Probe group and kit for detecting mutation of bacteria and drug-resistant genes thereof and application of probe group and kit

Abstract

The invention provides a probe group and a kit for detecting drug-resistant gene mutation of bacteria and application of the probe group and the kit. According to the present invention, the drug-resistant gene mutation detection of various bacteria can be simultaneously performed, the mutation frequency can be accurately quantified, the drug-resistant gene subtype can be accurately identified, the drug-resistant heterogeneity can be accurately diagnosed, the sensitivity and the specificity are high, the detection time is short, and the cost is substantially reduced.

Description

Technical Field

[0001] The present disclosure belongs to the field of bioengineering technology, and particularly relates to a probe set, a kit and an application for detecting drug-resistant gene mutations of bacteria. Background Art

[0002] The treatment of infectious diseases mainly relies on antimicrobial drugs. However, with the widespread use of antimicrobial drugs, the number of bacteria with antimicrobial resistance (AMR) has been increasing continuously, which has brought great difficulties to clinical anti-infection treatment and become a worldwide medical problem. The monitoring data of the China Antimicrobial Resistance Surveillance Network in 2022 showed that the resistance rates of Klebsiella pneumoniae to imipenem and meropenem in China were 20.4% and 21.9% respectively. The methicillin resistance rate in Staphylococcus aureus was 28.7%. The resistance rates of Escherichia coli to ceftriaxone and ciprofloxacin were 51.3% and 61.5% respectively. The resistance rates of Pseudomonas aeruginosa to imipenem and meropenem were 22.1% and 17.6% respectively. Antimicrobial resistance is caused by the use of antimicrobial drugs, especially the improper use of antimicrobial drugs. When microorganisms mutate or acquire drug-resistant genes, drug resistance is produced.

[0003] Rapid and accurate detection of pathogenic bacteria and their drug resistance can timely analyze and master the sources and distributions of drug-resistant bacteria and the information on the changes in drug resistance, which is beneficial to epidemiological surveillance and hospital infection control.

[0004] At present, traditional phenotypic detection methods are often used for clinical bacterial drug resistance detection, including dilution methods, diffusion methods (paper disk methods), etc. The traditional antimicrobial susceptibility test is still the main basis for current anti-infection treatment, but the detection cycle is long. Even for fully automated microbial identification and drug sensitivity analysis systems, they also need to go through three steps: culturing, isolation and purification, and identification. These methods are not conducive to timely anti-infection treatment and infection control. Using molecular biology methods for drug resistance detection is faster, more sensitive and specific. However, most of the commonly used drug-resistant gene molecular diagnostic techniques are PCR techniques based on amplification. Since PCR specifically amplifies the target fragment in vitro, it may lead to false positives or false negatives in the results.

[0005] Compared with traditional detection methods, the second-generation sequencing (NGS) method has many advantages in terms of detection time, throughput and accuracy, so it has incomparable advantages over traditional methods for severe infections and ICU infections. Through NGS, Mycobacterium tuberculosis can be accurately identified. At the same time, sequencing of drug-resistant genes can accurately detect mutation sites on the genes, thus providing more effective guidance for clinical practice. However, direct sequencing of clinical samples is limited by the complex situation of clinical samples, human interference, and the load of drug-resistant bacteria.

[0006] Therefore, there is an urgent need for a detection method that can quickly and accurately detect drug-resistant gene mutations in bacteria. Summary of the Invention

[0007] To solve at least one of the above technical problems, the present disclosure provides a probe set, a kit, a detection method, and their applications for detecting drug-resistant gene mutations in bacteria.

[0008] According to a first aspect of the present disclosure, a probe set for identifying bacterial species is provided, characterized in that the probe set includes one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 36, or one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0009] In some embodiments, the probe set includes any one or more of the following groups:

[0010] A probe set for detecting Enterococcus faecium, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 3, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0011] A probe set for detecting Enterococcus faecalis, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 4 to 6, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0012] A probe set for detecting Staphylococcus aureus, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 7 to 9, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0013] A probe set for detecting Klebsiella pneumoniae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 10 to 12, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0014] A probe set for detecting Acinetobacter baumannii, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 13 to 15, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0015] A probe set for detecting Escherichia coli, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 16 to 18, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0016] A probe set for detecting Pseudomonas aeruginosa, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 19-21, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0017] A probe set for detecting Serratia marcescens, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 22-24, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0018] A probe set for detecting Proteus mirabilis, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 25-27, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0019] A probe set for detecting Salmonella enterica, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 28-30, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0020] A probe set for detecting Streptococcus pneumoniae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 31-33, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0021] A probe set for detecting Enterobacter cloacae, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 34-36, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0022] In some embodiments, the drug-resistant genes include one or more of the following: ctx_m gene, ges gene, kpc gene, mecA gene, tem gene, oxa-51 gene, aph gene or sul gene.

[0023] In some embodiments, the drug-resistant genes include one or more of the following: mecA gene of Staphylococcus aureus; kpc gene of Klebsiella pneumoniae; oxa-51 gene of Acinetobacter baumannii; ges gene and tem gene of Pseudomonas aeruginosa; aph gene, tem gene, sul gene and ctx_m gene of Proteus mirabilis.

[0024] In some embodiments, the probe set comprises one or more of the nucleotide sequences shown in SEQ ID NO: 37-60, or one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0025] In some embodiments, the probe set includes:

[0026] A probe set for detecting the ctx_m gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 37-39, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0027] A probe set for detecting the ges gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 40-42, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0028] A probe set for detecting the kpc gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 43-45, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0029] A probe set for detecting the mecA gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 46-48, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0030] A probe set for detecting the tem gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 49-51, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0031] A probe set for detecting the oxa_51 gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 52-54, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0032] A probe set for detecting the aph gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 55-57, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0033] A probe set for detecting the sul gene, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 58-60, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0034] In some embodiments, the drug-resistant genes further include one or more of the following: MREJ gene, ampc gene, gim gene, imi gene, mecC gene, ndm gene, shv gene, sme gene, spm gene, vanA gene, vanB gene, vim gene, oxa-23 gene, oxa-24 gene, oxa-48 gene, aac_3 gene, ant gene, ermA gene, ermB gene, ermC gene, mcr gene, mefA gene, mefE gene, msrA gene, qnrA gene, tetA gene, tetB gene, tetM gene, vanD gene, imp gene or veb gene.

[0035] In some embodiments, the drug-resistant genes further include one or more of the following: mecC gene, MREJ gene, msrA gene, ermC gene of Staphylococcus aureus; imp gene, ampc gene of Klebsiella pneumoniae; oxa-51 gene, oxa-24 gene, ndm gene of Acinetobacter baumannii; oxa-48 gene, oxa-23 gene, shv gene, tetA gene, tetB gene of Escherichia coli; gim gene, spm gene, vim gene or veb gene of Pseudomonas aeruginosa; sme gene of Serratia marcescens; tem gene of Proteus mirabilis; mcr gene of Salmonella enterica; mefA gene, mefE gene or tetM gene of Streptococcus pneumoniae; vanA gene, vanB gene, vanD gene of Enterococcus faecium, Enterococcus faecalis; ermA, ermB gene of Enterococcus faecium; ant gene of Enterococcus faecalis; imi gene, aac_3 gene, qnrA gene of Enterobacter cloacae.

[0036] The probe set includes one or more of the nucleotide sequences shown in SEQ ID NO: 61 to 150, or one or more of the nucleotide sequences having at least 60% sequence identity thereto.

[0037] In some embodiments, the probe set for detecting drug-resistant bacteria includes:

[0038] The probe set for detecting the MREJ gene, which includes one or more of the nucleotide sequences shown in SEQ ID NO: 61 to 63, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0039] The probe set for detecting the ampc gene, which includes one or more of the nucleotide sequences shown in SEQ ID NO: 64 to 66, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0040] A probe set for detecting the gim gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 67 to 69, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0041] A probe set for detecting the imi gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 70 to 72, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0042] A probe set for detecting the mecC gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 73 to 75, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0043] A probe set for detecting the ndm gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 76 to 78, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0044] A probe set for detecting the shv gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 79 to 81, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0045] A probe set for detecting the sme gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 82 to 84, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0046] A probe set for detecting the spm gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 85 to 87, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0047] A probe set for detecting the vanA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 88 to 90, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0048] A probe set for detecting the vanB gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 91 to 93, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0049] A probe set for detecting the vim gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 94 to 96, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0050] A probe set for detecting the oxa_48 gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 97 to 99, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0051] A probe set for detecting the aac_3 gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 100 to 102, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0052] A probe set for detecting the ant gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 103 to 105, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0053] A probe set for detecting the ermA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 106 to 108, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0054] A probe set for detecting the ermB gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 109 to 111, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0055] A probe set for detecting the mcr gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 112 to 114, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0056] A probe set for detecting the mefA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 115 to 117, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0057] A probe set for detecting the mefE gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 118 to 120, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0058] A probe set for detecting the msrA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 121 to 123, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0059] A probe set for detecting the qnrA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 124 to 126, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0060] A probe set for detecting the tetA gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 127 to 129, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0061] A probe set for detecting the tetB gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 130 to 132, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0062] A probe set for detecting the tetM gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 133 to 135, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0063] A probe set for detecting the vanD gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 136 to 138, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0064] A probe set for detecting the veb gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 139 to 141, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0065] A probe set for detecting the imp gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 142 to 144, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0066] A probe set for detecting the OXA-23 gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 145 to 147, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto;

[0067] A probe set for detecting the OXA-24 gene, which comprises any one or more of the nucleotide sequences shown in SEQ ID NO: 148 to 150, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith.

[0068] In some embodiments, each probe sequence in the probe set specifically binds to the target region of each microorganism and cannot bind to any region of any other species.

[0069] In some embodiments, the probe is modified with biotin. After biotin modification, the probe sequence that has enriched the microorganisms in the sample is captured and enriched by streptavidin magnetic beads. Subsequently, the captured sample is eluted at a high temperature with a washing solution to remove non-specifically captured sequences, and then subjected to PCR specific amplification and then sequenced on a machine, thereby achieving the purpose of rapidly, highly sensitively and specifically enriching and detecting pathogenic microorganisms in the sample.

[0070] In some embodiments, the drug resistance gene mutation results in resistance to one or more of the following drugs:

[0071] Methicillin, β-lactam antibiotics, macrolides, lincosamides, streptogramins, tetracyclines, aminoglycosides, glycopeptides, sulfonamides, carbapenems, penicillins, cephalosporins, polymyxins or fluoroquinolones.

[0072] According to a second aspect of the present disclosure, there is provided a kit for detecting drug resistance gene mutations in bacteria, characterized in that the kit comprises the probe set described in the first aspect.

[0073] According to a third aspect of the present disclosure, there is provided a method for detecting drug resistance gene mutations in bacteria using the probe set described in the first aspect or the kit described in the second aspect, the method comprising the following steps:

[0074] 1) Extract nucleic acid from the sample,

[0075] 2) Construct a library for the extracted nucleic acid,

[0076] 3) Hybridization capture of the target sequence using the probe set,

[0077] 4) Sequence the captured product and perform data analysis.

[0078] In some embodiments, the sample comprises one or more of the following: cells, tissues, saliva, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, feces, tears, bronchoalveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge and their processed products.

[0079] In some embodiments, the sample includes one or more of the following: bronchoalveolar lavage fluid, sputum, nasopharyngeal swab, or oral swab fluid.

[0080] According to a fourth aspect of the present disclosure, a system for detecting drug-resistant gene mutations of bacteria is provided. The system includes:

[0081] (1) A detection module; and

[0082] (2) A judgment module.

[0083] In some embodiments, the system is used to perform the method described in the third aspect.

[0084] According to a fifth aspect of the present disclosure, an application of the probe set described in the first aspect in preparing a kit is provided.

[0085] In some embodiments, the probe set is used to detect one or more of the following drug-resistant bacteria: Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Salmonella enterica, Streptococcus pneumoniae, or Enterobacter cloacae.

[0086] In some embodiments, the kit is used to detect drug-resistant gene mutations of drugs, and the drugs include one or more of the following: methicillin, β-lactams, macrolides, lincosamides, streptogramins, tetracyclines, aminoglycosides, glycopeptides, sulfonamides, carbapenems, penicillins, cephalosporins, polymyxins, or fluoroquinolones.

[0087] In some embodiments, the drug-resistant gene mutations include ctx_m gene, ges gene, kpc gene, mecA gene, tem gene, oxa-51 gene, aph gene, sul gene, MREJ gene, ampc gene, gim gene, imi gene, mecC gene, ndm gene, shv gene, sme gene, spm gene, vanA gene, vanB gene, vim gene, oxa-23 gene, oxa-24 gene, oxa-48 gene, aac_3 gene, ant gene, ermA gene, ermB gene, ermC gene, mcr gene, mefA gene, mefE gene, msrA gene, qnrA gene, tetA gene, tetB gene, tetM gene, vanD gene, imp gene or veb gene.

[0088] Using the probe set, kit, method and application thereof described in the present disclosure, the drug-resistant gene mutations of multiple bacteria can be detected simultaneously, and the mutation frequency can be accurately quantified, the drug-resistant gene subtypes can be identified, the drug-resistant heterogeneity can be diagnosed, with high sensitivity and specificity, short detection time and greatly reduced cost. Detailed implementation manners

[0089] The present disclosure provides a probe set, a kit and an application thereof for detecting drug-resistant gene mutations of bacteria.

[0090] The present disclosure provides a probe set for detecting drug-resistant gene mutations of bacteria through screening, which has nucleotide sequences shown in SEQ ID NO: 1 to 150. It can comprehensively, rapidly and accurately detect drug-resistant gene mutations of bacteria.

[0091] The bacteria detected by the present disclosure include at least one of Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Salmonella enterica, Streptococcus pneumoniae, Enterococcus faecium and Enterococcus faecalis.

[0092] The probes for detecting bacteria and their drug-resistant genes screened by the present disclosure are shown in Table 1.

[0093] Table 1. Probes for detecting bacteria and their drug-resistant genes screened by the present disclosure

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] The drug-resistant genes are shown in Table 2.

[0103] Table 2. Drug-resistant genes to be detected in the present disclosure and detection probes

[0104]

[0105]

[0106] Among them, the full name of the CARD database is The Comprehensive Antibiotic Research Database, and the address is http: / / arpcard.mcmaster.ca.

[0107] ARO is the Antibiotic Resistance Ontology.

[0108] In some embodiments, the present invention provides a method for analyzing Mycobacterium tuberculosis, related drug-resistant genes, and variant DNA in bronchoalveolar lavage fluid or sputum samples. The process includes:

[0109] Take a certain amount of biological fluid samples (bronchoalveolar lavage fluid, sputum, etc.), remove host nucleic acids, then lyse them, and extract Mycobacterium tuberculosis complex DNA.

[0110] Prepare a library for the extracted DNA, including fragmentation, end repair, and addition of "A", then add adapters to the ends of the DNA fragments and amplify them using PCR to obtain a pre-library. The pre-library with qualified quantification and fragment quality control is prepared for hybridization with probes: use the above-mentioned probe set for detecting Mycobacterium tuberculosis complex, related drug-resistant genes, and variants to hybridize with the qualified library, capture the target sequence, elute the non-target sequence and impurities, then amplify and purify the captured target sequence to obtain a final library. After qualified quantification and quality control, sequence the final library and perform bioinformatics analysis.

[0111] To make the objectives, technical solutions and advantages of the present disclosure more clear and understandable, the present disclosure will be further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0112] Definition

[0113] Unless otherwise defined, all technical terms and scientific and technical terms used in the present disclosure have the same meaning as commonly used in the field to which the present disclosure belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0114] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural references.

[0115] The expression "about" used herein is as understood by those of ordinary skill in the art and varies within a certain range according to the context in which it is used. If those of ordinary skill in the art do not understand the use of this term according to the context in which it is used, "about" will mean at most plus or minus 10% of a specific value.

[0116] In the present disclosure, the terms "pathogen" or "pathogenic microorganism" are used to refer to viruses, bacteria, yeasts, and other microorganisms that can cause diseases in a subject.

[0117] In the present disclosure, the term "RNA", full name "ribonucleic acid" (RiboNucleic Acid, abbreviated as RNA), is one of the four biological macromolecules contained in biological cells, a type of nucleic acid. RNA is a macromolecular polymer composed of nucleotides. Nucleotides are composed of bases, ribose, and phosphoric acid. Among them, there are 4 types of bases: adenine (A), guanine (G), uracil (U), and cytosine (C).

[0118] In the present disclosure, the term "DNA", full name "deoxyribonucleic acid" (DeoxyriboNucleic Acid, abbreviated as DNA), is one of the four biological macromolecules contained in biological cells, a type of nucleic acid. DNA carries the genetic information necessary for the synthesis of RNA and proteins and is an essential biological macromolecule for the development and normal operation of organisms. DNA is a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of bases, deoxyribose, and phosphoric acid. Among them, there are 4 types of bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0119] In the present disclosure, the term "probe" refers to a primer labeled with a capture label or a detection label to detect primer products. The probe sequence is used to hybridize with the sequence generated by the primer sequence and typically hybridizes with a sequence that does not include the primer sequence. Similar to the primer sequence, the probe sequence is also labeled with a capture label or a detection label. It should be noted that when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa. The appropriate length of the probe depends on the intended use of the probe and is typically in the range of 80 to 200 nucleotides (nt). Preferably, the appropriate length of the primer includes 100 to 150 nucleotides. In the present disclosure, the probe can be one of a fluorescent probe and a hybridization probe.

[0120] In the present disclosure, the term "probe set" generally refers to a collection of more than one probe, which locates and / or quantifies the target nucleic acid by recognizing and binding to the target sequence (by means of hybridization pairing). Each probe in the probe combination is usually an oligonucleotide, such as a single-stranded DNA molecule or RNA.

[0121] In the present disclosure, the term "NT library (Nucleotide Sequence Database)" refers to a nucleic acid sequence database. The NT database is the nucleic acid sequence database of NCBI. The NT library belongs to a non-redundant nucleic acid sequence database, and the data comes from GenBank, EMBL, and DDBJ. The NT library stores the karyotype DNA sequence information of known species and all human DNA information. By comparing with the NT library, it can be known which species our data comes from (the species to which it is aligned is that species).

[0122] In the present disclosure, "alignment" refers to the process of comparing a read or tag with a reference sequence and thereby determining whether the reference sequence contains the read sequence. If the reference sequence contains the read, the read can be mapped to the reference sequence, or in some embodiments, to a specific position in the reference sequence.

[0123] In the present disclosure, the term "sequence identity" refers to the "percent sequence identity" or "percent identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for the optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since gaps are not nucleotides, gaps present in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted since target sequence nucleotides are counted and reference sequence nucleotides are not. At least 60% sequence identity includes contiguous segments having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the full length of the sequence.

[0124] The percent sequence identity can be calculated by the following process: Determine the number of positions at which the same amino acid residue or nucleic acid base appears in both of the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percent sequence identity. The comparison of sequences and the determination of the percent sequence identity between two sequences can be done using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for the alignment of protein and nucleotide sequences. One suitable program for determining the percent sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website of the National Center for Biotechnology Information of the US government (blast.ncbi.nlm.nih.gov). Bl2seq uses the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0125] In the present disclosure, the term "hybridization" or "specific hybridization" refers to the ability of a molecule to bind, duplex, or hybridize only to a particular polynucleotide sequence under stringent conditions, as it exists in a complex mixture (e.g., total cellular) DNA or RNA.

[0126] In the present disclosure, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide strands or between two regions of the same polynucleotide strand. It is known that an adenine base in a first region of a polynucleotide can form specific hydrogen bonds ("base pairs") with a base (if that base is thymine or uracil) in a second region of a polynucleotide that is antiparallel to the first region. Similarly, it is known that a cytosine base in a first polynucleotide strand can base pair with a base (if that base is guanine) in a second polynucleotide strand that is antiparallel to the first region. Two regions are complementary if at least one nucleotide in the first region can base pair with a base in the second region when the first region of the polynucleotide is arranged antiparallel to the second region of the same or another different polynucleotide. Thus, two complementary polynucleotides do not need to base pair at every nucleotide position. "Complementary" refers to 100% or "perfect" complementarity of a first polynucleotide with a second polynucleotide and thus base pairing at every nucleotide site. "Complementary" also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70%, or 60%, or 50% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotides are capable of hybridizing to each other under highly stringent hybridization conditions.

[0127] In the present disclosure, the term "stringent hybridization conditions" refers to conditions under which a probe hybridizes to its target subsequence, typically in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, under defined ionic strength and pH, the stringent conditions selected are about 5 - 10 °C lower than the thermal melting point (Tm) of a particular sequence. Tm is the temperature (at a specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (since the target sequence is in excess, at Tm, 50% of the probe is occupied at equilibrium). Stringent conditions can also be achieved by adding destabilizing agents (e.g., formamide). For selective or specific hybridization, the positive signal is at least twice the background, preferably 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C, or 5×SSC, 1% SDS, incubated at 65 °C, with washes in 0.2×SSC and 0.1% SDS at 65 °C.

[0128] In the present disclosure, the term "sequencing" refers to a technique for determining the sequence (e.g., the identity and order of monomeric units) of a biomolecule, such as a nucleic acid, such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy chain termination sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short-read sequencing, single molecule sequencing, synthesis sequencing, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa genome analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), combinatorial probe-anchor synthesis (cPAS), and combinations thereof. In some embodiments, sequencing can be performed using a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or BGI Genomics Co., Ltd. For example, BGI DNBseq sequencing platforms such as BGISEQ-500, BGISEQ-50, MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-T20X2, or Illumina's HiSeq 2000, HiSeq 2500, HiSeq 4000, HiSeq X10, NovaSeq 6000, etc.

[0129] In the present disclosure, the term "targeted sequencing" refers to a technique of capturing and sequencing a target fragment in a DNA sample using a biotin-labeled DNA or RNA probe. The probe can be labeled with biotin. Each nucleotide in the probe of the present disclosure can be chemically synthesized using, for example, a general DNA synthesizer (e.g., Model 394 manufactured by Applied Biosystems). Any other method well known in the art can also be used to synthesize oligonucleotides, such as probes.

[0130] Examples are provided below to facilitate understanding of the present disclosure. It should be understood that these examples are only used to illustrate the present disclosure, but do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0131] Example

[0132] Example 1 A method for detecting pathogenic microorganisms

[0133] The steps are as follows:

[0134] 1. DNA extraction

[0135] The applicable sample scope includes bronchoalveolar lavage fluid, sputum, swab fluid, etc.

[0136] Take bronchoalveolar lavage fluid, sputum, and swab fluid and, according to the instruction manual of the Magnetic Pathogen Microorganism DNA / RNA Kit extraction reagent, use a lysis method combining chemical and mechanical methods to extract DNA and RNA, use silica-based magnetic beads to quickly separate and purify nucleic acids, and use dsDNA HS Assay Kit, RNA HS Assay Kit for nucleic acid quantification.

[0137] 2. cDNA synthesis

[0138] Take the extracted nucleic acid and make up to 15 μL with nuclease-free water, and perform cDNA synthesis according to Hieff ds-cDNA Synthesis Kit, including RNA denaturation and the synthesis of the first and second strands.

[0139] 3. Library construction

[0140] Use Hieff C37P4 OnePot cDNA&gDNA Library Prep Kit for library construction. It can be understood that the library construction reagent is not limited by the sequencing platform and can be prepared by conventional methods according to needs.

[0141] ① Fragmentation, end repair, and addition of "A"

[0142] Prepare the fragmentation, end repair, and addition of "A" reaction solution according to Table 3

[0143] Table 3. Fragmentation, end repair, and addition of "A" reaction system

[0144] Component Single reaction volume (μL) Second-strand cDNA 36 Nuclease-free water 10 Smearase buffer 10 Smearase enzyme 5 Total volume 61

[0145] Vortex and briefly centrifuge the reaction solution, then place it in a PCR instrument and set the reaction program shown in Table 4.

[0146] Table 4. Fragmentation, End Repair, and A-adding Reaction Program

[0147] Temperature Time 4℃ 1 min 30℃ 20 min 72℃ 20 min 4℃ Hold

[0148] * Set the hot lid temperature to 80 °C

[0149] ② Adapter Ligation

[0150] Prepare the adapter ligation reaction solution according to Table 5.

[0151] Table 5. Adapter Ligation Reaction System

[0152] Component Single reaction volume (μL) A-tailed DNA 61 Ligation Enhancer 30 Novel T4 DNA Ligase 5 Adapter (3 μM) 5 Total volume 101

[0153] Vortex and briefly centrifuge the reaction solution, then place it in a PCR instrument and set the reaction program shown in Table 6 to perform adapter ligation.

[0154] Table 6. Adapter Ligation Reaction Program

[0155] Temperature Time 20℃ 15 min 4℃ Hold

[0156] After the ligation reaction, use magnetic beads to purify the adapter ligation product, and finally resuspend it in 22 μL of nuclease-free water.

[0157] ③ Pre-Capture PCR (Non-C-PCR) to Introduce Index

[0158] Prepare the library amplification reaction solution according to Table 7.

[0159] Table 7. Library Amplification Reaction System

[0160] Component Single reaction volume (μL) Forward / Reverse primer (F / R primer, 15 μM) 5 2× Ultima HF Amplification Mix 25 Adapter-Ligated library 20 Total volume 50

[0161] Vortex and briefly centrifuge the reaction solution, then place it in a PCR instrument and set the reaction program shown in Table 8.

[0162] Table 8. Library Amplification Reaction Program

[0163]

[0164] After the reaction, use magnetic beads to purify the PCR product, and finally resuspend it in 27 μL of Hyb buffer.

[0165] Quantify the purified product using dsDNA HS Assay Kit and perform fragment quality control using Labchip.

[0166] 4. Target Sequence Enrichment

[0167] ① Hybridization

[0168] After the library quality control is qualified, enrichment probes (with nucleotide sequences shown in SEQ ID NO: 1 to 150, specific information is shown in Table 1 and Table 2) and hybridization elution reagents are used for hybridization capture and elution. Specifically, when implementing, hybridization capture can be performed on a single library, or input according to the corresponding required library volume, and the hybridization capture effect can also be achieved.

[0169] Prepare the hybridization reaction solution according to Table 9.

[0170] Table 9. Hybridization reaction system

[0171] Component Single reaction volume (μL) Library 26 Cot-1 DNA 5 Blocker 2 Probe 1 Total volume 34

[0172] After the reaction solution is shaken and mixed evenly, centrifuge it instantaneously, place it in a PCR instrument, and set the reaction program shown in Table 10.

[0173] Table 10. Hybridization reaction program

[0174] Temperature Time 95℃ 30s 65℃ 1h 65℃ hold

[0175] * Set the hot lid temperature to 100 °C

[0176] ② Magnetic bead capture

[0177] After the reaction ends, add streptavidin magnetic beads to capture the PCR product, and set the capture program according to Table 11.

[0178] Table 11. Streptavidin magnetic bead capture program

[0179] Temperature Time 65℃ 15 min 65℃ Hold

[0180] * Set the hot lid temperature to 70 °C, and resuspend and mix evenly once every 7 minutes during the 65 °C incubation.

[0181] ③ Elution

[0182] After the incubation is completed, perform the hot elution and room temperature elution processes according to Table 12.

[0183] Table 12. Elution system

[0184]

[0185] After discarding the eluate, add 20 μL of nuclease-free water to resuspend the magnetic beads.

[0186] ④ Amplification of hybridization capture products

[0187] Prepare the amplification reaction solution according to Table 13, add it to the magnetic bead suspension from the previous step, vortex and mix evenly, and centrifuge instantaneously.

[0188] Table 13. Amplification System for Hybrid Capture Products

[0189]

[0190] Place the PCR tube in a PCR instrument, set the reaction program shown in Table 14, and perform library amplification.

[0191] Table 14. Amplification Program for Hybrid Capture Products

[0192]

[0193] *Set the hot lid temperature to 105 °C.

[0194] ⑤ Purification of Amplification Products

[0195] Add 60 μL of magnetic beads to purify the PCR products, and finally resuspend them in 22 μL of TE buffer.

[0196] For the purified products, use the dsDNA HS Assay Kit for quantification.

[0197] 5. Sequencing on the Machine

[0198] Use the Gene+seq100 sequencer for sequencing on the machine. The sequencing experiment operation is carried out according to the operation manual provided by the manufacturer.

[0199] 6. Analyze the sequencing data of the capture library using a bioinformatics analysis pipeline to obtain the pathogen detection results. Among them, the subtypes of genes are typed according to the gene mutation types, such as kpc, ndm, tem.

[0200] Example 2 Accuracy Experiment of Standard Strains

[0201] 1. Method

[0202] 1.1 Preparation of Simulated Samples

[0203] The simulated mixed samples contain human A549 cells, and each simulated mixed sample contains 1×10 5 A549 cells / mL, and finally the pathogen concentrations are shown in Table 15.

[0204] Among them, P1 is prepared using a Klebsiella pneumoniae strain with a kpc-2 drug-resistant gene shown by first-generation sequencing results, P2 is prepared using a Klebsiella pneumoniae strain with a genotype of kpc-33, P3 is prepared by mixing two strains with different drug-resistant gene types at a ratio of 1:1, and P4 is prepared using a Staphylococcus aureus strain with a mecA drug-resistant gene.

[0205] Table 15. Pathogens and Concentrations in Simulated Mixed Samples

[0206]

[0207] 1.2 Library preparation

[0208] Take 400 μl of each simulated sample, break the cell wall with a homogenizer, and extract and purify DNA and RNA with nucleic acid extraction or purification reagents. Perform library construction and hybridization capture according to the method described in Example 1, and run the NGS sequencing program on the Gene+seq100 sequencing platform for each capture library, and sequence according to the required data volume of 5M.

[0209] 2. Test results

[0210] The captured library was analyzed by bioinformatics process, and the number of sample call sequences was counted. The results are shown in Tables 16 and 17.

[0211] Table 16. Detection results of pathogenic microorganisms in simulated samples

[0212] Sample number Detected pathogen Species detection result Detected drug resistance gene P1 Klebsiella pneumoniae Klebsiella pneumoniae positive kpc P2 Klebsiella pneumoniae Klebsiella pneumoniae positive kpc P2 Klebsiella pneumoniae Klebsiella pneumoniae positive kpc P4 Staphylococcus aureus Staphylococcus aureus positive mecA

[0213] Table 17. KPC genotyping results detected in simulated samples

[0214] Sample number Gene name Gene sequencing depth Detected mutation Mutation frequency P1 kpc 404.77 None 0％ P2 kpc 268.43 kpc-c.532G>T 100％ P3 kpc 695.35 kpc-c.532G>T 44％

[0215] The above results show that this process can accurately detect drug-resistant genes in standard products, and can distinguish subtypes of KPC genes based on point mutations. At the same time, the proportion of the two genotypes, kpc-2 and kpc-33, can be calculated by mutation frequency, which is consistent with the pre-mixed ratio.

[0216] Example 3 Clinical sample detection

[0217] 1. Methods

[0218] 1.1 Randomly collect clinical specimens (including bronchoalveolar lavage fluid and sputum) from 7 patients with clinically confirmed infection with drug-resistant bacteria.

[0219] 1.2 Library preparation

[0220] Take 400 μl of each clinical sample, break the cell wall with a homogenizer, and extract and purify DNA and RNA with nucleic acid extraction or purification reagents. According to the method described in Example 1, library construction and hybridization capture were performed, and the conventional library before hybridization (prelibrary) and the corresponding capture library after hybridization (final library) were run on the Gene+seq 100 sequencing platform for NGS sequencing. The prelibrary was sequenced according to the required data volume of each 50M, and the final library was sequenced according to the required data volume of each 5M.

[0221] 2. Test results

[0222] The detection results are shown in Table 18.

[0223] Table 18. Genotyping results of drug-resistant genes detected in clinical samples

[0224]

[0225] In the detection of the above 7 samples, all target pathogenic microorganisms can be detected, and drug-resistant genes with a high degree of correlation with the drug-resistant phenotype can be detected simultaneously, which is consistent with the mNGS detection results. From the above results, it can be seen that this method can simultaneously complete pathogen detection and drug-resistant gene detection in clinical samples, and has high clinical value.

[0226] The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solutions of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. A probe set for identifying bacterial species, characterized in that, The probe set includes one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 36, or one or more of the nucleotide sequences having at least 60% sequence identity thereto.

2. The probe set according to claim 1, wherein The probe set includes any one or more of the following groups: A probe set for detecting Enterococcus faecium, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 1 to 3, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Enterococcus faecalis, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 4 to 6, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Staphylococcus aureus, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 7 to 9, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Klebsiella pneumoniae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 10 to 12, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Acinetobacter baumannii, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 13 to 15, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Escherichia coli, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 16 to 18, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Pseudomonas aeruginosa, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 19 to 21, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Serratia marcescens, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 22 to 24, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Proteus mirabilis, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 25 to 27, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Salmonella enterica, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 28 to 30, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting Streptococcus pneumoniae, which includes any one or more of the nucleotide sequences shown in SEQ ID NO: 31 to 33, or any one or more of the nucleotide sequences having at least 60% sequence identity thereto; A probe set for detecting *Enterobacter cloacae*, which comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 34 to 36, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith.

3. The probe set according to claim 1, wherein The probe set further comprises a probe set for detecting drug-resistant gene mutations, and the drug-resistant genes include one or more of the following: ctx_m gene, ges gene, kpc gene, mecA gene, tem gene, oxa-51 gene, aph gene, sul gene, MREJ gene, ampc gene, gim gene, imi gene, mecC gene, ndm gene, shv gene, sme gene, spm gene, vanA gene, vanB gene, vim gene, oxa-23 gene, oxa-24 gene, oxa-48 gene, aac_3 gene, ant gene, ermA gene, ermB gene, ermC gene, mcr gene, mefA gene, mefE gene, msrA gene, qnrA gene, tetA gene, tetB gene, tetM gene, vanD gene, imp gene or veb gene.

4. The probe set according to claim 3, wherein The probe set comprises any one or more of the nucleotide sequences shown in SEQ ID NOs: 37 to 150, or any one or more of the nucleotide sequences having at least 60% sequence identity therewith.

5. The probe set according to any one of claims 1 to 4, characterized in that The probe is modified with biotin.

6. A kit for detecting drug-resistant gene mutations of bacteria, characterized in that, The kit comprises the probe set according to any one of claims 1 to 5.

7. A method for detecting drug-resistant gene mutations of bacteria using the probe set according to any one of claims 1 to 5 or the kit according to claim 6, the method comprising the following steps: 1) Extracting nucleic acid from a sample; 2) Constructing a library for the extracted nucleic acid; 3) Hybridization capture of a target sequence using the probe set; 4) Sequencing the captured product and performing data analysis.

8. Use of the probe set according to claims 1 to 5 in the preparation of a kit.

9. The application according to claim 8, characterized in that, The probe set is used to detect one or more of the following drug-resistant bacteria: Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, Salmonella enterica, Streptococcus pneumoniae or Enterobacter cloacae.

10. The application according to claim 8, wherein The probe set is used to detect drug-resistant mutations to drugs, where the drugs include one or more of the following items: methicillin, β-lactams, macrolides, lincosamides, streptogramins, tetracyclines, aminoglycosides, glycopeptides, sulfonamides, carbapenems, penicillins, cephalosporins, polymyxins or fluoroquinolones.

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