probe comprises a probe as shown in SEQ ID NO:57; A PCR primer and probe combination for specifically amplifying Candida albicans, wherein the primers comprise a forward primer as shown in SEQ ID NO:58 and a reverse primer as shown in SEQ ID NO:59, and the probe comprises a probe as shown in SEQ ID NO:

60.

7. The combination according to claim 5, wherein the primers and probes for the drug-resistant gene include any one, two or three of the following three groups of primers and probes, wherein The first set of primers and probes are: a PCR primer and probe combination for specifically amplifying mecA, the primers comprising an upstream primer as shown in SEQ ID NO: 67 and a downstream primer as shown in SEQ ID NO: 68, and the probe comprising a probe as shown in SEQ ID NO: 69; a PCR primer and probe combination for specifically amplifying vanA, the primers comprising an upstream primer as shown in SEQ ID NO: 73 and a downstream primer as shown in SEQ ID NO: 74, and the probe comprising a probe as shown in SEQ ID NO: 75; A PCR primer and probe combination for specifically amplifying MCR-1, wherein the primers comprise an upstream primer as shown in SEQ ID NO:79 and a downstream primer as shown in SEQ ID NO:80, and the probe comprises a probe as shown in SEQ ID NO:81; A PCR primer and probe combination for specifically amplifying blaKPC, wherein the primers comprise an upstream primer as shown in SEQ ID NO:82 and a downstream primer as shown in SEQ ID NO:83, and the probe comprises a probe as shown in SEQ ID NO:84; A PCR primer and probe combination for specifically amplifying blaNDM-1, wherein the primers comprise an upstream primer as shown in SEQ ID NO:91 and a downstream primer as shown in SEQ ID NO:92, and the probe comprises a probe as shown in SEQ ID NO:93; A PCR primer and probe combination for specifically amplifying blaIMP-1, wherein the primers comprise an upstream primer as shown in SEQ ID NO:106 and a downstream primer as shown in SEQ ID NO:107, and the probe comprises a probe as shown in SEQ ID NO:108; A PCR primer and probe combination for specifically amplifying SmeDEF, wherein the primers comprise an upstream primer as shown in SEQ ID NO:112 and a downstream primer as shown in SEQ ID NO:113, and the probe comprises a probe as shown in SEQ ID NO:114; A PCR primer and probe combination for specifically amplifying Sul1, wherein the primers comprise an upstream primer as shown in SEQ ID NO:115 and a downstream primer as shown in SEQ ID NO:116, and the probe comprises a probe as shown in SEQ ID NO:117; A PCR primer and probe combination for specifically amplifying QnrB, wherein the primer comprises an upstream primer shown in SEQ ID NO: 121 and a downstream primer shown in SEQ ID NO: 122, and the probe comprises a probe shown in SEQ ID NO: 123; A PCR primer and probe combination for specifically amplifying gyrB, wherein the primer comprises an upstream primer shown in SEQ ID NO: 124 and a downstream primer shown in SEQ ID NO: 125, and the probe comprises a probe shown in SEQ ID NO: 126; A PCR primer and probe combination for specifically amplifying Aac(6’)-Ib-cr, wherein the primer comprises an upstream primer shown in SEQ ID NO: 130 and a downstream primer shown in SEQ ID NO: 131, and the probe comprises a probe shown in SEQ ID NO: 132; The second group of primers and probes are: A PCR primer and probe combination for specifically amplifying mecC, wherein the primer comprises an upstream primer shown in SEQ ID NO: 70 and a downstream primer shown in SEQ ID NO: 71, and the probe comprises a probe shown in SEQ ID NO: 72; A PCR primer and probe combination for specifically amplifying blaCTX-M, wherein the primer comprises an upstream primer shown in SEQ ID NO: 85 and a downstream primer shown in SEQ ID NO: 86, and the probe comprises a probe shown in SEQ ID NO: 87; A PCR primer and probe combination for specifically amplifying blaTEM, wherein the primer comprises an upstream primer shown in SEQ ID NO: 94 and a downstream primer shown in SEQ ID NO: 95, and the probe comprises a probe shown in SEQ ID NO: 96; A PCR primer and probe combination for specifically amplifying blaVIM-2, wherein the primer comprises an upstream primer shown in SEQ ID NO: 103 and a downstream primer shown in SEQ ID NO: 104, and the probe comprises a probe shown in SEQ ID NO: 105; A PCR primer and probe combination for specifically amplifying blaVIM-1, wherein the primer comprises an upstream primer shown in SEQ ID NO: 100 and a downstream primer shown in SEQ ID NO: 101, and the probe comprises a probe shown in SEQ ID NO: 102; A PCR primer and probe combination for specifically amplifying gyrA, wherein the primer comprises an upstream primer shown in SEQ ID NO: 127 and a downstream primer shown in SEQ ID NO: 128, and the probe comprises a probe shown in SEQ ID NO: 129; A PCR primer and probe combination for specifically amplifying ermB, wherein the primer comprises an upstream primer shown in SEQ ID NO: 133 and a downstream primer shown in SEQ ID NO: 134, and the probe comprises a probe shown in SEQ ID NO: 135; A PCR primer and probe combination for specifically amplifying aac(3’)-VI, wherein the primer comprises an upstream primer shown in SEQ ID NO: 142 and a downstream primer shown in SEQ ID NO: 143, and the probe comprises a probe shown in SEQ ID NO: 144; A PCR primer and probe combination for specifically amplifying cfr, wherein the primer comprises an upstream primer shown in SEQ ID NO: 139 and a downstream primer shown in SEQ ID NO: 140, and the probe comprises a probe shown in SEQ ID NO: 141; A PCR primer and probe combination for specifically amplifying catA1, wherein the primer comprises an upstream primer shown in SEQ ID NO: 154 and a downstream primer shown in SEQ ID NO: 155, and the probe comprises a probe shown in SEQ ID NO: 156; A PCR primer and probe combination for specifically amplifying TetB, wherein the primer comprises an upstream primer shown in SEQ ID NO: 160 and a downstream primer shown in SEQ ID NO: 161, and the probe comprises a probe shown in SEQ ID NO: 162; The third group of primers and probes is: A PCR primer and probe combination for specifically amplifying vanB, wherein the primer comprises an upstream primer shown in SEQ ID NO: 76 and a downstream primer shown in SEQ ID NO: 77, and the probe comprises a probe shown in SEQ ID NO: 78; A PCR primer and probe combination for specifically amplifying blaOXA-23, wherein the primer comprises an upstream primer shown in SEQ ID NO: 88 and a downstream primer shown in SEQ ID NO: 89, and the probe comprises a probe shown in SEQ ID NO: 90; A PCR primer and probe combination for specifically amplifying blaSHV, wherein the primer comprises an upstream primer shown in SEQ ID NO: 97 and a downstream primer shown in SEQ ID NO: 98, and the probe comprises a probe shown in SEQ ID NO: 99; A PCR primer and probe combination for specifically amplifying blaIMP-4, wherein the primer comprises an upstream primer shown in SEQ ID NO: 109 and a downstream primer shown in SEQ ID NO: 110, and the probe comprises a probe shown in SEQ ID NO: 111; A PCR primer and probe combination for specifically amplifying QnrA, wherein the primer comprises an upstream primer shown in SEQ ID NO: 118 and a downstream primer shown in SEQ ID NO: 119, and the probe comprises a probe shown in SEQ ID NO: 120; A PCR primer and probe combination for specifically amplifying TetA, wherein the primer comprises an upstream primer shown in SEQ ID NO: 157 and a downstream primer shown in SEQ ID NO: 158, and the probe comprises a probe shown in SEQ ID NO: 159; A PCR primer and probe combination for specifically amplifying aph(3’)-VI, wherein the primer comprises an upstream primer shown in SEQ ID NO: 145 and a downstream primer shown in SEQ ID NO: 146, and the probe comprises a probe shown in SEQ ID NO: 147; A PCR primer and probe combination for specifically amplifying MefA, wherein the primer comprises an upstream primer shown in SEQ ID NO: 136 and a downstream primer shown in SEQ ID NO: 137, and the probe comprises a probe shown in SEQ ID NO: 138; A PCR primer and probe combination for specifically amplifying ant(2”)-Ia, wherein the primer comprises an upstream primer shown in SEQ ID NO: 148 and a downstream primer shown in SEQ ID NO: 149, and the probe comprises a probe shown in SEQ ID NO: 150; A PCR primer and probe combination for specifically amplifying DfrA1, wherein the primer comprises an upstream primer shown in SEQ ID NO: 151 and a downstream primer shown in SEQ ID NO: 152, and the probe comprises a probe shown in SEQ ID NO: 153; A PCR primer and probe combination for specifically amplifying B2M, wherein the primer comprises an upstream primer shown in SEQ ID NO: 163 and a downstream primer shown in SEQ ID NO: 164, and the probe comprises a probe shown in SEQ ID NO:

165.

8. The PCR primer and probe combination according to claim 6 or 7, wherein the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end of the probe sequence is labeled with a fluorescent quenching group; when the probes are in the same reaction system, the spectral ranges of the fluorescent reporter groups for screening different types of pathogens or drug resistance genes are different.

9. The PCR primer and probe combination according to claim 8, wherein the fluorescent reporter group is selected from FAM, VIC, ROX, Cy5, and / or HEX; the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3, and / or Eclipse.

10. The PCR primer and probe combination according to claim 6, wherein the probes used and their fluorescent reporter groups labeled at the 5' end and fluorescent quenching groups labeled at the 3' end are as follows: The probe for screening Bacteroides fragilis is shown in SEQ ID NO: 3, with its 5'-end fluorescently labeled with FAM and its 3'-end fluorescently labeled with BHQ1; the probe for screening Staphylococcus epidermidis is shown in SEQ ID NO: 6, with its 5'-end fluorescently labeled with HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Enterococcus faecalis is shown in SEQ ID NO: 9, with its 5'-end fluorescently labeled with ROX and its 3'-end fluorescently labeled with BHQ2; the probe for screening Streptococcus pneumoniae is shown in SEQ ID NO: 12, with its 5'-end fluorescently labeled with Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Acinetobacter baumannii is shown in SEQ ID NO: 15, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1 / BHQ2; the probe for screening Enterobacter cloacae is shown in SEQ ID NO: 18, with its 5'-end fluorescently labeled with HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Enterococcus faecium is shown in SEQ ID NO: 21, with its 5'-end fluorescently labeled with ROX and its 3'-end fluorescently labeled with BHQ2; the probe for screening Staphylococcus aureus is shown in SEQ ID NO: 24, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Klebsiella pneumoniae is shown in SEQ ID NO: 27, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Pseudomonas aeruginosa is shown in SEQ ID NO: 30, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Escherichia coli is shown in SEQ ID NO: 33, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Staphylococcus capitis is shown in SEQ ID NO: 36, with its 5'-end fluorescently labeled with FAM and its 3'-end fluorescently labeled with BHQ1; the probe for screening Stenotrophomonas maltophilia is shown in SEQ ID NO: 39, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Haemophilus influenzae is shown in SEQ ID NO: 42, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Serratia marcescens is shown in SEQ ID NO: 45, with its 5'-end fluorescently labeled with Cy5.

5. The 3'-end is fluorescently labeled with BHQ3; the probe for screening Candida tropicalis is shown in SEQ ID NO: 48, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Candida krusei is shown in SEQ ID NO: 51, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Candida parapsilosis is shown in SEQ ID NO: 54, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Candida glabrata is shown in SEQ ID NO: 57, with its 5'-end fluorescently labeled with FAM / HEX and its 3'-end fluorescently labeled with BHQ1; the probe for screening Candida albicans is shown in SEQ ID NO: 60, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2; the probe for screening Cryptococcus neoformans is shown in SEQ ID NO: 63, with its 5'-end fluorescently labeled with ROX / Cy5 and its 3'-end fluorescently labeled with BHQ2.

11. The PCR primer and probe combination according to claim 7, wherein the probes used and their fluorescent reporter groups labeled at the 5' end and fluorescent quenching groups labeled at the 3' end are as follows: The probe for screening the mecA gene is shown in SEQ ID NO:69, with the 5'-end fluorescently labeled with FAM and the 3'-end fluorescently labeled with BHQ1; the probe for screening the mecC gene is shown in SEQ ID NO:72, with the 5'-end fluorescently labeled with FAM and the 3'-end fluorescently labeled with BHQ1; the probe for screening the vanA gene is shown in SEQ ID NO:75, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the vanB gene is shown in SEQ ID NO:78, with the 5'-end fluorescently labeled with FAM and the 3'-end fluorescently labeled with BHQ1; the probe for screening the MCR-1 gene is shown in SEQ ID NO:81, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaKPC gene is shown in SEQ ID NO:84, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaCTX-M gene is shown in SEQ ID NO:87, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaOXA-23 gene is shown in SEQ ID NO:24, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaNDM-1 gene is shown in SEQ ID NO:93, with the 5'-end fluorescently labeled with HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaTEM gene is shown in SEQ ID NO:96, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaSHV gene is shown in SEQ ID NO:99, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaVIM-1 gene is shown in SEQ ID NO:102, with the 5'-end fluorescently labeled with HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaVIM-2 gene is shown in SEQ ID NO:105, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the blaIMP-1 gene is shown in SEQ ID NO:108, with the 5'-end fluorescently labeled with ROX and the 3'-end fluorescently labeled with BHQ2; the probe for screening the blaIMP-4 gene is shown in SEQ ID NO:111, with the 5'-end fluorescently labeled with FAM / HEX and the 3'-end fluorescently labeled with BHQ1; the probe for screening the SmeDEF gene is shown in SEQ ID NO:114, with the 5'-end fluorescently labeled with ROX / CY5 and the 3'-end fluorescently labeled with BHQ2; the probe for screening the Sul1 gene is shown in SEQ ID NO:117, with the 5'-end fluorescently labeled with ROX / CY5 and the 3'-end fluorescently labeled with BHQ2;The probe for screening the QnrA gene is shown in SEQ ID NO: 120, with a CY5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the QnrB gene is shown in SEQ ID NO: 123, with a ROX / CY5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the gyrB gene is shown in SEQ ID NO: 126, with a Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the gyrA gene is shown in SEQ ID NO: 129, with a ROX fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the Aac(6')-Ib-cr gene is shown in SEQ ID NO: 132, with a Cy5.5 fluorescent label at the 5'-end and a BHQ3 fluorescent label at the 3'-end; the probe for screening the ermB gene is shown in SEQ ID NO: 135, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the MefA gene is shown in SEQ ID NO: 138, with a Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the cfr gene is shown in SEQ ID NO: 141, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the aac(3')-VI gene is shown in SEQ ID NO: 144, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the aph(3')-VI gene is shown in SEQ ID NO: 147, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the ant(2")-Ia gene is shown in SEQ ID NO: 150, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the DfrA1 gene is shown in SEQ ID NO: 153, with a ROX / Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the catA1 gene is shown in SEQ ID NO: 156, with a Cy5 fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the TetA gene is shown in SEQ ID NO: 159, with a ROX fluorescent label at the 5'-end and a BHQ2 fluorescent label at the 3'-end; the probe for screening the TetB gene is shown in SEQ ID NO: 162, with a Cy5.5 fluorescent label at the 5'-end and a BHQ3 fluorescent label at the 3'-end; the probe for screening the B2M gene is shown in SEQ ID NO: 165, with a Cy5.5 fluorescent label at the 5'-end and a BHQ3 fluorescent label at the 3'-end.; 12. The combination according to any one of claims 1-11, which is a combination used for digital PCR (dPCR) reaction.

13. A digital PCR reaction premix for specifically detecting a target gene, wherein the digital PCR reaction premix contains the combination according to any one of claims 1-12.

14. The digital PCR reaction premix according to claim 13, wherein the digital PCR reaction premix further comprises one, two, three, four, five, six, seven or eight components selected from the following: hot start Taq enzyme, UDG enzyme, dNTPs, bovine serum albumin, glycerol, betaine, NH4 + , and Mg 2+ ions.

15. A digital PCR microfluidic cartridge, wherein the digital PCR microfluidic cartridge includes the digital PCR reaction premix according to claim 13 or claim 14.

16. The microfluidic cartridge according to claim 15, wherein the microfluidic cartridge includes at least two wells.

17. A kit for detecting a target gene, wherein the kit includes the digital PCR (dPCR) reaction premix according to claim 13 or 14.

18. The kit according to claim 17, wherein the kit further includes a positive control, a negative control, and / or droplet generation oil.

19. The kit according to claim 18, wherein the kit further includes the digital PCR microfluidic cartridge according to claim 15.

20. A method for detecting a target gene for non-disease diagnosis purposes, the method comprising the following steps: (1) Releasing the nucleic acid of the sample to be tested; (2) Preparing a digital PCR amplification mixture containing the nucleic acid of the sample to be tested provided in step (1), the combination according to any one of claims 1-12, and / or the digital PCR premix according to claim 13 or 14; (3) Preparing droplets in a digital PCR microfluidic cartridge and performing a PCR amplification reaction; (4) Reading the fluorescence signal after the droplet PCR amplification reaction using a digital PCR microfluidic cartridge reader.

21. The method for detecting a target gene for non-disease diagnosis purposes according to claim 20, wherein the sample to be tested is selected from whole blood, serum, plasma, throat swab, sputum, urine, feces, cerebrospinal fluid, body fluid, or ascites.

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