CN112522378B - Kit for detecting MCR gene, detection method and application thereof - Google Patents
Kit for detecting MCR gene, detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting MCR genes, a detection method and application thereof. The kit comprises the following 5 groups of primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2, the primer pairs shown in SEQ ID No. 3 and SEQ ID No. 4, the primer pairs shown in SEQ ID No. 5 and SEQ ID No. 6, the primer pairs shown in SEQ ID No. 7 and SEQ ID No. 8 and the primer pairs shown in SEQ ID No. 7 and SEQ ID No. 9. The invention willmcr‑8And (3) withmcr‑10After the unified pre-primer sequence SEQ ID No. 7 is adopted for the specific primer pair, not only the differentiation and parting of small differences are finished through an HRM method, but also a multiplex PCR system is effectively simplified, and a good amplification effect can be achieved; at the same time for mixing withmcr‑1Andmcr‑3the clinical samples of the invention can be rapidly and effectively detected and identified by adopting the multiplex PCR kit. Makes up for the blank that a multiplex PCR kit for realizing the effective typing and detection of all MCR genes in clinical samples does not appear in the market at present, and particularly can effectively identify typingmcr‑8Andmcr‑10blank of a functional multiplex PCR kit.
Description
Technical Field
The invention belongs to the fields of medical and health and microorganism detection, and particularly relates to a kit for detecting an MCR gene, a detection method and application thereof.
Background
Polymyxin (polymyxin) is found in Paenibacillus polymyxa @Paenibacilluspolymyxa) Among these, five kinds of antibiotic polypeptides, colistin and polymyxin B (PMB) are the most commonly used polymyxins in clinic, and A, B, C, D, E are used as antibiotic polypeptides in the culture solution. The clinical use of polymyxin is severely limited due to its neurotoxicity, but polymyxin is widely used in the livestock industry in asia, europe and north america.
In recent years, the metastatic resistance MCR (mobile colistin resistance) gene to polymyxin resistance has been found in clinical, breeding plants, animal foods, and even healthy human samples. Self-supportingmcr-1Since the first report, 10 MCR genes are found in clinical and environmental samples for many times and are distributed worldwidemcr-1、mcr-2、mcr-3、mcr- 4、mcr-5、mcr-6、mcr-7、mcr-8、mcr-9、mcr-10) Is reported to find that each gene may contain a plurality of gene variation types, whereinmcr-1Up to 30 types of genetic variation have been found. Due to the wide use of polymyxins in the farming industry, polymyxin drug resistance genes enter healthy people through the food chain from animal foods to people. The transferability, high carrying rate and wide spread distribution of MCR genes have attracted great social attention and anxiety. Coliform bacteria resistant to polymyxin are constantly present in medical clinics and spread in parallel, becoming a focus of attention for global health workers.
However, only those found in clinical specimens at presentmcr-1、mcr-3、mcr-8Andmcr-10a sample alone, andmcr-1andmcr-3the simultaneous presence of samples indicates that part of MCR genotypes do not have the ability to spread among the population, but that the "superbacteria" carrying MCR types that are prevalent in the clinic seriously threatens the role of polymyxin as the "last line of defense" in clinical treatment. However, for the clinical detection and early warning of the MCR gene and specific typing, the traditional microorganism culture method and drug sensitivity test method cannot be popularized and used clinically on a large scale due to the long time consumption, complicated operation process, high requirements on instruments and equipment and the like. Thus, a kind of energy is soughtA method for effectively identifying superbacteria carrying various MCR genes is urgent, and simultaneously clinically identifying and monitoring the type of high-risk MCR currently popular in clinicmcr-1、mcr-3、mcr-8Andmcr-10) Is particularly important.
Disclosure of Invention
The primary object of the present invention is to provide a kit for detecting a mobile polymyxin drug resistance MCR gene.
It is another object of the present invention to provide a method for detecting MCR gene for non-disease diagnosis and therapeutic use.
It is still another object of the present invention to provide an application of the kit for detecting MCR gene as described above in non-disease diagnosis and therapeutic use in clinical or environmental monitoring.
It is a further object of the present invention to provide a multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples.
The aim of the invention is achieved by the following technical scheme:
in a first aspect, a kit for detecting an MCR gene comprises 5 primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2, 3 and 4, 5 and 6, 7 and 8, and 7 and 9.
As a preferred embodiment of the kit for detecting MCR gene provided by the present invention, the kit further comprises: 2 XPCR buffer, taq enzyme, dNTPs, mgCl 2 Fluorescent dye EvaGreen, FTA test paper, 10% SDS solution, TE buffer.
As a preferred implementation mode of the kit for detecting the MCR gene, which is provided by the invention, the kit is a kit of 20-50 mu L fluorescent PCR reaction system, and the components and the contents thereof are as follows:
| PCR reaction system | |
| Component (A) | Final concentration |
| PCR buffer | 1× |
| Mg 2＋ Concentration of | 2.0mmol/L |
| dNTPs (containing dUTP) | 0.15mmol/L |
| Taq enzyme | 2U |
| 20 XEvaGreen fluorescent dye | 1× |
| SEQ ID No. 1 primer | 0.6μmol/L |
| Primers shown in SEQ ID No. 2 | 0.6μmol/L |
| Primers shown in SEQ ID No. 3 | 0.05μmol/L |
| Primers shown in SEQ ID No. 4 | 0.05μmol/L |
| SEQ ID No. 5 primer | 0.15μmol/L |
| SEQ ID No. 6 primer | 0.15μmol/L |
| SEQ ID No. 7 primer | 0.20μmol/L |
| SEQ ID No. 8 primer | 0.10μmol/L |
| Primers shown in SEQ ID No. 9 | 0.10μmol/L |
| DNA template | 2μL |
| Moisturizing to | 20~50μL |
In a second aspect, a method for detecting MCR genes for non-disease diagnostic and therapeutic uses, comprising the steps of:
(1) Extracting DNA in a sample to be detected;
(2) Respectively configuring PCR reaction systems with the 5 groups of primer pairs;
(3) Adding the DNA extracted in the step (1) as a template into a PCR reaction system, performing PCR amplification reaction and performing fluorescence PCR detection; when the Tm value of the actual dissolution curve of the PCR reaction system is consistent with the Tm value of the standard dissolution curve of any MCR-like gene, the sample to be tested has the MCR gene; wherein any of the MCR genes refers tomcr-1、mcr-3、mcr-8Andmcr-10any of the classes of genes.
As a preferred embodiment of the method for detecting MCR gene for non-disease diagnosis and treatment provided by the present invention, step (1) specifically comprises the steps of: taking 20 mu L of a sample to be tested, placing the sample into a centrifuge tube with a filter membrane with the diameter of 2.0mmFTA, drying at 56 ℃, adding 200 mu L of 10% SDS solution into the dried filter membrane, boiling for 10min, washing for 2 times by using a special buffer solution for FTA, washing for two times by using a TE buffer solution, and drying at 56 ℃ to be used as a PCR reaction template.
As a preferred embodiment of the method for detecting MCR gene for non-disease diagnosis and treatment provided by the present invention, the kit further comprises: 2 XPCR buffer, taq enzyme, dNTPs, mgCl2, fluorescent dye EvaGreen, FTA test paper, 10% SDS solution, TE buffer.
As a preferred implementation mode of the method for detecting the MCR gene for non-disease diagnosis and treatment, which is provided by the invention, the PCR reaction system is a 20-50 mu L fluorescent PCR reaction system, and the components and the content thereof are as follows:
| PCR reaction system | |
| Component (A) | Final concentration |
| PCR buffer | 1× |
| Mg 2＋ Concentration of | 2.0mmol/L |
| dNTPs (containing dUTP) | 0.15mmol/L |
| Taq enzyme | 2U |
| 20 XEvaGreen fluorescent dye | 1× |
| SEQ ID No. 1 primer | 0.6μmol/L |
| Primers shown in SEQ ID No. 2 | 0.6μmol/L |
| Primers shown in SEQ ID No. 3 | 0.05μmol/L |
| Primers shown in SEQ ID No. 4 | 0.05μmol/L |
| SEQ ID No. 5 primer | 0.15μmol/L |
| SEQ ID No. 6 primer | 0.15μmol/L |
| SEQ ID No. 7 primer | 0.20μmol/L |
| SEQ ID No. 8 primer | 0.10μmol/L |
| Primers shown in SEQ ID No. 9 | 0.10μmol/L |
| DNA template | 2μL |
| Moisturizing to | 20~50μL |
In a third aspect, a kit for detecting MCR gene as described above is used for detecting MCR gene for non-disease diagnosis and therapeutic use in clinical or environmental monitoring.
In a fourth aspect, a multiplex PCR primer set for detecting and identifying MCR genotyping in a clinical sample, comprising: primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2, primer pairs shown in SEQ ID No. 7 and SEQ ID No. 8, and primer pairs shown in SEQ ID No. 7 and SEQ ID No. 9.
As a preferred embodiment of the multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples provided by the invention, the multiplex PCR primer set further comprises 2 primer pairs shown in SEQ ID No. 3 and SEQ ID No. 4 and primer pairs shown in SEQ ID No. 5 and SEQ ID No. 6.
Compared with the prior art, the invention has the following advantages and effects:
(1) Aiming at the type of MCR popular in clinical samples at presentmcr-1、mcr-3、mcr-8Andmcr-10) The prior art has no relevant report on the detection and identification typing multiplex PCR detection technology, and when a conventional multiplex PCR detection scheme is adopted (i.e. respectively designedmcr-1、mcr-3、mcr-8Andmcr-10the specific primer pairs of (a) are different, and the mutual interference exists between the primer pairs when the clinical samples are detected, so that the amplification efficiency is reduced, an effective amplification curve and a dissolution curve can not be obtained, the products in the samples can not be effectively detected, and the new technical problem that the products can not be distinguished can not be obtained is solved (see comparative example 1). In the HRM method (high resolution melting curve analysis), the difference of single base is distinguished by the difference of melting curve Tm value through the design of the primer, so that a technician can select amplified fragments with a longer distance during the design of the primer, namely, similar fragment sequences are avoided as far as possible in the detection and typing of multiplex PCR, so that the difference of the melting curve is larger and easier to distinguish. However, the present inventors have used the contrary design concept to make use of the difficulty in designing the original primermcr-8And (3) withmcr-10The specific primer pair of (a) adopts a uniform pre-primer sequence SEQ ID No. 7, and the unexpected discovery is that: will bemcr-8And (3) withmcr-10After the unified pre-primer sequence SEQ ID No. 7 is adopted for the specific primer pair, not only the differentiation and typing of small differences are finished through an HRM (high resolution melting curve analysis), but also a multiple PCR system is effectively simplified, and a good amplification effect can be achieved; at the same time for mixing withmcr-1Andmcr-3the clinical samples of the invention can be rapidly and effectively detected and identified by adopting the multiplex PCR kit. Make up that the effective typing and detection of all MCR genes in clinical samples are not realized in the market at presentBlank for multiplex PCR kit for detection, especially for efficient identification of typingmcr-8Andmcr-10blank of a functional multiplex PCR kit.
(2) The kit is applied to clinical scenes, can effectively detect MCR genes, can effectively identify all types of the MCR genes popular in clinical samples, and can rapidly trace the source to know main popular types.
(3) The kit is particularly suitable for medical clinical monitoring scenes, can rapidly, simply, conveniently and effectively detect and early warn MCR genes in stool samples, monitors PCR products in real time, greatly reduces the manpower and material resource consumption for monitoring movable polymyxin drug-resistant genes, and simplifies the spot check and monitoring of large-scale clinical samples in daily production and life, so the kit and the detection method thereof have larger application value in medical clinical monitoring scenes, and can also be applied to MCR types with high monitoring and early warning dangers in environmental monitoring scenesmcr-1、mcr-3、mcr-8Andmcr-10）。
(4) The primer provided by the invention has no amplification signal to a detection sample without mcr, which indicates that the primer has good specificity.
Drawings
FIG. 1 is a SeqMan sequence analysis of 46 MCR genes in NCBI database;
FIG. 2 shows a test using the kit of the present inventionmcr-1The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 3 and the primer shown in SEQ ID No. 4, and comprises the dissolution curve of three positive templates, and Tm #mcr-1)=84.8℃；
FIG. 3 shows a test using the kit of the present inventionmcr-3The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 5 and the primer shown in SEQ ID No. 6, and comprises the dissolution curve of three positive templates, and Tm #mcr-2)= 87.8℃；
FIG. 4 shows a test using the kit of the present inventionmcr-8The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 7 and the primer shown in SEQ ID No. 8, comprises the dissolution curves of three positive templates,Tm(mcr-8)= 88.2℃；
FIG. 5 shows a test using the kit of the present inventionmcr-10The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 7 and the primer shown in SEQ ID No. 9, and comprises the dissolution curve of three positive templates, and Tm #mcr-10)= 88.5℃；
FIG. 6 is a schematic view of a device withmcr-1、mcr-3The mixed template sample of the kit is adopted to detect the obtained dissolution curve;
FIG. 7 is a schematic view of a device havingmcr-1、mcr-3、mcr-8Andmcr-10the mixed template sample of the kit is adopted to detect the obtained dissolution curve;
FIG. 8 is a schematic representation of detection using multiplex PCRmcr-8The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 10 and the primer shown in SEQ ID No. 11, and comprises the dissolution curve of three positive templates, and Tm #mcr-8)= 87.7℃；
FIG. 9 is a schematic representation of detection using multiplex PCRmcr-10The standard dissolution curve obtained by the specific primer pair is the primer shown in SEQ ID No. 12 and the primer shown in SEQ ID No. 13, and comprises the dissolution curve of three positive templates, and Tm #mcr-10)= 75.1℃；
Wherein the ordinate of the dissolution profile is the Derivative of Derivative, in particular the change in fluorescence intensity, deriving the change in temperature, i.e. d (Fluorescence)/d (T) or d (fluorescence value)/d (temperature); the abscissa is the Temperature (. Degree. C.), i.e., each Temperature point of the dissolution process. At the peak is the dissolution temperature Tm.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
1. Primer sequence design in the kit: by analyzing the sequences of the MCR genes in all known clinical samples, carrying out sequence analysis on 59 genes by adopting SeqMan (figure 1), finding out a highly conserved segment through analysis, selecting a segment which has no secondary structure and is highly conserved through premier 3.0, and designing a plurality of groups of primers, wherein the length of the primers is generally about 20 bases, and the primers have no complementary sequences. Primer list 1 is shown below:
TABLE 1 primer and amplified fragment sequence List (both 5 '-3')
| Target gene | Primer sequence 5'-3' | SEQ ID | Product length (bp) |
| Universal primers | F: 5’AGCTACGTACTCTGCGTGTGCTA-3’ | 1 | |
| R: 5’GACGGTATTGACTGCTGTCTCG-3’ | 2 | ||
| mcr-1 | F:5’AGCACACGCAGAGTACGTAGCTCTCGTTGGCTTAGATGACT-3’ | 3 | mcr-1:216bp |
| R: 5’CGAGACAGCAGTCAATACCGTCAAGTGCGAACATCAGTCC-3’ | 4 | ||
| mcr-3 | F:5’ATATGGGGAGAAAGGAGTTTGAT-3’ | 5 | mcr-3:207bp |
| R:5’CACATGCTATGACGAGGTTGT-3’ | 6 | ||
| mcr-8 | F:5’TAGCACACGCAGAGTACGTAGCTCTGGTTAATACCTACGACAATAC-3’ | 7 | mcr-8:223bp |
| R:5’CAAACACACATCCCGATG-3’ | 8 | ||
| mcr-10 | F:5’TAGCACACGCAGAGTACGTAGCTCTGGTTAATACCTACGACAATAC-3’ | 7 | mcr-10:241bp |
| R:5’CGAGACAGCAGTCAATACCGTCCAGACGCACATCCCGATG-3’ | 9 |
And (3) establishing and optimizing a reaction system: the target region template used in the establishment and optimization of the reaction system is obtained by the following method: respectively take outmcr-1、mcr-3、mcr-8Andmcr-10the escherichia coli and klebsiella pneumoniae strains carrying strains are recovered and cultured for 48 hours, 1mL of culture solution is taken and is re-dissolved to 1mL after being washed by sterile water, a phenol-chloroform method or a kit is adopted to extract genome nucleic acid respectively, the primers are used for PCR amplification, and the Ct value between 24 and 27 is taken as a template for optimizing a subsequent reaction system. Wherein the initial reaction system is shown in Table 2:
TABLE 2 initially designed PCR reaction System
| PCR reaction system | |
| Component (A) | Final concentration |
| PCR buffer | 1× |
| Mg 2＋ Concentration of | 2.0mmol/L |
| dNTPs (containing dUTP) | 0.15mmol/L |
| Taq enzyme | 2U |
| 20 XEvaGreen fluorescent dye | 1× |
| SEQ ID No. 1 primer | 0.6μmol/L |
| Primers shown in SEQ ID No. 2 | 0.6μmol/L |
| Primers shown in SEQ ID No. 3 | 0.05μmol/L |
| Primers shown in SEQ ID No. 4 | 0.05μmol/L |
| SEQ ID No. 5 primer | 0.15μmol/L |
| SEQ ID No. 6 primer | 0.15μmol/L |
| SEQ ID No. 7 primer | 0.20μmol/L |
| SEQ ID No. 8 primer | 0.10μmol/L |
| Primers shown in SEQ ID No. 9 | 0.10μmol/L |
| Template | 2μL |
| Moisturizing to | 40μL |
2.1 optimization of primer concentration: in the reaction system, the primer concentration is continuously diluted from 0.1 mu mol/L to 0.8 mu mol/L, and then detection is carried out, and the optimal primer final concentration is determined as the reaction through analysis and comparison of test results: the concentration of the universal primers (primers shown as SEQ ID No. 1 and SEQ ID No. 2) is 0.6 mu mol/L, and the concentration of the specific primers (primers shown as SEQ ID No. 3 to SEQ ID No. 10) is 0.05-0.20 mu mol/L.
2.2 optimization of magnesium ion concentration: mgCl was added to the reaction system under the conditions of 0.2. Mu. Mol/L final primer concentration and the other conditions in the reaction system as shown in Table 2 2 The concentration of the magnesium ions in the reaction system of the kit is increased from 1 mmol/L to 2.5 mmol/L by 0.5 mmol/L, and 2.5 mmol/L is selected as the concentration of the magnesium ions in the reaction system of the kit through repeated experiments.
2.3 optimization of Taq DNA polymerase (Taq enzyme) dosage: under the precondition that the final concentration of the primer is 0.2 mu mol/L, the final concentration of magnesium ions is 2.5 mmol/L and other conditions in the reaction system are the same as those in Table 2, 2U is selected as the amount of Taq enzyme in the reaction system of the kit by comparing the optimized experimental results of the amount (calculated by Unit) of Taq enzyme.
2.4 optimization of dNTPs (deoxyribonucleoside triphosphates) concentration: on the premise that the final concentration of the primer is 0.2 mu mol/L, the final concentration of magnesium ions is 2.5 mmol/L and other conditions in the reaction system are the same as those in Table 2, detection is carried out by using dNTPs with different concentrations, and 0.2 mmol/L is selected as the using amount of dNTPs in the reaction system of the kit after comprehensive evaluation.
Because the primer and target fragments related by the method are numerous, the unsaturated dye SYBRGREEN has larger error and can not distinguish single-gene difference, the method adopts the saturated dye EvaGreen as the fluorescent dye in the PCR process. Evagreen has little interference to qPCR, no 'dye redistribution', saturated intercalation into DNA double strand, and far better resolution effect than SYBR Green. The above primers and fluorescent dye were used to build a reaction system, and finally, the fluorescent PCR reaction system was determined to be 40. Mu.L, and the required components and the corresponding concentrations are shown in Table 3.
TABLE 3 optimized PCR reaction System
| PCR reaction system | |
| Component (A) | Final concentration |
| PCR buffer | 1× |
| Mg 2＋ Concentration of | 2.0mmol/L |
| dNTPs (containing dUTP) | 0.15mmol/L |
| Taq enzyme | 2U |
| 20 XEvaGreen fluorescent dye | 1× |
| SEQ ID No. 1 primer | 0.6μmol/L |
| Primers shown in SEQ ID No. 2 | 0.6μmol/L |
| Primers shown in SEQ ID No. 3 | 0.05μmol/L |
| Primers shown in SEQ ID No. 4 | 0.05μmol/L |
| SEQ ID No. 5 primer | 0.15μmol/L |
| SEQ ID No. 6 primer | 0.15μmol/L |
| SEQ ID No. 7 primer | 0.20μmol/L |
| SEQ ID No. 8 primer | 0.10μmol/L |
| Primers shown in SEQ ID No. 9 | 0.10μmol/L |
| Template | 2μL |
| Moisturizing to | 40μL |
Note that: when the fluorescence PCR reaction volumes are different, the reagents should be adjusted proportionally.
3. The detection method of the kit of the invention specifically comprises the following steps:
(1) Extracting DNA in a sample to be detected;
because the known movable MCR genes are all on plasmids, DNA in a sample can be extracted by a relatively violent method such as a boiling method, and the specific steps are as follows: taking 20 mu L of fresh sample to be tested, such as a fecal sample, placing the sample into a centrifuge tube with a filter membrane with the diameter of 2.0mm, drying the sample at 56 ℃, adding 200 microliters of 10% SDS solution into the dried filter membrane, boiling the sample for 10min, washing the sample with a buffer solution special for FTA for 2 times, washing the sample with a TE buffer solution for two times, and drying the sample at 56 ℃ to be used as a PCR reaction template.
(2) Configuring a PCR reaction system with primers according to Table 3;
(3) Adding the DNA extracted in the step (1) as a template into a PCR reaction system according to a table 3, performing PCR amplification reaction and fluorescence PCR detection, and performing high-resolution dissolution analysis on a PCR amplification product in the PCR detection, wherein the procedure is as follows: 95 ℃ 15s,60 ℃ 1min,95 ℃ 15s,60 ℃ 15s, dissolution rate of 0.2 ℃/s; when the actual dissolution curve Tm value of any one of the PCR-A reaction system and the PCR-B reaction system is consistent with the standard dissolution curve Tm value of any MCR gene, the sample to be tested has the MCR gene.
Wherein, the PCR amplification reaction conditions are as follows: 2min at 95 ℃ for 1 cycle; 95 ℃ for 5 sec, 60 ℃ for 40 sec, 40 cycles.
In this example, a real-time fluorescent quantitative PCR apparatus of model Applied Biosystems ABI 7500 manufactured by Semer Feier technology Co., ltd was used for the fluorescent PCR detection, but the present invention is not limited thereto. Selecting a detection channel of an instrument: in the fluorescent PCR reaction, the collection of fluorescent signals of a reaction tube in the used instrument should be set, and the specific setting method of the selected fluorescent detection channel is different from instrument to instrument and reference should be made to the instruction of the instrument.
Example 2
The primer pair in the table 1 in the example 1 is selected, and the genome DNA of bacteria in various source samples is extracted from the culture solution of 33 strains of bacteria to be detected and other non-target strains by using a phenol-chloroform method or a kit. Wherein the standard strain is a food safety and detection laboratory purchase or preservation strain of university of south China university; the isolated strain is derived from the excrement of various farms, farmers markets and healthy people in Shenzhen city, and adopts Shen,et al.(2018) The presence or absence of the MCR gene in the strain is identified, and the strain is identified by 16S sequencing or mass spectrometry.
In a 40. Mu.L fluorescent PCR reaction system (prepared as in Table 3), 2. Mu.L of the genomic DNA of the different strains extracted above was added, and fluorescent PCR detection was performed according to the PCR reaction conditions in example 1.
The experimental results are shown in table 4 below.
TABLE 4 fluorescent quantitative qPCR results for different strains
| Strain | Source b | qPCR | Shen, et al.(2018) c |
| Target strain (n=14) | |||
| Klebsiella genusKlebsiella(n=3) | |||
| Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis | Fecal isolate 1 | ＋ a | ＋ |
| Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis | Fecal isolate 2 | ＋ | ＋ |
| Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis | Fecal isolate 3 | ＋ | ＋ |
| Escherichia coli genusEscherichiacoli(n=11) | ＋ | ＋ | |
| Coli bacteriumEscherichiacoli | Fecal isolate 1 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 2 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 3 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 4 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 5 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 6 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 7 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 8 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 9 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 10 | ＋ | ＋ |
| Coli bacteriumEscherichiacoli | Fecal isolate 11 | ＋ | ＋ |
| Non-target strain (n=19) | |||
| Enterobacter genusEnterbacter(n=4) | |||
| Enterobacter cloacaeEnterbacter cloacae | CICC No.21539 | - | - |
| Enterobacter sakazakiiEnterbactersakazakii | ATCC No.29544 | - | - |
| Enterobacter aerogenesEnterbacteraerogenes | CICC No.10293 | - | - |
| Enterobacter aerogenesEnterobacteraerogenes | ATCCNo.13408 | - | - |
| Escherichia coli genusEscherichiacoli(n=12) | |||
| Coli bacteriumE. coli O157:H7 | CICC No.21530 | - | - |
| Coli bacteriumE. coli O157:H7 | SZCIQ No.13813 | - | - |
| Coli bacteriumE. coli O157:H7 | ADCPC No.931 | - | - |
| Coli bacteriumEscherichiacoli | ATCC No.9637 | - | - |
| Coli bacteriumEscherichiacoli | SZCIQNo.eco3 | - | - |
| Coli bacteriumEscherichiacoli | SZCIQNo.eco5 | - | - |
| Coli bacteriumEscherichiacoli | SZCIQNo.jm109 | - | - |
| Coli bacteriumEscherichiacoli | CMCC No.44104 | - | - |
| Coli bacteriumEscherichiacoli | CMCC No.44105 | - | - |
| Coli bacteriumEscherichiacoli | CMCC No.44106 | - | - |
| Coli bacteriumEscherichiacoli | CMCC No.44111 | - | - |
| Coli bacteriumEscherichiacoli | CGMCC No.1.129 | - | - |
| Klebsiella genusKlebsiella(n=2) | |||
| Klebsiella pneumoniaeKlebsiellapneumonia | CMCC No.46102 | - | - |
| Klebsiella pneumoniaeKlebsiellapneumonia | CICC No.10781 | - | - |
| Salmonella genusSalmonella（n=1） | - | - | |
| Salmonella dublinSalmonella dublin | GZCDCNo.dbl1a | - | - |
a.+ -., positive; negative of
Atcc, american type culture collection, university of marasa denver, mevirginia, university of denver, post code 10801; CMCC, chinese medical microbiological bacterial collection center, bio-pharmaceutical industry base, huatuolu 31, postal code 102629, of the university district of beijing; CGMCC, china general microbiological culture Collection center, beijing city, kogyo district, north Chen Xiyu No. 1, 3, and post code 100101; CICC, industrial microorganism strain preservation management center, no. 6 building of 24 th yard of Jiuxian bridge in the Kogyang area of Beijing city, post code: 100015; SZCIQ, shenzhen exit and entrance inspection and quarantine bureau, shenzhen city forda area forciqu 1011 number, zip code 518000; ADCPC, chongqing animal disease control and prevention center, chongqing Chongjiang two-way No. 8, post code: 400042. wherein the strain from ATCC, CMCC, CGMCC, CICC is commercially available. Strains from SZCIQ and adcc are donated by these mechanisms separately; these strains are disclosed in the literature "Xing-long Xiao, li Zhang, hui Wu et. al, simultaneous Detection ofSalmonella, literiamonostomies, andStaphylococcusaureusby Multiplex Real-Time PCR Assays Using High-Resolution Melting [ J ]. Food Analytical Methods, 2014,7 (10): 1960-1972".
c. MCR detection methods reference "Yingbo, s., hongwei, z., jiao, x., yongqiang, w., qijin, z., & Walsh, t.r., et al (2018) Anthropogenic and environmental factors associated with high incidence of MCR-1 carriage in humans across china.Nature Microbiology").
By detection, if the culture medium to be detected contains MCR variants (gene variants of MCR, namelymcr-1、mcr-3、mcr-8Andmcr-10) Any one or more of them shows a positive amplification curve, and the Tm value and the dissolution curve aremcr-1、mcr-3、mcr-8Andmcr-10any standard dissolution profileTm values match, whereinmcr-1、mcr-3、mcr-8Andmcr-10the standard dissolution curves of (2) are shown in FIGS. 2-5; if the culture solution to be detected does not contain MCR varians, no amplification signal exists, which indicates that the primer pair has good sensitivity and specificity. The detection of the strains in Table 4 shows that the strains containing the target genes are detected, and the non-target strains are negative in the amplification-free curve, thus the method has good specificity.
In order to determine the MCR species in the sample to be tested, it is necessary to identify the sample to be tested by means of a melting curve. Using the Tm values as the interpretation criteria, as shown in FIGS. 2 to 5, peaks of standard dissolution curves corresponding to different types of MCR genes, when the measured Tm values are 84.8, 87.8, 88.2, 88.5, the corresponding drug-resistant genes are respectivelymcr-1、mcr-3、mcr-8Andmcr-10the method has good resolution.
For simultaneous occurrence in clinical samplesmcr-1、mcr-3In the case of (2), the mixed DNA template was further examined, as shown in FIG. 6, whenmcr-1Andmcr-3in the presence of the same time, the PCR reaction system can obtain T m Bimodal dissolution profile with values of 84.8 ℃ and 87.8 ℃ and T m (mcr-1) And T is m (mcr-3) And consistent.
In conclusion, the detection method of the kit of the invention not only can effectively detect the MCR found in all clinical samples in the samples to be detectedmcr-1、mcr-3、mcr-8Andmcr-10) When present in a clinical samplemcr-1And (3) withmcr-3In this case, the type of the MCR gene can be detected and identified effectively.
Further, in order to verify the kit of the present invention, the kit may be usedmcr-1、mcr-3、mcr-8Andmcr-10the inventor can artificially detect and pre-warn the MCR genes effectively by any combinationmcr-1、mcr-3、mcr-8Andmcr-10the mixed DNA templates of 4 mcr gene types are formed by mixing, and a bimodal dissolution curve (shown in figure 7) with Tm value of 84.8 ℃ and 88.5 ℃ is obtained by the PCR reaction system of the invention, and the Tm (mcr-1) and Tm (mcr-10) are consistent, so that whenmcr-1、mcr-3、mcr-8Andmcr-10when the kit is simultaneously present, the functions of a plurality of MCR genotypes can be detected simultaneously, and the function of effective detection and early warning is achieved.
Example 3
The excrement is 112 parts of fresh excrement samples of healthy people, which are collected by adopting the Huada Zhi excrement collection set, are suspended in a preservation solution and are preserved at the temperature of 4 ℃ for no more than one week.
The primer set of Table 1 in example 1 was selected, and total DNA of fecal bacteria extracted by the boiling method described in example 1 and the use of Shen described in example 2 was used,et althe DNA templates extracted by the method of (2018) were used as different DNA templates for PCR detection comparison. As a result, it was found that the number of the samples of the Shen,et althe method of (2018) detected a positive rate of 12.5% (14/112), whereas the method detected a positive rate of 13.4% (15/112). The method has the advantage that the DNA in the sample to be detected is extracted by adopting the boiling method and is used as a template, so that the method has good detection rate.
It should be noted that: FIGS. 2-7 are all 2-3 parallel experiments, and although the dissolution curves in each graph are not overlapped, the Tm of each dissolution curve automatically calculated according to the software of a PCR detection instrument is the same, so that the subtle differences of the forms of the 3 dissolution curves of the 3 parallel experiments of the same DNA sample are normal in the field.
Comparative example 1
This comparative example differs from example 1 in that: table 1 was modified as table 5 below, specifically,
TABLE 5 primer and amplified fragment sequence List (both 5 '-3')
| Target gene | Primer sequence 5'-3' | SEQ ID | Product length (bp) |
| Universal primers | F: 5’AGCTACGTACTCTGCGTGTGCTA-3’ | 1 | |
| R: 5’GACGGTATTGACTGCTGTCTCG-3’ | 2 | ||
| mcr-1 | F:5’AGCACACGCAGAGTACGTAGCTCTCGTTGGCTTAGATGACT-3’ | 3 | 216bp |
| R: 5’CGAGACAGCAGTCAATACCGTCAAGTGCGAACATCAGTCC-3’ | 4 | ||
| mcr-3 | F:5’ATATGGGGAGAAAGGAGTTTGAT-3’ | 5 | 207bp |
| R:5’CACATGCTATGACGAGGTTGT-3’ | 6 | ||
| mcr-8 | F:5’AGCTACGTACTCTGCGTGTGCTACAGTCTGCCAATATTTCTTTTCTGCT-3’ | 10 | 214bp |
| R: 5’GACGGTATTGACTGCTGTCTCGGTTTTGCACCATGCTGCGGTC -3’ | 11 | ||
| mcr-10 | F:5’AGCTACGTACTCTGCGTGTGCTACGCTACCTGCTCAAACCCTTCTTTGCCCTGT-3’ | 12 | 232bp |
| R: 5’GACGGTATTGACTGCTGTCTCGGAATGCCCATGAAGACCAGCCACAGCA -3’ | 13 |
Will be described in Table 5mcr-1、mcr-3、mcr-8Andmcr-10when the combination of each specific primer and the universal primer is used for single PCR detection, the target fragment can be specifically identified in a single PCR system, whereinmcr-8Andmcr-10the Tm values obtained by single PCR detection are 87.7 and 75.1 respectively, andmcr-8andmcr-10tm value of standard dissolution curve is consistentmcr-8Andmcr-10the standard dissolution curves of (a) are shown in FIGS. 8 and 9), the corresponding drug resistance genes are respectivelymcr-8Andmcr-10. To better facilitate typing by Tm values, the method is aimed atmcr-8Andmcr-10the primer pairs of (a) are selected for amplified fragments that are far apart. However, when the primer set combinations of table 5 were applied to a multiplex PCR system, it was found that there was a mutual interference between the primer sets, resulting in a decrease in amplification efficiency, and an effective amplification curve and dissolution curve could not be obtained, and products in the sample could not be detected effectively, and typing could not be distinguished between the products.
Although there are multiple PCR techniques for detection and early warning of various types of MCR genes in the prior art, primers for detecting each type of MCR gene are different and amplified fragments far apart are selected so as to facilitate detection or typing by the Tm values of the HRM method (high resolution melting curve analysis). But specific to the type of MCR currently popular in clinicmcr-1、mcr-3、mcr-8Andmcr-10) Is subjected to detection, identification and typing, and is screened outmcr-1、mcr-3、mcr-8Andmcr-10the corresponding specific primer pairs (the single PCR detection can well detect and identify the MCR genes) can be combined, and mutual interference exists among the primer pairs when the multiplex PCR detection is carried out, so that the amplification efficiency is reduced, an effective amplification curve and a dissolution curve can not be obtained, products in a sample can not be effectively detected, and the parting can not be distinguished among the products.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A method for detecting MCR gene for non-disease diagnosis and therapeutic use, characterized in that the method for detecting MCR gene comprises the steps of:
(1) Extracting DNA in a sample to be detected;
the method comprises the following specific steps: taking 20 mu L of fresh sample to be tested, placing the sample into a centrifuge tube with a filter membrane with the diameter of 2.0mmFTA, drying at 56 ℃, adding 200 mu L of 10% SDS solution into the dried filter membrane, boiling for 10min, washing for 2 times by using a special buffer solution for FTA, washing twice by using a TE buffer solution, and drying at 56 ℃ to serve as a PCR reaction template;
(2) Configuring a PCR reaction system;
the PCR reaction system comprises the following components in percentage by weight:
(3) Adding the DNA extracted in the step (1) as a template into the PCR reaction system in the step (2), performing PCR amplification reaction and fluorescence PCR detection, and performing high-resolution melting analysis on a PCR amplification product in the PCR detection, wherein the procedure is as follows: 95 ℃ 15s,60 ℃ 1min,95 ℃ 15s,60 ℃ 15s, the melting rate is 0.2 ℃/s; when the Tm value of the actual melting curve in the PCR reaction system is consistent with the Tm value of the standard melting curve of any MCR-like gene, the sample to be detected has the MCR gene;
the MCR gene of any kind refers to any kind of MCR-1, MCR-3, MCR-8 and MCR-10 genes;
wherein, the PCR amplification reaction procedure is as follows: 2min at 95 ℃ for 1 cycle; 95℃for 5s and 60℃for 40 s.
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