Nucleic acid compositions for detecting drug resistance genes, kits containing them, and their uses.

Abstract

This application discloses a nucleic acid composition for detecting drug resistance genes, a kit containing the composition, and their uses. The nucleic acid composition includes one or more sets of primer-probe sets 1 to 9. Using this nucleic acid composition or kit for drug resistance gene detection shortens the reaction time and simplifies the operation steps; moreover, in some embodiments, only one nucleic acid extraction and one PCR detection are needed to obtain the detection results for six drug resistance genes, with no interference during the PCR process.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to a nucleic acid composition for detecting drug resistance genes, a kit containing the same, and its uses. Background Technology

[0002] After repeated exposure to a certain drug, pathogens gradually develop the ability to become insensitive to or resistant to that drug. Individuals carrying mutated genes that resist the drug's mechanism of action are more likely to survive and reproduce during drug action, posing a major challenge to public health.

[0003] Extensive genetic diversity exists within drug-resistant pathogen populations. For example, the qnr gene in urinary tract pathogens encodes proteins that protect bacterial DNA gyrase and topoisomerase IV from quinolone antibiotics, leading to antibiotic resistance. Clinical implications include reduced efficacy of quinolone treatment for certain bacterial infections, increased treatment difficulty, and higher healthcare costs. The ErmA and ErmB genes encode methyltransferases that methylate specific nucleotides on bacterial ribosomal 23S rRNA, preventing macrolides, lincosamides, and streptozotocins (MLS) from binding to ribosomes and exerting their effects. This results in antibiotic resistance, limiting treatment options and potentially leading to treatment failure and persistent infection. The dfr gene encodes a variant of dihydrofolate reductase (DHFR) that induces trimethoprim (TMP) resistance. This variant DHFR more effectively catalyzes the reduction of dihydrofolate to tetrahydrofolate, thus reducing the inhibitory effect of the drug. The tet(M) and tet(B) genes function by encoding different efflux pump proteins or ribosome protection proteins, enabling bacteria to expel tetracycline antibiotics or protect ribosomes from the effects of tetracycline, thereby developing resistance to tetracycline drugs. The emergence of resistance genes reduces the effectiveness of tetracycline drugs in clinical treatment and increases the difficulty of treating bacterial infections.

[0004] The presence of these drug-resistant genes makes the treatment of urinary tract infections more difficult, and these drug-resistant genes have also been detected in some pathogens in vaginal swabs and skin lesion swabs. Therefore, accurate detection of drug-resistant genes is crucial for the rational selection of antibiotics and improving treatment efficacy. Combined detection of drug-resistant genes is of great significance, as detailed below:

[0005] 1) Rational selection of antibiotics: By detecting multiple drug resistance genes, it is possible to clarify the resistance of pathogens to different types of antibiotics, helping doctors to accurately select sensitive antibiotics for treatment and avoid treatment failure and delay in diagnosis caused by empirical drug use.

[0006] 2) Early detection of drug-resistant bacteria: Combined testing can quickly and accurately identify pathogens carrying drug-resistant genes, facilitating early isolation measures and preventing the spread of drug-resistant bacteria within hospitals or the community. Timely identification of drug-resistant bacteria can remind medical staff to strengthen infection control measures, such as strict hand hygiene, environmental disinfection, and appropriate patient placement, reducing the risk of drug-resistant bacteria transmission.

[0007] 3) Monitoring drug resistance trends: Regular joint testing of drug resistance genes can monitor the drug resistance trends of pathogens, providing a basis for public health departments to formulate antimicrobial drug management policies. By understanding the prevalence of drug resistance genes, antibiotic use strategies can be adjusted, the use of certain highly resistant antibiotics can be restricted, and rational drug use can be promoted, thereby slowing down the emergence and spread of drug-resistant bacteria.

[0008] 4) Understanding drug resistance mechanisms: Joint detection of drug resistance genes helps in-depth research into the drug resistance mechanisms of pathogens, providing direction for the development of novel antibacterial drugs. By analyzing the mechanisms of action of different drug resistance genes, novel drugs that can overcome existing drug resistance mechanisms can be developed in a targeted manner, improving the ability to combat drug-resistant bacteria.

[0009] However, there are currently no kits or methods in this field for the combined detection of the aforementioned multiple drug resistance genes. Sequencing, microarrays, and multiplex PCR are the main methods for detecting multiple drug resistance genes. However, sequencing methods are complex, costly, and time-consuming for data analysis; microarrays require immobilizing a large number of oligonucleotide probes on the chip, which is costly and may result in false positives due to cross-hybridization. Multiplex PCR has high detection efficiency, but the design and optimization of primers and probes are difficult, and they are prone to dimerization and non-specific amplification.

[0010] Therefore, there is an urgent need in this field to develop such drug resistance gene detection kits and methods. Summary of the Invention

[0011] Therefore, it is necessary to provide at least one nucleic acid composition for detecting drug resistance genes, a kit containing the composition, and its uses.

[0012] In a first aspect of this application, a nucleic acid composition for detecting drug resistance genes is provided, said nucleic acid composition comprising one or more groups selected from the following primer and probe sets:

[0013] Primer and probe set 1: Sequences shown in SEQ ID NO: 1~3 are used for upstream and downstream primers and probes to detect gene qnrA;

[0014] Primer and probe set 2: Sequences shown in SEQ ID NO: 4~6 are used for upstream and downstream primers and probes for detecting gene qnrB;

[0015] Primer and probe set 3: Sequences shown in SEQ ID NO: 7~9 are used for upstream and downstream primers and probes for detecting the qnrS gene;

[0016] Primer and probe set 4: The sequences of the upstream and downstream primers and probes for detecting the ErmA gene are shown in SEQ ID NO: 10~12;

[0017] Primer and probe set 5: The sequences of the upstream and downstream primers and probes for detecting the ErmB gene are shown in SEQ ID NO: 13~15;

[0018] Primer and probe set 6: Sequences shown in SEQ ID NO: 16~18 are used for upstream and downstream primers and probes for detecting the dfrA1 gene;

[0019] Primer and probe set 7: Sequences shown in SEQ ID NO: 19~21 are used for upstream and downstream primers and probes to detect the dfrA5 gene;

[0020] Primer and probe set 8: The sequences of the upstream and downstream primers and probes for detecting the gene tet(M) are shown in SEQ ID NO: 22~24;

[0021] Primer and probe set 9: The sequences of upstream and downstream primers and probes for detecting the gene tet(B) are shown in SEQ ID NO: 25~27.

[0022] In some embodiments, the nucleic acid composition further includes primer and probe set 10: upstream and downstream primers and probes with sequences as shown in SEQ ID NO: 28-30 for detecting GAPDH.

[0023] In a second aspect of this application, a kit is provided comprising the nucleic acid composition as described in the first aspect.

[0024] In some embodiments, the probes in the primer-probe sets for detecting the qnr gene, the primer-probe set for detecting the dfr gene, the primer-probe set for detecting the ErmA gene, the primer-probe set for detecting the ErmB gene, the primer-probe set for detecting the tet(M) gene, and the primer-probe set for detecting the tet(B) gene are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other.

[0025] The qnr gene includes at least one of the qnrA, qnrB, and qnrS genes; the dfr gene includes at least one of the dfrA1 and dfrA5 genes.

[0026] In some embodiments, the probes in primer-probe set 10 are labeled with fluorescent reporter groups, and the fluorescent reporter groups labeled with the probes in primer-probe sets 1 to 9 are different from those labeled with fluorescent reporter groups and do not interfere with each other.

[0027] In some embodiments, the probes in primer-probe sets 1-3 are labeled with the same fluorescent reporter group.

[0028] In some implementations, the probes of primer probe sets 6 and 7 are labeled with the same fluorescent reporter group.

[0029] In some embodiments, the fluorescent reporter group includes one or more of FAM, HEX, VIC, ROX, CY5, CY7, ATTO425, and Quasar 705.

[0030] In some embodiments, the kit also includes a positive control and / or a negative control.

[0031] In some embodiments, the kit further includes a nucleic acid release reagent, a nucleic acid extraction reagent, UNG enzyme, DNA polymerase, dNTPs (U), PCR buffer, and Mg. 2+ At least one of them.

[0032] In a third aspect of this application, a method for detecting drug resistance genes is provided, the method comprising the following steps:

[0033] Nucleic acid is extracted from the sample to be tested to obtain a nucleic acid sample;

[0034] The nucleic acid sample was PCR amplified using the nucleic acid composition as described in the first aspect, or the kit as described in the second aspect; and,

[0035] Obtain and analyze the results.

[0036] In some implementations, in each reaction system for PCR amplification, the initial system concentration of each primer is independently 125 nM to 500 nM, and the initial system concentration of each probe is independently 60 nM to 250 nM.

[0037] In some embodiments, in each reaction system for PCR amplification, the initial concentration of the DNA polymerase is 0.1 U / μL to 0.5 U / μL, the initial concentration of the UNG enzyme is 0.005 U / μL to 0.015 U / μL, and the Mg... 2+ The initial system concentration was 5 mM to 10 mM, and the initial system concentration of dNTP(U)s was 2 mM to 3 mM.

[0038] In some implementations, the procedure for performing PCR amplification includes:

[0039] UNG enzyme treatment, 50 ℃~65 ℃, 1 min~5 min;

[0040] Pre-denaturation, 92 ℃~98 ℃, 5 min~10 min;

[0041] Denaturation, 92 ℃~98 ℃, 15 s~30 s; annealing, extension and fluorescence detection, 55 ℃~60 ℃, 30 s~60 s; 35~50 cycles. The detection method in this application is based on real-time fluorescent PCR, integrating PCR, molecular hybridization and photochemistry, combining PCR sensitivity with probe specificity. This significantly overcomes the shortcomings of traditional PCR, shortens reaction time, and simplifies operation. The entire process of PCR amplification and product analysis is performed under closed conditions, avoiding false negatives and environmental contamination caused by cross-contamination between samples.

[0042] The nucleic acid composition and kit in this application, through multiplex PCR technology, can greatly improve detection efficiency, shorten the detection cycle, reduce detection costs, shorten the time for judging the drug resistance of pathogens, and accelerate the implementation of countermeasures.

[0043] In some implementations, drug resistance genes qnr, ErmA, ErmB, dfr, tet(M), and tet(B) can be detected simultaneously in a single reaction tube. A single sample requires only one nucleic acid extraction and one simultaneous PCR test to obtain results for all six drug resistance genes, with no interference during the PCR process and accurate results. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0045] Figure 1 This is a diagram showing the detection of six drug resistance genes within the detection range using the kit developed in one embodiment of this application.

[0046] Figure 2 This is a specific detection diagram of the PCR reaction system in one embodiment of this application.

[0047] Figure 3 This is a graph showing the detection sensitivity of the drug resistance gene qnrA at 2000 copies / mL in one embodiment of this application.

[0048] Figure 4This is a graph showing the detection sensitivity of the drug resistance gene qnrB at 2000 copies / mL in one embodiment of this application.

[0049] Figure 5 This is a graph showing the detection sensitivity of the drug resistance gene qnrS at 2000 copies / mL in one embodiment of this application.

[0050] Figure 6 This is a graph showing the detection sensitivity of the drug resistance gene ErmA at 2000 copies / mL in one embodiment of this application.

[0051] Figure 7 This is a graph showing the detection sensitivity of the drug resistance gene ErmB at 2000 copies / mL in one embodiment of this application.

[0052] Figure 8 This is a detection graph showing the sensitivity of the drug resistance gene dfrA1 at 2000 copies / mL in one embodiment of this application.

[0053] Figure 9 This is a detection graph showing the sensitivity of the drug resistance gene dfrA5 at 2000 copies / mL in one embodiment of this application.

[0054] Figure 10 This is a detection graph showing the sensitivity of the drug resistance gene tet(M) at 2000 copies / mL in one embodiment of this application.

[0055] Figure 11 This is a detection graph of the drug resistance gene tet(B) at a sensitivity of 2000 copies / mL in one embodiment of this application.

[0056] Figure 12 The results show the detection of six target drug resistance genes using control primers and probes. Detailed Implementation

[0057] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0059] In this application, unless otherwise specified, "one or more groups" means any group or combination of the listed items. Similarly, "one or more" and other expressions that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0060] The terms “combination,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0061] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0063] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0064] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0065] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0066] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0067] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0068] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0069] When detecting multiple pathogens simultaneously, the mutual interference between primers and probes is a technical issue that needs to be considered.

[0070] Due to the complementary base pairing principle, primers and / or probes can form dimers, which must be ruled out from the initial design stage. This is especially true when detecting multiple pathogens simultaneously, where there are numerous primers and probes, and dimers are more likely to form between primers, probes, or other probes. Ensuring design conservation (crucial for detection accuracy) while also considering interference between different primers and probes requires meticulous and creative design.

[0071] In a first aspect of this application, a nucleic acid composition for detecting drug resistance genes is provided, said nucleic acid composition comprising one or more of the following primer and probe sets:

[0072] Primer and probe set 1: Sequences shown in SEQ ID NO: 1-3 are used for detecting upstream and downstream primers and probes for gene qnrA; Primer and probe set 2: Sequences shown in SEQ ID NO: 4-6 are used for detecting upstream and downstream primers and probes for gene qnrB; Primer and probe set 3: Sequences shown in SEQ ID NO: 7-9 are used for detecting upstream and downstream primers and probes for gene qnrS; Primer and probe set 4: Sequences shown in SEQ ID NO: 10-12 are used for detecting upstream and downstream primers and probes for gene ErmA; Primer and probe set 5: Sequences shown in SEQ ID NO: 13-15 are used for detecting upstream and downstream primers and probes for gene ErmB; Primer and probe set 6: Sequences shown in SEQ ID NO: 16-18 are used for detecting upstream and downstream primers and probes for gene dfrA1; Primer and probe set 7: Sequences shown in SEQ ID NO: 19-21 are used for detecting upstream and downstream primers and probes for gene dfrA5; Primer and probe set 8: Sequences shown in SEQ ID NO: Primers and probes for detecting gene tet(M) are shown in SEQ ID NO: 25-27; primer and probe set 9: primers and probes for detecting gene tet(B) are shown in SEQ ID NO: 25-27.

[0073] The nucleic acid composition may contain any two, three, four, five, six, seven, or eight primer and probe sets from the above nine primer and probe sets, or may contain all nine primer and probe sets.

[0074] In some embodiments, the nucleic acid composition simultaneously comprises primer-probe sets 1 to 9. The drug resistance genes qnr, ErmA, ErmB, dfr, tet(M), and tet(B) can be detected simultaneously in a single reaction tube.

[0075] In some embodiments, the nucleic acid composition further includes a probe set 10: upstream and downstream primers and probes with sequences as shown in SEQ ID NO: 28-30 for detecting GAPDH. While not wishing to be limited by any theoretical framework, it is believed that the housekeeping gene GAPDH, acting as an internal standard quality control, participates in the extraction of urethral pathogen nucleic acid from biological samples and the PCR reaction process to determine whether the sampling, sample processing, and PCR amplification system are functioning correctly. Simultaneously with sample detection, it can monitor the collection of urine samples and the nucleic acid purification process in the samples, avoiding false negatives, increasing the reliability of the detection results, and making the entire experimental process more accurate and reliable.

[0076] In a second aspect of this application, a kit is provided comprising the nucleic acid composition as described in the first aspect.

[0077] In some embodiments, the nucleic acid composition comprises the nine primer-probe sets described above.

[0078] In some embodiments, the probes in the primer-probe sets for detecting the qnr gene, the primer-probe set for detecting the dfr gene, the primer-probe set for detecting the ErmA gene, the primer-probe set for detecting the ErmB gene, the primer-probe set for detecting the tet(M) gene, and the primer-probe set for detecting the tet(B) gene are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other.

[0079] The qnr gene includes at least one of the qnrA, qnrB, and qnrS genes; the dfr gene includes at least one of the dfrA1 and dfrA5 genes.

[0080] In some embodiments, the probes in the primer-probe set 10 are labeled with fluorescent reporter groups, and the fluorescent reporter groups labeled with the probes in primer-probe sets 1 to 9 are different from those labeled with fluorescent reporter groups and do not interfere with each other.

[0081] In some embodiments, the probes in primer-probe sets 1-3 are labeled with the same fluorescent reporter group.

[0082] In some implementations, the probes of primer probe sets 6 and 7 are labeled with the same fluorescent reporter group.

[0083] In some embodiments, the fluorescent reporter group includes one or more of FAM, HEX, VIC, ROX, CY5, CY7, ATTO425, and Quasar 705.

[0084] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 1, as shown in SEQ ID NO: 3, is FAM.

[0085] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 2, as shown in SEQ ID NO: 6, is FAM.

[0086] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 3, as shown in SEQ ID NO: 9, is FAM.

[0087] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 4, as shown in SEQ ID NO: 12, is HEX.

[0088] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 5, such as those shown in SEQ ID NO: 15, is ROX.

[0089] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 6, as shown in SEQ ID NO: 18, is QUASAR705.

[0090] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 7, such as those shown in SEQ ID NO: 21, is QUASAR705.

[0091] In some embodiments, the fluorescent reporter group labeled with the probes in primer probe set 8, such as those shown in SEQ ID NO: 24, is ATTO425.

[0092] In some embodiments, the probes in primer probe set 9 with sequences such as SEQ ID NO: 27 are labeled with a fluorescent reporter group of CY7.

[0093] In this application, the kit may comprise different units. Each unit may be contained in an independent small package or in a separate compartment. In some embodiments, each unit comprises different primer-probe sets. Each unit may also comprise different subunits, which may be, for example, upstream primers, downstream primers, or probes from the aforementioned primer-probe sets. For example, a primer with the sequence shown in SEQ ID NO: 1 may be in dry powder or liquid form as one subunit. A primer with the sequence shown in SEQ ID NO: 2 may be in dry powder or liquid form as another subunit.

[0094] In some embodiments, the kit further comprises one or more of a positive control and a negative control.

[0095] In some embodiments, the kit further includes a nucleic acid release reagent, a nucleic acid extraction reagent, UNG enzyme, DNA polymerase, dNTPs (U), PCR buffer, and Mg. 2+ At least one of the following. In some embodiments, the kit includes a nucleic acid release reagent, a nucleic acid extraction reagent, UNG enzyme, DNA polymerase, dNTPs (U), PCR buffer, and Mg. 2+ .

[0096] In a third aspect of this application, a method for detecting drug resistance in environmental pathogens is provided, the method comprising the following steps:

[0097] Nucleic acid is extracted from the sample to be tested to obtain a nucleic acid sample;

[0098] The nucleic acid sample was PCR amplified using the nucleic acid composition as described in the first aspect, or the kit as described in the second aspect; and,

[0099] Obtain and analyze the results.

[0100] The method described in this application may optionally be used for non-disease diagnostic purposes.

[0101] Unless otherwise specified, the term "sample" in this application can refer to any item that may contain urethral pathogens, such as contaminated water sources, everyday items (such as toilets, bedpans), etc. In some embodiments, the sample is a culture. Exemplarily, in some cases, the content of urethral pathogens in the original sample is extremely low, below or even far below the detection limit of the detection method to be used. In such cases, it is selectively necessary to culture the small amount of pathogens in the original sample. In this case, the sample is a pure culture sample.

[0102] In some implementations, in each reaction system for PCR amplification, the initial system concentration of each primer is independently 125 nM to 500 nM, and the initial system concentration of each probe is independently 60 nM to 250 nM.

[0103] In some embodiments, in each reaction system for PCR amplification, the initial concentration of the DNA polymerase is 0.1 U / μL to 0.5 U / μL, the initial concentration of the UNG enzyme is 0.005 U / μL to 0.015 U / μL, and the Mg... 2+ The initial system concentration was 5 mM to 10 mM, and the initial system concentration of dNTP(U)s was 2 mM to 3 mM.

[0104] In some embodiments, the initial concentration of the DNA polymerase in each reaction system for PCR amplification is 0.1 U / μL, 0.2 U / μL, 0.3 U / μL, 0.4 U / μL, 0.5 U / μL, or any range or value between two values.

[0105] In some embodiments, the initial concentration of the UNG enzyme in each reaction system for PCR amplification is 0.005 U / μL, 0.006 U / μL, 0.007 U / μL, 0.008 U / μL, 0.009 U / μL, 0.01 U / μL, 0.011 U / μL, 0.012 U / μL, 0.013 U / μL, 0.014 U / μL, 0.015 U / μL, or any range or value between two such values.

[0106] In some implementations, in each reaction system undergoing PCR amplification, Mg 2+ The initial system concentration is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, or any range or value between two values.

[0107] In some implementations, the initial concentration of dNTP(U)s in each reaction system for PCR amplification is 2 mM, 2.5 mM, 3 mM, or any range or value between two values.

[0108] In some implementations, the procedure for performing PCR amplification includes:

[0109] UNG enzyme treatment, 50 ℃~65 ℃, 1 min~5 min;

[0110] Pre-denaturation, 92 ℃~98 ℃, 5 min~10 min;

[0111] Denaturation, 92 ℃~98 ℃, 15 s~30 s, annealing, extension and fluorescence detection, 55 ℃~60 ℃, 30 s~60 s, 35~50 cycles.

[0112] In some implementations, the procedure for performing PCR amplification includes:

[0113] UNG enzyme treatment, 50℃, 2 min;

[0114] Pre-denaturation, 95℃, 5 min;

[0115] Denaturation, 95℃, 15s, annealing, extension and fluorescence detection, 57℃, 30s, 45 cycles.

[0116] The following are some examples.

[0117] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0118] Example 1

[0119] The following provides a method for detecting the presence of six drug resistance genes—qnr, ErmA, ErmB, dfr, tet(M), and tet(B)—in biological samples. This type of method involves at least one cyclic step, which may include an amplification step. The amplification step typically involves contacting the sample with a pair of primers for the aforementioned six pathogens to generate pathogen amplification products when pathogen nucleic acid molecules are present in the sample. Detection and differentiation of the amplification products can be performed using various methods (such as sequencing of the amplification products, agarose gel electrophoresis analysis, SYBR fluorescent dye method, TaqMan probe method, etc.). In this application, TaqMan probes are used to detect the presence of drug resistance genes in the amplification products. An increase in fluorescence signal indicates the presence of the corresponding drug resistance gene in the sample, while the absence of fluorescence signal indicates the absence of the aforementioned six drug resistance genes in the sample. To differentiate the detected drug resistance genes qnr, ErmA, ErmB, dfr, tet(M), and tet(B), different fluorescent groups can be labeled onto different probes to distinguish the signals.

[0120] The primer and probe sequences used are shown in Table 1.

[0121] Table 1

[0122]

[0123] Note: Y represents C / T, M represents A / C, R represents A / G, and S represents C / G.

[0124] The probes for detecting qnrA, qnrB, and qnrS genes were labeled with FAM fluorescent reporter groups; the probes for detecting ErmA genes were labeled with HEX fluorescent groups; the probes for detecting ErmB genes were labeled with ROX fluorescent groups; the probes for detecting dfrA1 and dfrA5 genes were labeled with QUASAR 705 fluorescent groups; the probes for detecting tet(M) genes were labeled with ATTO425 fluorescent groups; the probes for detecting tet(B) genes were labeled with CY7 fluorescent groups; and the probes for detecting internal standards were labeled with CY5 fluorescent groups.

[0125] The PCR reaction system (20 µL) consists of: 14.5 µL PCR reaction solution + 1.5 µL enzyme mixture + 4 µL sample.

[0126] The PCR reaction solution formulation is shown in Table 2 (single dose).

[0127] Table 2

[0128]

[0129] Preparation of enzyme mixture:

[0130] The enzyme mixture consists of UNG enzyme and Taq enzyme. UNG enzyme (2 U / μL) and Taq enzyme (5 U / μL) are mixed at a ratio of 1:14 (each person's dose consists of 0.1 μL of UNG enzyme and 1.4 μL of Taq enzyme).

[0131] The PCR reaction solution and enzyme mixture were prepared according to the above protocol. A negative control was prepared using sterile saline as a matrix, and a positive control was prepared using plasmids positive for drug resistance genes of six urethral pathogens within the detection range. This constituted the detection kit. To monitor sample collection, extraction, and PCR amplification and avoid false negative results, this application uses the human housekeeping gene as an internal control. The detection of the internal control is performed simultaneously with the detection of drug resistance genes of urethral pathogens in the sample using the corresponding primers (SEQ ID NO:28, SEQ ID NO:29) and probes (SEQ ID NO:30).

[0132] Test methods and reaction conditions:

[0133] 1. Reagent preparation:

[0134] Based on the number of samples to be tested, positive controls, and negative controls, take the corresponding amounts of PCR reaction solution and enzyme mixture according to the ratio (14.5 μL / person for PCR reaction solution + 1.5 μL / person for enzyme mixture), mix thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds, and set aside for later use.

[0135] 2. Sample processing and loading

[0136] Take 300 μL of the test sample, negative control, and positive control into a 1.5 mL centrifuge tube, and perform nucleic acid extraction using the nucleic acid extraction or purification reagents from Sansure Biotech Inc. in accordance with their instructions.

[0137] Take 4 μL each of the above-processed sample, negative control, and positive control and add them to the corresponding 0.2 mL PCR reaction tubes. Add 16 μL of PCR mixture to each tube and cap the tubes.

[0138] 3. PCR amplification

[0139] PCR amplification was performed on the SLAN-96H fully automated medical PCR analysis system according to a specific temperature and time setting program. The optional protocols for this application are shown in Table 3.

[0140] Table 3

[0141]

[0142] 4. Interpretation of test results

[0143] If the sample shows obvious S-shaped amplification curves in the FAM, HEX, ROX, QUASAR705, ATTO425, and CY7 channels, and the Ct value is ≤40, it is considered positive. If the sample shows no amplification curves (No Ct) or a Ct value >40 in the FAM, HEX, ROX, QUASAR705, ATTO425, and CY7 channels, and the CY5 internal standard channel is positive (Ct value ≤40), it is considered negative. See Table 4 for details.

[0144] Table 4

[0145]

[0146] The kit described in this application can accurately detect and differentiate six target drug resistance genes: qnr, ErmA, ErmB, dfr, tet(M), and tet(B). Results can be found in [link to kit]. Figure 1 .

[0147] Specificity experiments have shown that the method in this application has no cross-reactivity with common urethral pathogen resistance genes (vanA, vanB, mecA, CTX-M, KPC, IMP, NDM, VIM, OXA), and the results can be found in [link to relevant documentation]. Figure 2 .

[0148] The results of the sensitivity analysis showed that the detection limits for the drug resistance genes qnr, ErmA, ErmB, dfr, tet(M), and tet(B) were all 2000 copies / mL. (See attached image for details.) Figures 3-11 .

[0149] Comparative Example

[0150] Primers and probes with good single-gene detection efficacy were combined, and the method described in Example 1 was used for combined detection of six target drug resistance genes. The detection results are as follows: Figure 12 As shown, after assembling the combined detection reagent, some curves showed poor morphology and did not achieve the expected S-shape. At the same time, the Ct values ​​of each target were significantly delayed compared with those of the embodiments in this application, and some channels even failed to form lines, indicating that the amplification efficiency of the comparative composition was poor.

[0151] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A nucleic acid composition for detecting drug resistance genes, characterized in that, The nucleic acid composition includes the following primer and probe set: Primer and probe set 1: Sequences shown in SEQ ID NO: 1~3 are used for upstream and downstream primers and probes to detect gene qnrA; Primer and probe set 2: Sequences shown in SEQ ID NO: 4~6 are used for upstream and downstream primers and probes for detecting gene qnrB; Primer and probe set 3: Sequences shown in SEQ ID NO: 7~9 are used for upstream and downstream primers and probes for detecting the qnrS gene; Primer and probe set 4: The sequences of the upstream and downstream primers and probes for detecting the ErmA gene are shown in SEQ ID NO: 10~12; Primer and probe set 5: The sequences of the upstream and downstream primers and probes for detecting the ErmB gene are shown in SEQ ID NO: 13~15; Primer and probe set 6: Sequences shown in SEQ ID NO: 16~18 are used for upstream and downstream primers and probes for detecting the dfrA1 gene; Primer and probe set 7: Sequences shown in SEQ ID NO: 19~21 are used for upstream and downstream primers and probes to detect the dfrA5 gene; Primer and probe set 8: The sequences of the upstream and downstream primers and probes for detecting the gene tet(M) are shown in SEQ ID NO: 22~24; Primer and probe set 9: The sequences of upstream and downstream primers and probes for detecting the gene tet(B) are shown in SEQ ID NO: 25~27.

2. The nucleic acid composition according to claim 1, characterized in that, It also includes primer and probe set 10: upstream and downstream primers and probes with sequences as shown in SEQ ID NO: 28~30 for detecting GAPDH.

3. A reagent kit, characterized in that, It comprises the nucleic acid composition as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that, The probes in the primer and probe sets for detecting the qnr gene, the primer and probe sets for detecting the dfr gene, the primer and probe sets for detecting the ErmA gene, the primer and probe sets for detecting the ErmB gene, the primer and probe sets for detecting the tet(M) gene, and the primer and probe sets for detecting the tet(B) gene are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other; the qnr gene includes at least one of the qnrA, qnrB, and qnrS genes; the dfr gene includes at least one of the dfrA1 and dfrA5 genes.

5. The kit according to claim 4, characterized in that, The probes in primer-probe set 10 are labeled with fluorescent reporter groups, and the fluorescent reporter groups labeled with the probes in primer-probe sets 1 to 9 are different from those labeled with fluorescent reporter groups and do not interfere with each other.

6. The reagent kit as described in claim 5, characterized in that, The probes in primer-probe sets 1-3 are labeled with the same fluorescent reporter group.

7. The kit according to claim 5, characterized in that, The probes in primer and probe sets 6 and 7 are labeled with the same fluorescent reporter group.

8. The reagent kit as described in claim 5, characterized in that, The fluorescent reporter group includes one or more of FAM, HEX, VIC, ROX, CY5, CY7, ATTO425, and Quasar 705.

9. The kit according to any one of claims 3 to 8, characterized in that, The kit also includes positive and / or negative controls.

10. A method for detecting drug resistance in environmental pathogens, characterized in that, The method includes the following steps: Nucleic acid is extracted from the sample to be tested to obtain a nucleic acid sample; The nucleic acid sample was PCR amplified using the nucleic acid composition as described in claim 1 or 2, or the kit as described in any one of claims 3 to 9; and, Obtain and analyze the results.

11. The method as described in claim 10, characterized in that, In each reaction system for PCR amplification, the initial concentration of each primer was independently 125 nM to 500 nM, and the initial concentration of each probe was independently 60 nM to 250 nM.

12. The method as described in claim 10 or 11, characterized in that, The procedure for PCR amplification includes: UNG enzyme treatment, 50 ℃~65 ℃, 1 min~5 min; Pre-denaturation, 92 ℃~98 ℃, 5 min~10 min; Denaturation, 92 ℃~98 ℃, 15 s~30 s, annealing, extension and fluorescence detection, 55 ℃~60 ℃, 30 s~60 s, 35~50 cycles.

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