Detection method of aminoglycoside drug resistance gene armA

Abstract

The invention belongs to the technical field of molecular diagnosis / detection, and particularly discloses a detection method of an aminoglycoside drug resistance gene armA. Comprising the following steps: extracting genome DNA from a sample to be detected; the method comprises the following steps: in the presence of Bst DNA polymerase, an MCDA primer and a reaction buffer solution, carrying out MCDA isothermal amplification on DNA to be detected to obtain an amplification product containing a PAM site; the obtained amplification product is mixed with Cas12a protein, gRNA molecules, a double-labeled DNA probe and a reaction buffer solution, and CRISPR / Cas12a reaction is carried out in the same reaction system; and detecting a fluorescence signal of the reaction system in the step 3, and judging that the armA gene exists in the sample when an effective fluorescence signal is generated. The invention provides a method (MCDA-CRISPR / Cas12a) capable of rapidly, sensitively and specifically detecting armA by combining an MCDA isothermal amplification technology and a CRISPR / Cas12a fluorescence detection technology.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics / detection technology, and more specifically, relates to a method for detecting the aminoglycoside drug resistance gene armA. Background Technology

[0002] In recent years, due to the irrational use of antibiotics and other reasons, bacterial resistance has become a thorny problem in clinical practice, posing a huge challenge to anti-infective treatment worldwide. Aminoglycosides, with their broad antibacterial spectrum, high efficacy, and rapid bactericidal effects, are important anti-infective drugs in clinical practice, especially indispensable for treating Gram-negative bacterial infections and tuberculosis. However, with the widespread use of these antibiotics, the number of resistant strains is constantly increasing, greatly limiting the choice of clinical drugs, making infection treatment very difficult, and also posing a severe challenge to the prevention and control of nosocomial infections.

[0003] Research has found that, generating Methyltransferases are an important mechanism of bacterial resistance to aminoglycoside antibiotics, among which the armA gene is the most widely distributed. One of the methyltransferase genes. The armA gene was first discovered in a strain of Klebsiella pneumoniae in France in 2003. The armA gene hosts almost all clinically significant Gram-negative bacteria, including Enterobacteriaceae, Weedentelella, Klebsiella, Serratia, Shigella flexneri, and non-fermenting genera such as Acinetobacter and Pseudomonas. The armA gene is not only found clinically but also in many livestock, poultry, animal-derived foods, and environmental water bodies. In 2015-2016, *Salmonella Indiana* and *Salmonella Californiae* carrying the armA gene were detected in chicken and chicken farm samples collected from different provinces in my country. The gene encoding this enzyme is mostly located on plasmids. Plasmids can carry drug resistance genes that spread between different bacterial genera and enhance the drug resistance of pathogens, leading to the widespread dissemination of multidrug-resistant bacteria.

[0004] Currently, common methods for detecting the armA gene include molecular detection technologies such as polymerase chain reaction (PCR), quantitative real-time PCR, high-throughput sequencing, and gene chips. However, PCR and quantitative real-time PCR methods are limited by the high cost of equipment, the expense of probes, and the long processing time, making them unsuitable for rapid and emergency detection. Therefore, developing a rapid, sensitive, and specific method for detecting armA is imperative.

[0005] The system (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated proteins system) is a revolutionary technology with broad application prospects and has already been applied in fields such as gene editing and nucleic acid detection. However, simply using... The sensitivity of the system for nucleic acid detection is not high, so it is usually combined with nucleic acid amplification methods to effectively optimize the detection sensitivity. The rapid development of isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and multiple cross displacement amplification (MCDA), has further simplified nucleic acid amplification techniques. These techniques do not require complex equipment or strict experimental environments and can achieve nucleic acid detection in a short time. Among them, MCDA technology is characterized by its speed, high sensitivity, high specificity, short processing time, and simple operation, making it applicable to a wider range of fields. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a method for detecting the aminoglycoside drug resistance gene armA, which combines multiple cross-permutation amplification (MCDA) technology with... The nucleic acid detection system is deeply coupled and combined with specificity. The invention, along with dual-labeled fluorescent quenching probes, enables a rapid, sensitive, specific, and visualized detection system for the aminoglycoside resistance gene armA. It can quickly complete the entire process from sample processing to result interpretation without the need for large-scale instruments and equipment, and has significant improvements in sensitivity, specificity, and application adaptability compared to traditional PCR or real-time fluorescent PCR methods.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for detecting the aminoglycoside drug resistance gene armA is provided, comprising the following steps: Step 1: Extract genomic DNA from the sample to be tested; Step two, in In the presence of polymerase, MCDA primers, and reaction buffer, the DNA to be tested is subjected to isothermal MCDA amplification to obtain an amplification product containing PAM sites. Step 3, mix the amplification product obtained in Step 2 with... protein, The molecules, dual-labeled DNA probes, and reaction buffer are mixed and carried out in the same reaction system. reaction; Step four: Detect the fluorescence signal of the reaction system from step three. When a valid fluorescence signal is generated, it indicates the presence of the armA gene in the sample.

[0008] As a further preferred embodiment, the MCDA primers include: Replace primers F1 and F2; Cross primers CP1 and CP2; Amplification primers C1 and C2, D1 and D2, R1 and R2.

[0009] As a further preferred embodiment, the The nucleotide sequence is SEQ ID NO.3.

[0010] As a further preferred embodiment, the sequence of the dual-labeled DNA probe is selected from SEQ ID NO.4, and its End marker FAM, End marker BHQ1.

[0011] As a further preferred embodiment, the temperature for the isothermal amplification of MCDA is 60-67℃, and more preferably, the temperature for the isothermal amplification of MCDA is 63℃.

[0012] As a further preferred embodiment, the The reaction temperature is 37℃ and the reaction time is 5-15 minutes.

[0013] As a further preferred option, the armA gene can be combined with NDM-1, IMP-4, KPC-2, OXA-1, TEM-1B, , The sul1 drug resistance gene can be distinguished to achieve specific detection of armA.

[0014] According to another aspect of the invention, a method for detecting the armA gene is also provided. The kit includes: The MCDA amplification system components include MCDA primers, 2× reaction buffer, and Bst DNA polymerase. The components of the detection system, The components of the detection system include: protein, Double-labeled fluorescence quenching Probe, 10× reaction buffer; and Table of positive plasmid control and negative control.

[0015] As a further preferred embodiment, the feature is that the The sequence is SEQ ID NO.3; The sequence of the dual-labeled DNA probe is selected from SEQ ID NO.4, and its End marker FAM, End marker BHQ1; The positive plasmid is a standard plasmid containing the armA gene sequence; The kit also includes a lysis buffer or DNA extraction reagent for sample genome extraction; The kit is suitable for 63℃ isothermal amplification devices and fluorescence detection equipment.

[0016] According to another aspect of the present invention, a method for detecting the aminoglycoside drug resistance gene armA, as described in any of the above embodiments or combinations thereof, is also provided for use in screening for resistance to aminoglycoside antibiotics, for detecting the presence of the armA gene in a sample.

[0017] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: 1. This invention combines multiple cross-permutation amplification (MCDA) technology with... The nucleic acid detection system is deeply coupled and combined with specificity. With dual-labeled fluorescent quenching probes, a rapid, sensitive, specific, and visualized detection system for the aminoglycoside resistance gene armA was realized. It can quickly complete the entire process from sample processing to result interpretation without the need for large-scale instruments and equipment, and has significant improvements in sensitivity, specificity, and application adaptability compared with traditional PCR or real-time fluorescent PCR methods.

[0018] 2. This invention utilizes MCDA technology for rapid isothermal amplification of the target sequence, introducing PAM sites into the amplicon and significantly increasing the target nucleic acid copy number; based on this, The system passes This technology identifies specific target sequences in amplicones and triggers paracleavage activity, enabling efficient cleavage of fluorescence-quenched probes. The synergy of these two technologies significantly improves detection sensitivity, allowing the detection limit to reach [a certain level]. It is far superior to traditional PCR (detection limit is approximately...). This meets the need for rapid identification of low-abundance drug resistance genes.

[0019] 3. This invention designs MCDA primer combinations targeting multiple regions of the armA gene and highly specific primers. This ensures that both the amplification and recognition stages are based on the unique sequence regions of the armA gene. This approach can effectively distinguish between cells carrying NDM-1, IMP-4, KPC-2, OXA-1, TEM-1B, and others. , This invention enables accurate identification of armA in strains containing drug-resistant genes such as sul1. Experimental results show that this invention has extremely high specificity and can significantly reduce the risks of non-specific amplification and misjudgment common in traditional amplification methods. Attached Figure Description

[0020] Figure 1 As per the embodiments of the present invention Technical fluorescence signal detection diagram; Figure 2 As per the embodiments of the present invention Technical principle diagram; Figure 3 As per the embodiments of the present invention Schematic diagram of the armA technical testing workflow; Figure 4 As per the embodiments of the present invention A schematic diagram showing the position and orientation of the technical primers; Figure 5 As per the embodiments of the present invention Confirmation diagram of detection technology; Figure 6 This is an electrophoresis pattern obtained by testing the optimal reaction temperature of the standard MCDA involved in the embodiments of the present invention; Figure 7 As per the embodiments of the present invention A spectrum for evaluating the sensitivity of plasmid DNA in this technology; Figure 8 As per the embodiments of the present invention The specific detection and evaluation spectrum of the technology; Figure 9 This is an electrophoresis pattern used in the embodiment of the present invention to evaluate the sensitivity of conventional PCR detection of the armA gene. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0022] This invention provides a method for detecting the aminoglycoside resistance gene armA, comprising the following steps: Step 1: Extract genomic DNA from the sample to be tested; Step 2: In the presence of Bst DNA polymerase, MCDA primers, and reaction buffer, the DNA to be tested is amplified by MCDA at a constant temperature to obtain an amplification product containing PAM sites. Step 3, mix the amplification product obtained in Step 2 with... protein, The molecules, dual-labeled DNA probes, and reaction buffer are mixed and carried out in the same reaction system. reaction; Step four: Detect the fluorescence signal of the reaction system in step three. When an effective fluorescence signal is generated, it is determined that the armA gene is present in the sample.

[0023] As a further preferred embodiment, the MCDA primers include: Replace primers F1 and F2; Cross primers CP1 and CP2; Amplification primers C1 and C2, D1 and D2, R1 and R2.

[0024] More specifically, the sequences of the MCDA primers are selected from SEQ ID NO.5–SEQ ID NO.14, as shown in Table 1. The nucleotide sequence of the substitution primer F1 is SEQ ID NO.5, the nucleotide sequence of the substitution primer F2 is SEQ ID NO.6, the nucleotide sequence of the cross primer CP1 is SEQ ID NO.7, the nucleotide sequence of the cross primer CP2 is SEQ ID NO.8, the nucleotide sequence of the amplification primer C1 is SEQ ID NO.9, the nucleotide sequence of the amplification primer C2 is SEQ ID NO.10, the nucleotide sequence of the amplification primer D1 is SEQ ID NO.11, the nucleotide sequence of the amplification primer D2 is SEQ ID NO.12, the nucleotide sequence of the amplification primer R1 is SEQ ID NO.13, and the nucleotide sequence of the amplification primer R2 is SEQ ID NO.14.

[0025] As a further preferred embodiment, the The nucleotide sequence is SEQ ID NO.3.

[0026] As a further preferred embodiment, the sequence of the dual-labeled DNA probe is selected from SEQ ID NO.4, and its End marker FAM, End marker BHQ1.

[0027] As a further preferred embodiment, the temperature for the isothermal amplification of MCDA is 60-67℃, and more preferably, the temperature for the isothermal amplification of MCDA is 63℃.

[0028] As a further preferred embodiment, the The reaction temperature is 37℃ and the reaction time is 5-15 minutes.

[0029] As a further preferred option, the armA gene can be combined with NDM-1, IMP-4, KPC-2, OXA-1, TEM-1B, , The sul1 drug resistance gene can be distinguished to achieve specific detection of armA.

[0030] The present invention provides A method for detecting the armA gene was developed, which exhibits excellent detection sensitivity and a detection limit of [value missing]. It also has a fast detection speed, and a visual amplification curve can be obtained in just 70 minutes.

[0031] Strains that do not carry the armA gene, such as Klebsiella pneumoniae producing carbapenemase NDM-1, Enterobacter cloacae producing carbapenemase IMP-4, Klebsiella pneumoniae producing carbapenemase KPC-2, Escherichia coli producing carbapenemase OXA-1, Klebsiella pneumoniae producing extended-spectrum β-lactamase TEM-1B, and those carrying quinolone resistance genes. Escherichia coli carrying aminoglycoside resistance genes Klebsiella pneumoniae, Pseudomonas aeruginosa carrying the sulfonamide resistance gene sul1, and other strains not carrying the above-mentioned resistance genes (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, etc.), as well as armA-positive strains were used as templates for evaluation. The technology is specific. The results show that... The technology can accurately identify armA, indicating that The method exhibits good specificity. This method requires only a constant temperature of 63℃ to achieve the entire reaction process, and the results can be interpreted using a fluorescence reading device, making it suitable for rapid detection in areas with limited experimental conditions.

[0032] More specifically, the following details a method for detecting the aminoglycoside drug resistance gene armA, which relates to this invention.

[0033] 1. Amplification Principles and Procedures (1) Constructing detectable products by MCDA amplification The MCDA reaction comprises 10 primers that recognize 10 regions of the target sequence, including two substitution primers F1 and F2, two crossover primers CP1 and CP2, and six amplification primers C1, C2, D1, D2, R1, and R2. Substitution primers F1 and F2 play a substitution role in the MCDA reaction, replacing crossover primers CP1 and CP2. The six amplification primers C1, C2, D1, D2, R1, and R2 accelerate the MCDA reaction and increase the product yield. Using this primer combination and Bst DNA polymerase, highly efficient amplification of the target gene can be achieved, and a PAM (protospace radjacent motif) site (TTTN, where N represents any single base) can be added to the amplicon.

[0034] (2) Detection of amplification products The system's reaction principle: When CRISPR edits genes, it requires... nucleases and (guide RNA) has two components, which are present when the reaction begins. and Nucleases form complexes, guide Locate the target DNA sequence near the target editing sequence and identify the target sequence upstream of PAM. After binding to the target sequence, The protein induces cleavage of the target sequence, leading to double-stranded DNA breaks. In addition to inducing cis-cleavage of the target sequence, it also initiates unlimited shearing of free single-stranded DNA within the system. Based on this principle, single-stranded DNA is fabricated into fluorescently quenched probes, enabling specific detection of the target sequence through fluorescence signal monitoring.

[0035] (3) Interpretation of fluorescence results: The fluorescence signal is detected in real time using a quantitative real-time PCR instrument. When a valid fluorescence signal is detected, it is considered a positive result. Figure 1 (A) When no valid fluorescence signal is detected, it is considered a negative result. Figure 1 (B in the middle).

[0036] (4) A schematic diagram of the principle of technology amplification is shown below. Figure 2 The target sequence containing the PAM (TTTN) site is specifically amplified by the MCDA reaction. The complex binds to the amplicon. The protein is induced to nonspecifically cleave single-stranded DNA. (Both ends are labeled with fluorescent groups).

[0037] (5) The technical testing armA workflow is as follows Figure 3 As shown. The entire workflow of the method consists of four steps: rapid DNA extraction (15 min), MCDA reaction (40 min), Shearing (5 min), fluorescence detection (10 min), the entire detection process can be completed within 70 min.

[0038] 2. Reagents and instruments involved in the embodiments of this invention: The reagents involved in the embodiments of this invention are: bacterial genome extraction kit, DL100 DNA Marker, and deoxyribonucleic acid isothermal amplification kit (including reaction buffer and Bst DNA polymerase). Protein, armA-positive plasmid, primers, The probe and all other reagents were commercially available analytical grade products.

[0039] The main instruments used in the experiments of this invention are: constant temperature heater (MTH-100), electrophoresis equipment (Universal), UV transilluminator (UVsolo touch), and Nanodrop instrument (ND-2000).

[0040] 3. The methods involved in the embodiments of the present invention Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit, following the instructions. The concentration and purity of the DNA were determined using a UV spectrophotometer. The armA-positive plasmid was synthesized by Beijing Qingke Biotechnology Co., Ltd., using the PUC57 vector and competent cells from the Escherichia coli TOP10 strain. The concentration of the synthesized plasmid was [missing information]. The converted copy number is The armA-positive plasmid DNA was serially diluted with double-distilled water (diluted sequentially by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ... , , , , , , , , , , After aliquoting each genomic DNA, store at -30°C for later use.

[0041] Serially diluted armA-positive plasmid DNA was used to establish the MCDA amplification system. DNA from common bacterial strains carrying other drug-resistant genes was used as a template for evaluation. The technology is specific. See Table 2 for strain information.

[0042] 4. The primers involved in the embodiments of the present invention, and probe design This invention establishes a sensitive, specific, and rapid method targeting the armA gene. The testing system is used for verification and evaluation. Technology. In this invention, a set of MCDA amplification primers based on the armA gene (GenBank accession number: KU984333.1) was designed using Primer Premier 6.0 software, and its specificity was analyzed using BLAST. Simultaneously, a primer for... of And a DNA probe with modified ends. End-labeled with the fluorescent group FAM, End-labeled with the fluorescence quencher group BHQ1. This was intended to verify... The feasibility and reliability of the technology. See the primer design diagram. Figure 4 Primers, The probe sequences and modifications are shown in Table 1.

[0043] According to another aspect of the invention, a method for detecting the armA gene is also provided. The kit includes: The MCDA amplification system components include MCDA primers, 2× reaction buffer, and Bst DNA polymerase. The components of the detection system, The components of the detection system include: protein, Double-labeled fluorescence quenching Probe, 10× reaction buffer; and Table of positive plasmid control and negative control.

[0044] As a further preferred embodiment, the feature is that the The sequence is SEQ ID NO.3; The sequence of the dual-labeled DNA probe is selected from SEQ ID NO.4, and its End marker FAM, End marker BHQ1; The positive plasmid is a standard plasmid containing the armA gene sequence; The kit also includes a lysis buffer or DNA extraction reagent for sample genome extraction; The kit is suitable for 63℃ isothermal amplification devices and fluorescence detection equipment.

[0045] According to another aspect of the present invention, a method for detecting the aminoglycoside drug resistance gene armA, as described in any of the above embodiments or combinations thereof, is also provided for use in screening for resistance to aminoglycoside antibiotics, for detecting the presence of the armA gene in a sample.

[0046] Example 1. Feasibility of amplification Standard MCDA reaction system: Reaction buffer (BF buffer) Polymerase, Indicators, cross-primers CP1 and CP2 each (concentration is) ), replacing primers F1 and F2 each (concentration is) ), amplification primers C1, C2, D1, D2, R1, and R2 each (concentration is) ), Template, add deionized water to The entire reaction was kept at 63℃ for 40 minutes.

[0047] After MCDA amplification, two detection methods were used for MCDA amplification discrimination: (1) MCDA products were detected by agarose gel electrophoresis; (2) by... The product is tested.

[0048] Electrophoresis detection: The MCDA amplification results are interpreted by electrophoresis. A positive reaction will show a specific amplification band at the target location, while a negative reaction and a blank control will not show a specific amplification band at the target location. Figure 5 (B), thus verifying the feasibility of the MCDA primers designed in this invention, which can be used for target sequence amplification and detection. Figure 5 In section B, lanes 1-3 represent concentrations of... armA-positive plasmid DNA, negative control (NDM-1-positive Klebsiella pneumoniae WHCDC-FY57), deionized water.

[0049] Detection: (1) Reaction system: 5 μL 10× buffer, , probe ( ), Protein (20 nM), Add deionized water to the product to make up to the final volume. (2) React at 37℃ for 5 min; (3) Fluorescence detection: Place the reaction solution from step (2) into a real-time PCR instrument, keep it at 37℃ for 10 minutes, and collect the fluorescence signal. When an effective fluorescence signal is detected, it is considered a positive result. Figure 5 C); when no valid fluorescence signal is detected, it is considered a negative result. Figure 5 C). Figure 5 In C, 1-3 represent concentrations of... armA-positive plasmid DNA, negative control (NDM-1-positive Klebsiella pneumoniae WHCDC-FY57), deionized water.

[0050] Example 2. Determination of the optimal reaction temperature for MCDA technology Under standard reaction conditions, armA-positive plasmid DNA template and the designed corresponding primers were added, resulting in a template concentration of [missing information]. The reaction was carried out under constant temperature (60-67℃), and the results were obtained using gel electrophoresis. Electrophoretic bands of varying brightness were obtained at different temperatures (see...). Figure 6 ). Figure 6 Lanes 1-8 show the electrophoresis results of armA-positive plasmid DNA at 60-67℃, lane 9 is the negative control (NDM-1 positive Klebsiella pneumoniae WHCDC-FY57), and lane 10 is deionized water. The figures show that the optimal reaction temperature for MCDA primers is 61-67℃. In subsequent experiments, this invention selected 63℃ for the MCDA reaction.

[0051] Example 3. Detection sensitivity Using serially diluted armA-positive plasmid DNA ( , , , , , , To evaluate Method sensitivity. Standardization. After the reaction, fluorescence detection showed that armA The detection limit of the technology is Positive amplification curves were observed. Figure 7 When the DNA mass in the reaction system decreases to If no amplification curve appears in the following cases, it indicates a negative result. Figure 7 ). Figure 7 Numbers 1-7 represent the template amount of armA-positive plasmid DNA, respectively. , , , , , , Number 8 is the negative control (NDM-1 positive Klebsiella pneumoniae WHCDC-FY57), and number 9 is deionized water.

[0052] Example 4. Measurement The specificity of technology Genomic DNA was extracted from 22 strains carrying the armA gene and 19 strains not carrying the armA gene to evaluate... Specificity of armA gene detection. Strains not carrying the armA gene include *Klebsiella pneumoniae* producing carbapenemase NDM-1, *Enterobacter cloacae* producing carbapenemase IMP-4, *Klebsiella pneumoniae* producing carbapenemase KPC-2, *Escherichia coli* producing carbapenemase OXA-1, and *Extremely broad-spectrum* bacteria. Klebsiella pneumoniae carrying the TEM-1B lactamase and quinolone resistance gene Escherichia coli carrying aminoglycoside resistance genes The results showed that *Klebsiella pneumoniae*, *Pseudomonas aeruginosa* carrying the sulfonamide resistance gene *sul1*, and other strains not carrying the above-mentioned resistance genes (*Pseudomonas aeruginosa*, *Escherichia coli*, *Acinetobacter baumannii*, etc.) were included (strain information is detailed in Table 2). The technology can accurately identify the armA gene, indicating that the method has excellent specificity. Figure 8 ). Figure 8 In the table, numbers 1-8 represent strains carrying the armA gene; numbers 9-14 represent strains not carrying the armA gene; and number 15 represents deionized water.

[0053] Example 5. Sensitivity of conventional PCR detection of the armA gene Using serially diluted armA-positive plasmid DNA ( , , , , , , , , , , After performing a standard PCR reaction, the amplification products were detected by agarose gel electrophoresis to evaluate the sensitivity of conventional PCR for detecting the armA gene. Electrophoresis showed that the detection range of conventional PCR was [missing information]. Positive amplification bands were observed. Figure 9 When the DNA mass in the reaction system decreases to If no amplification band appears in the following cases, it indicates a negative result. Figure 9). Figure 9 Numbers 1-11 represent the template amount of armA-positive plasmid DNA, respectively. , , , , , , , , , , Number 12 was the negative control (NDM-1 positive Klebsiella pneumoniae WHCDC-FY57), and number 13 was deionized water. Results showed... The method demonstrates superior sensitivity and shorter detection time compared to conventional PCR for identifying the armA gene.

[0054] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gene for resistance to aminoglycoside drugs The detection method is characterized by, Includes the following steps: Step 1: Extract genomic DNA from the sample to be tested; Step two, in In the presence of polymerase, MCDA primers, and reaction buffer, the DNA to be tested is subjected to isothermal MCDA amplification to obtain an amplification product containing PAM sites. Step 3, mix the amplification product obtained in Step 2 with... protein, The molecules, dual-labeled DNA probes, and reaction buffer are mixed and carried out in the same reaction system. reaction; Step four: Detect the fluorescence signal of the reaction system from step three. When a valid fluorescence signal is generated, it is determined that the sample contains [the virus / organism]. Gene.

2. An aminoglycoside drug resistance gene as described in claim 1 The detection method is characterized by, The MCDA primers include: Replace primers F1 and F2; Cross primers CP1 and CP2; Amplification primers C1 and C2, D1 and D2, R1 and R2.

3. An aminoglycoside drug resistance gene as described in claim 1 The detection method is characterized by, The The nucleotide sequence is a sequence .

4. An aminoglycoside drug resistance gene as described in claim 1 The detection method is characterized by, The sequence of the dual-labeled DNA probe is selected from... And its End marker FAM, End marker BHQ1.

5. An aminoglycoside drug resistance gene as described in claim 1 The detection method is characterized by, The isothermal amplification temperature of MCDA is 60-67℃, preferably 63℃.

6. An aminoglycoside drug resistance gene as described in claim 1 The detection method is characterized by, The The reaction temperature is 37℃ and the reaction time is 5-15 minutes.

7. An aminoglycoside drug resistance gene according to any one of claims 1-6 The detection method is characterized by, Able to Genes and NDM-1 Drug resistance gene differentiation, to achieve Specific detection.

8. A method for detecting Genetic The reagent kit is characterized by, include: The MCDA amplification system consists of MCDA primers, 2× reaction buffer, and other components. Polymerase; The components of the detection system, The components of the detection system include: protein, Double-labeled fluorescence quenching Probe, 10× reaction buffer; and Table of positive plasmid control and negative control.

9. A method for detecting according to claim 8 Genetic The reagent kit is characterized by, The The sequence is ; The sequence of the dual-labeled DNA probe is selected from... And its End marker FAM, End marker BHQ1; The positive plasmid contains Standard plasmids for gene sequences; The kit also includes a lysis buffer or DNA extraction reagent for sample genome extraction; The kit is suitable for 63℃ isothermal amplification devices and fluorescence detection equipment.

10. An aminoglycoside drug resistance gene as described in any one of claims 1-7 The detection method is used in the screening of aminoglycoside antibiotic resistance to detect the presence of [specific antibiotics] in samples. Gene.

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