Nucleic acid composition for detecting drug-resistant gene, kit containing nucleic acid composition and application of nucleic acid composition

Abstract

The invention discloses a nucleic acid composition for detecting a drug-resistant gene, a kit containing the nucleic acid composition and application of the nucleic acid composition. The nucleic acid composition comprises one or more of a primer probe group 1 to a primer probe group 9. When the nucleic acid composition or the kit is used for detecting the drug resistance gene, the reaction time is shortened, and the operation steps are simplified; in some embodiments, detection results of the six drug-resistant genes can be obtained only by performing nucleic acid extraction once and PCR detection once, and the PCR process is free of interference.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a nucleic acid composition for detecting drug-resistant genes, a kit containing the same, and uses thereof. Background Art

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

[0003] There is extensive genetic diversity in the population of resistant pathogens. For example, the proteins encoded by the qnr genes in urinary pathogens can protect the bacterial DNA gyrase and topoisomerase IV from the effects of quinolones, leading to bacterial resistance to quinolones. Clinical impacts include: making quinolones less effective in treating certain bacterial infections, increasing the difficulty of treatment and medical costs. The ErmA and ErmB genes can encode methylases that methylate specific nucleotides on the bacterial ribosome 23S rRNA, thereby preventing macrolide, lincosamide and streptogramin (MLS) antibiotics from binding to the ribosome to exert their effects, leading to bacterial resistance to these antibiotics, limiting treatment options and potentially leading to treatment failure and continued spread of infection. The mechanism of action of the dfr gene is to encode a variant dihydrofolate reductase (DHFR) that makes bacteria resistant to trimethoprim (TMP); this variant DHFR can more effectively catalyze the reduction of dihydrofolate to tetrahydrofolate, thereby reducing the inhibitory effect of the drug. The mechanism of action of tet(M) and tet(B) genes is to encode different efflux pump proteins or ribosome protection proteins, which enable bacteria to excrete tetracycline antibiotics or protect ribosomes from the effects of tetracycline, thereby producing 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 some pathogens in vaginal swabs and skin lesion swabs also detect the above-mentioned drug-resistant genes. Therefore, accurate detection of drug-resistant genes is crucial for the rational selection of antibiotics and improving treatment effects. The combined detection of drug-resistant genes is of great significance, as follows:

[0005] 1) Rational selection of antibiotics: By detecting multiple drug-resistant genes, we can clarify the resistance of pathogens to different types of antibiotics, help doctors accurately select sensitive antibiotics for treatment, and avoid treatment failure and delays caused by empirical medication.

[0006] 2) Early detection of drug-resistant bacteria: Combined testing can quickly and accurately detect pathogens carrying drug-resistant genes, which helps to take isolation measures early to prevent the spread of drug-resistant bacteria in hospitals or communities. Timely identification of drug-resistant bacteria can remind medical staff to strengthen infection control measures, such as strict hand hygiene, environmental disinfection, and reasonable patient placement, to reduce the risk of spreading drug-resistant bacteria.

[0007] 3) Monitoring drug resistance trends: Regular joint detection of drug resistance genes can monitor the drug resistance trends of pathogens and provide a basis for public health departments to formulate antimicrobial management policies. By understanding the prevalence of drug resistance genes, it is possible to adjust the use of antibiotics, limit the use of certain highly resistant antibiotics, promote rational use of drugs, and thus slow down the generation and spread of drug-resistant bacteria.

[0008] 4) Understanding the mechanism of drug resistance: Joint detection of drug-resistant genes helps to further study the drug resistance mechanism of pathogens and provide direction for the development of new antimicrobial drugs. By analyzing the mechanism of action of different drug-resistant genes, new drugs that can overcome existing drug resistance mechanisms can be developed in a targeted manner to improve the ability to fight drug-resistant bacteria.

[0009] However, there is no kit and method for joint detection of the above-mentioned multiple drug-resistant genes in the art. Sequencing, microarray chips and multiplex PCR are currently the main means of detecting multiple drug-resistant genes. However, the sequencing method is complex for data analysis, high cost and long time; microarray chips require a large number of oligonucleotide probes to be fixed on the chip, which is expensive and may cause false positives due to cross-hybridization. Multiplex PCR has a high detection efficiency, but the design and optimization of primers and probes are difficult, and dimers are easily formed, resulting in non-specific amplification.

[0010] Therefore, there is an urgent need in the art to develop such drug-resistant gene joint detection kits and methods. Summary of the invention

[0011] Based on this, it is necessary to provide at least a nucleic acid composition for detecting drug-resistant genes, a kit containing the same, and uses thereof.

[0012] In the first aspect of the present application, a nucleic acid composition for detecting drug-resistant genes is provided, wherein the nucleic acid composition comprises one or more groups selected from the following primer probe groups:

[0013] Primer probe set 1: The sequences are shown in SEQ ID NOs: 1 to 3, which are used to detect the upstream and downstream primers and probes of the gene qnrA;

[0014] Primer probe set 2: The sequences are shown in SEQ ID NOs: 4 to 6, which are used to detect upstream and downstream primers and probes of the gene qnrB;

[0015] Primer probe set 3: The sequences are shown in SEQ ID NOs: 7 to 9, which are used to detect the upstream and downstream primers and probes of the gene qnrS;

[0016] Primer probe set 4: the sequences are shown in SEQ ID NOs: 10 to 12, which are used for detecting the upstream and downstream primers and probes of the gene ErmA;

[0017] Primer probe set 5: the sequences are shown in SEQ ID NOs: 13 to 15, which are used for detecting upstream and downstream primers and probes of the gene ErmB;

[0018] Primer probe set 6: the sequences are shown in SEQ ID NOs: 16 to 18, which are used for detecting upstream and downstream primers and probes of gene dfrA1;

[0019] Primer probe set 7: the sequences are shown in SEQ ID NOs: 19 to 21, which are used to detect upstream and downstream primers and probes of gene dfrA5;

[0020] Primer probe set 8: The sequences are shown in SEQ ID NOs: 22 to 24, which are upstream and downstream primers and probes for detecting the gene tet (M);

[0021] Primer probe set 9: The sequences are shown in SEQ ID NOs: 25 to 27, which are used for detecting upstream and downstream primers and probes of the gene tet (B).

[0022] In some embodiments, the nucleic acid composition further comprises a primer probe set 10: upstream and downstream primers and probes for detecting GAPDH, whose sequences are shown in SEQ ID NOs: 28 to 30.

[0023] In a second aspect of the present 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 set 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] Among them, the qnr gene includes at least one of qnrA, qnrB and qnrS genes; the dfr gene includes at least one of dfrA1 and dfrA5.

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

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

[0028] In some embodiments, 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 further comprises a positive quality control and / or a negative quality control.

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

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

[0033] Extracting nucleic acid from the sample to be tested to obtain a nucleic acid sample;

[0034] Using the nucleic acid composition as described in the first aspect or the kit as described in the second aspect to perform PCR amplification on the nucleic acid sample; and,

[0035] Obtain and analyze results.

[0036] In some embodiments, 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 system concentration of the DNA polymerase is 0.1 U / μL~0.5 U / μL, the initial system concentration of the UNG enzyme is 0.005 U / μL~0.015 U / μL, and the Mg is 0. 2+ The initial system concentration of is 5 mM~10 mM, and the initial system concentration of dNTP(U)s is 2 mM~3 mM.

[0038] In some embodiments, the procedure for performing PCR amplification comprises:

[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, cycle 35 times ~ 50 times. The detection method in this application can be based on real-time fluorescence PCR, integrating PCR, molecular hybridization and photochemistry, combining PCR sensitivity with probe specificity, and greatly changing the defects of traditional PCR, shortening the reaction time, and simplifying the operation steps. The entire process of PCR amplification and product analysis is carried out under closed conditions, avoiding false negatives and environmental contamination caused by cross-samples.

[0042] The nucleic acid composition and kit in the present application can greatly improve the detection efficiency, shorten the detection cycle, reduce the detection cost, shorten the time for judging the drug resistance of pathogens, and speed up the implementation of response measures through multiple PCR technology.

[0043] In some embodiments, the drug resistance genes qnr, ErmA, ErmB, dfr, tet(M) and tet(B) can be detected simultaneously in one tube of reaction solution. For one specimen, only one nucleic acid extraction and one PCR test are required to obtain the test results of six drug resistance genes, and there is no interference in the PCR process, so the test results are accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. 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 explaining the present application.

[0045] Figure 1 This is a detection diagram of six drug-resistant genes within the detection range of the kit established in one embodiment of the present application.

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

[0047] Figure 3 This is a detection diagram of the drug-resistant gene qnrA with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0048] Figure 4This is a detection diagram of the drug-resistant gene qnrB with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0049] Figure 5 This is a detection diagram of the drug-resistant gene qnrS with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0050] Figure 6 This is a detection graph of the drug-resistant gene ErmA with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0051] Figure 7 This is a detection graph of the drug-resistant gene ErmB with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0052] Figure 8 This is a detection chart of the drug-resistant gene dfrA1 with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0053] Fig. 9 This is a detection chart of the drug-resistant gene dfrA5 with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0054] Fig.10 This is a detection graph of the drug-resistant gene tet (M) with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0055] Fig.11 This is a detection graph of the drug-resistant gene tet (B) with a sensitivity of 2000 copies / mL in one embodiment of the present application.

[0056] Fig.12 The results are from the joint detection of 6 target drug-resistance genes using control primers and probes. DETAILED DESCRIPTION

[0057] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0059] In this application, unless otherwise specified, "one or more groups" means any group of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

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

[0061] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means 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", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0063] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0064] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0065] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

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

[0067] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each 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 range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0068] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0069] In the joint detection of multiple pathogens, the mutual interference between primers and probes is a technical issue that needs special consideration.

[0070] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, and this possible result needs to be excluded at the beginning of the design. Especially when multiple pathogens are detected together, there are many primers and probes, and dimers are more likely to occur between primers and primers, probes and probes, or primers and probes. It is necessary to ensure the conservatism of the design (conservatism is crucial to the accuracy of the test) and consider the mutual interference between different primers and probes. Creative labor is needed to make particularly careful designs of primers and probes.

[0071] In a first aspect of the present application, a nucleic acid composition for detecting drug-resistant genes is provided, wherein the nucleic acid composition comprises one or more of the following primer probe sets:

[0072] Primer probe set 1: the upstream and downstream primers and probes for detecting the gene qnrA as shown in SEQ ID NOs: 1 to 3; Primer probe set 2: the upstream and downstream primers and probes for detecting the gene qnrB as shown in SEQ ID NOs: 4 to 6; Primer probe set 3: the upstream and downstream primers and probes for detecting the gene qnrS as shown in SEQ ID NOs: 7 to 9; Primer probe set 4: the upstream and downstream primers and probes for detecting the gene ErmA as shown in SEQ ID NOs: 10 to 12; Primer probe set 5: the upstream and downstream primers and probes for detecting the gene ErmB as shown in SEQ ID NOs: 13 to 15; Primer probe set 6: the upstream and downstream primers and probes for detecting the gene dfrA1 as shown in SEQ ID NOs: 16 to 18; Primer probe set 7: the upstream and downstream primers and probes for detecting the gene dfrA5 as shown in SEQ ID NOs: 19 to 21; Primer probe set 8: the upstream and downstream primers and probes for detecting the gene dfrA5 as shown in SEQ ID NOs: 22 to 24 are upstream and downstream primers and probes for detecting the gene tet (M); Primer probe set 9: the sequences are shown in SEQ ID NO: 25 to 27, which are upstream and downstream primers and probes for detecting the gene tet (B).

[0073] The nucleic acid composition may include any two, any three, any four, any five, any six, any seven, any eight of the above 9 primer probe sets, or all 9 primer probe sets.

[0074] In some embodiments, the nucleic acid composition comprises primer probe set 1 to primer probe set 9. In one tube of reaction solution, the drug resistance genes qnr, ErmA, ErmB, dfr, tet (M) and tet (B) can be detected simultaneously.

[0075] In some embodiments, the nucleic acid composition further comprises a probe group 10: the upstream and downstream primers and probes for detecting GAPDH are shown in SEQ ID NO: 28-30. Without wishing to be limited by any theory, it is believed that the housekeeping gene GAPDH is used as an internal standard quality control, and participates in the extraction and PCR reaction process of urinary pathogen nucleic acid in biological samples to determine whether the sampling, specimen processing and PCR amplification system are normal. While testing the specimen, the collection of urine specimens and the purification process of nucleic acids in the specimens can be monitored to avoid false negatives, increase the credibility of the test results, and make the entire experimental process more accurate and reliable.

[0076] In a second aspect of the present 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 9 primer probe sets as described above.

[0078] In some embodiments, the probes in the primer probe set 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] Among them, the qnr gene includes at least one of qnrA, qnrB and qnrS genes; the dfr gene includes at least one of dfrA1 and dfrA5.

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

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

[0082] In some embodiments, 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 probe whose sequence in primer probe set 1 is shown in SEQ ID NO: 3 is FAM.

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

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

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

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

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

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

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

[0092] In some embodiments, the fluorescent reporter group labeled with the probe in primer probe set 9 whose sequence is shown in SEQ ID NO: 27 is CY7.

[0093] In the present application, the kit may include different units. Each unit may be contained in an independent small package, or in a separate partition space. In some embodiments, each unit includes a different primer probe set. Wherein, each unit may also include different subunits, and the subunits may be, for example, upstream primers, downstream primers or probes in the primer probe set described above. For example, a primer with a sequence such as SEQ ID NO: 1 is in the form of a dry powder or a liquid storage as one of the subunits. A primer with a sequence such as SEQ ID NO: 2 is in the form of a dry powder or a liquid storage 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 comprises a nucleic acid release reagent, a nucleic acid extraction reagent, UNG enzyme, DNA polymerase, dNTP(U)s, PCR buffer and Mg. 2+ In some embodiments, the kit includes a nucleic acid release reagent, a nucleic acid extraction reagent, a UNG enzyme, a DNA polymerase, dNTP(U)s, a PCR buffer, and Mg. 2+ .

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

[0097] Extracting nucleic acid from the sample to be tested to obtain a nucleic acid sample;

[0098] Using the nucleic acid composition as described in the first aspect or the kit as described in the second aspect to perform PCR amplification on the nucleic acid sample; and,

[0099] Obtain and analyze results.

[0100] The method described in the present application can optionally be used for non-disease diagnosis purposes.

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

[0102] In some embodiments, 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 system concentration of the DNA polymerase is 0.1 U / μL~0.5 U / μL, the initial system concentration of the UNG enzyme is 0.005 U / μL~0.015 U / μL, and the Mg is 0. 2+ The initial system concentration of is 5 mM~10 mM, and the initial system concentration of dNTP(U)s is 2 mM~3 mM.

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

[0105] In some embodiments, in each reaction system for PCR amplification, the initial system concentration of the UNG enzyme 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 a range or value between any two values.

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

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

[0108] In some embodiments, the procedure for performing PCR amplification comprises:

[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, cycle 35 times ~ 50 times.

[0112] In some embodiments, the procedure for performing PCR amplification comprises:

[0113] UNG enzyme treatment, 50°C, 2 min;

[0114] Pre-denaturation, 95°C, 5 min;

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

[0116] Some examples are provided below.

[0117] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0118] Example 1

[0119] The following provides a method for detecting whether six drug resistance genes, qnr, ErmA, ErmB, dfr, tet(M) and tet(B), are present in a biological sample. Such a method includes performing at least one cycle step, which may include an amplification step. The amplification step generally includes contacting the sample with a pair of the six pathogen primers mentioned above so as to produce pathogen amplification products when pathogen nucleic acid molecules are present in the sample. The detection and resolution of the amplification products can be carried out by a variety of methods (such as sequencing of the amplification products, agarose gel electrophoresis analysis, SYBR fluorescent dye method, TaqMan probe method, etc.). In the present application, TaqMan probes are used to detect whether there are drug resistance genes in the amplification products. When there is an increase in the fluorescent signal, it indicates that the corresponding drug resistance genes are present in the sample. When there is no increase in the fluorescent signal, it indicates that the above six drug resistance genes are not present in the sample. For the drug resistance genes qnr, ErmA, ErmB, dfr, tet(M) and tet(B) to be detected, different fluorescent groups can be used to label different probes to distinguish the signals.

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

[0121] Table 1

[0122]

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

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

[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 formula 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 (2U / μL) and Taq enzyme (5U / μL) are mixed at a ratio of 1:14 (0.1μL UNG enzyme and 1.4μL Taq enzyme per person).

[0131] According to the above scheme, a PCR reaction solution and an enzyme mixture are prepared, and a negative control with sterile saline as the matrix is ​​prepared, and a positive plasmid of urethral pathogen resistance genes within the six detection ranges is used as a positive control to form a detection kit. Among them, in order to monitor sample collection, extraction and PCR amplification to avoid false negative results, this application uses human housekeeping genes as internal standard quality control, and the detection of internal standard quality control is carried out simultaneously with the detection of urethral pathogen resistance genes in the specimen through the corresponding primers (SEQ ID NO: 28, SEQ ID NO: 29) and probes (SEQ ID NO: 30).

[0132] Test method and reaction conditions:

[0133] 1. Reagent preparation:

[0134] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the proportion (PCR reaction solution 14.5μL / person + enzyme mixture 1.5μL / person), mix them thoroughly to form a PCR mixture, and centrifuge at 2000rpm for 10s before use.

[0135] 2. Sample processing and sample addition

[0136] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. according to its instructions to extract nucleic acid.

[0137] 4 μL of the above-treated samples, negative controls and positive controls were pipetted and added to the corresponding 0.2 mL PCR reaction tubes, 16 μL of PCR mixture was added to each tube, and the tubes were capped.

[0138] 3. PCR amplification

[0139] On the SLAN-96H fully automatic medical PCR analysis system, PCR amplification was performed according to a certain temperature and time setting program. The optional schemes of this application are shown in Table 3.

[0140] Table 3

[0141]

[0142] 4. Interpretation of test results

[0143] If the sample has an obvious S-shaped amplification curve in the FAM, HEX, ROX, QUASAR705, ATTO425 and CY7 channels, and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM, HEX, ROX, QUASAR705, ATTO425 and CY7 channels, and the CY5 internal standard channel is positive (Ct value ≤40), it is judged as negative. The details are shown in Table 4.

[0144] Table 4

[0145]

[0146] The kit of the present application can accurately detect and distinguish the six target drug resistance genes: qnr, ErmA, ErmB, dfr, tet(M) and tet(B). The results can be found in Figure 1 .

[0147] Specific experiments have shown that the method in this application has no cross-reaction to common urinary tract pathogen resistance genes (vanA, vanB, mecA, CTX-M, KPC, IMP, NDM, VIM, OXA). The results can be found in Figure 2 .

[0148] The results of the analytical sensitivity test showed that the detection limit of the drug resistance genes qnr, ErmA, ErmB, dfr, tet(M) and tet(B) could reach 2000 copies / mL. Figure 3~Figure 11 .

[0149] Comparative Example

[0150] The primers and probes with good single-detection effects for other drug-resistant genes were combined, and the method of Example 1 was used to perform joint detection on the six target drug-resistant genes. The detection results were as follows: Fig.12 After the combined test reagents were formed, some curves had poor morphology and did not reach the expected S-shape. At the same time, the Ct values ​​of each target were seriously delayed compared with the examples of the present application, and even some channels did not start, indicating that the amplification efficiency of the comparative composition was poor.

[0151] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A nucleic acid composition for detecting drug resistance genes, characterized in that: The nucleic acid composition comprises one or more groups selected from the following primer probe groups: Primer probe set 1: The sequences are shown in SEQ ID NOs: 1 to 3, which are used to detect the upstream and downstream primers and probes of the gene qnrA; Primer probe set 2: The sequences are shown in SEQ ID NOs: 4 to 6, which are used to detect upstream and downstream primers and probes of the gene qnrB; Primer probe set 3: The sequences are shown in SEQ ID NOs: 7 to 9, which are used to detect the upstream and downstream primers and probes of the gene qnrS; Primer probe set 4: the sequences are shown in SEQ ID NOs: 10 to 12, which are used for detecting the upstream and downstream primers and probes of the gene ErmA; Primer probe set 5: the sequences are shown in SEQ ID NOs: 13 to 15, which are used for detecting upstream and downstream primers and probes of the gene ErmB; Primer probe set 6: the sequences are shown in SEQ ID NOs: 16 to 18, which are used for detecting upstream and downstream primers and probes of gene dfrA1; Primer probe set 7: the sequences are shown in SEQ ID NOs: 19 to 21, which are used to detect upstream and downstream primers and probes of gene dfrA5; Primer probe set 8: The sequences are shown in SEQ ID NOs: 22 to 24, which are upstream and downstream primers and probes for detecting the gene tet (M); Primer probe set 9: The sequences are shown in SEQ ID NOs: 25 to 27, which are used for detecting upstream and downstream primers and probes of the gene tet (B).

2. The nucleic acid composition according to claim 1, characterized in that It also includes a primer probe set 10: upstream and downstream primers and probes for detecting GAPDH, whose sequences are shown in SEQ ID NOs: 28-30.

3. A kit, characterized in that: It comprises the nucleic acid composition according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The probes in the primer probe set for detecting qnr gene, the primer probe set for detecting dfr gene, the primer probe set for detecting ErmA gene, the primer probe set for detecting ErmB gene, the primer probe set for detecting tet(M) gene and the primer probe set for detecting 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 qnrA, qnrB and qnrS genes; the dfr gene includes at least one of dfrA1 and dfrA5 genes; Optionally, the probes in the primer probe set 10 are labeled with a fluorescent reporter group and are different from the fluorescent reporter groups labeled with the probes in the primer probe sets 1 to 9 and do not interfere with each other; Optionally, the probes in primer probe sets 1 to 3 are labeled with the same fluorescent reporter group; Optionally, the probes of primer probe sets 6 and 7 are labeled with the same fluorescent reporter group.

5. The kit according to claim 4, characterized in that The fluorescent reporter group includes one or more of FAM, HEX, VIC, ROX, CY5, CY7, ATTO425 and Quasar 705.

6. The kit according to any one of claims 3 to 5, characterized in that The kit further comprises a positive quality control product and / or a negative quality control product; And / or, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, UNG enzyme, DNA polymerase, dNTP(U)s, PCR buffer and Mg 2+ At least one of .

7. A method for detecting drug resistance of environmental pathogens, characterized in that: The method comprises the following steps: Extracting nucleic acid from the sample to be tested to obtain a nucleic acid sample; Performing PCR amplification on the nucleic acid sample using the nucleic acid composition according to claim 1 or 2, or the kit according to any one of claims 3 to 6; and, Obtain and analyze results.

8. The method according to claim 7, characterized in that 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.

9. The method according to claim 7 or 8, characterized in that In each reaction system for PCR amplification, the initial system concentration of the DNA polymerase is 0.1 U / μL~0.5 U / μL, the initial system concentration of the UNG enzyme is 0.005 U / μL~0.015 U / μL, and the Mg is 0. 2+ The initial system concentration of is 5 mM~10 mM, and the initial system concentration of dNTP(U)s is 2 mM~3 mM.

10. The method according to claim 9, characterized in that The procedure for performing 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, cycle 35 times ~ 50 times.

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