阅读说明：本技术 一种检测mcr基因的试剂盒、检测方法及其应用 (Kit for detecting MCR gene, detection method and application thereof ) 是由 胡双芳 柯跃斌 吕子全 彭长凤 项秋梅 汪洋 沈建忠 于 2020-11-18 设计创作，主要内容包括：本发明公开了一种检测MCR基因的试剂盒、检测方法及其应用。该试剂盒包括如下的5组引物对,SEQ ID No:1和SEQ ID No:2所示的引物对、SEQ ID No:3和SEQ ID No:4所示的引物对、SEQ ID No:5和SEQ ID No:6所示的引物对、SEQ ID No:7和SEQ ID No:8所示的引物对以及SEQ ID No:7和SEQ ID No:9所示的引物对。本发明将mcr-8与mcr-10的特异性引物对采用了统一的前引物序列SEQ ID No:7后,不仅通过HRM法完成细小的差异的区分分型,还有效的简化了多重PCR体系,能达到良好的扩增效果；同时对于混合有mcr-1和mcr-3的临床样品采用本发明的多重PCR试剂盒也能够快速有效地检测及鉴别。弥补了目前市面上还没有出现实现临床样本中所有MCR基因的有效分型与检测的多重PCR试剂盒的空白,特别是能有效鉴别分型mcr-8和mcr-10功能的多重PCR试剂盒的空白。(The invention discloses a kit for detecting MCR gene, a detection method and application thereof. The kit comprises 5 groups of primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2, primer pairs as shown in SEQ ID No. 3 and SEQ ID No. 4, primer pairs as shown in SEQ ID No. 5 and SEQ ID No. 6, primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 9. The invention is to mcr‑8 And mcr‑10 after the specific primer pair of (1) adopts a unified pre-primer sequence SEQ ID No. 7, not only the differential typing of small differences is completed by an HRM method, but also a multiple PCR system is effectively simplified, and a good amplification effect can be achieved; at the same time as for mixing mcr‑1 And mcr‑3 the clinical samples can be quickly and effectively detected and identified by adopting the multiplex PCR kit. Makes up for the fact that the effective typing and detection of all MCR genes in clinical samples do not appear in the market at presentBlanks of the multiplex PCR kit, in particular for efficient differential typing mcr‑8 And mcr‑10 blank for functional multiplex PCR kit.)

1. A kit for detecting MCR genes is characterized by comprising 5 primer pairs, namely a primer pair shown in SEQ ID No. 1 and SEQ ID No. 2, a primer pair shown in SEQ ID No. 3 and SEQ ID No. 4, a primer pair shown in SEQ ID No. 5 and SEQ ID No. 6, a primer pair shown in SEQ ID No. 7 and SEQ ID No. 8 and a primer pair shown in SEQ ID No. 7 and SEQ ID No. 9.

2. The kit for detecting an MCR gene according to claim 1, further comprising: 2 XPCR buffer, Taq enzyme, dNTP, MgCl₂Fluorescent dye EvaGreen and FTA test paper, 10% SDS solution and TE buffer solution.

3. The kit for detecting the MCR gene according to claim 2, wherein the kit is a 20-50 μ L kit of a fluorescent PCR reaction system, and the components and the content thereof are as follows:

PCR reaction system Components Final concentration PCR buffer solution 1× Mg^2＋Concentration of 2.0mmol/L dNTPs (containing dUTP) 0.15mmol/L Taq enzyme 2U 20 × EvaGreen fluorescent dye 1× Primer shown as SEQ ID No. 1 0.6μmol/L Primer shown as SEQ ID No. 2 0.6μmol/L Primer shown as SEQ ID No. 3 0.05μmol/L Primer shown as SEQ ID No. 4 0.05μmol/L Primer shown as SEQ ID No. 5 0.15μmol/L Primer shown as SEQ ID No. 6 0.15μmol/L Primer shown as SEQ ID No. 7 0.20μmol/L Primer shown as SEQ ID No. 8 0.10μmol/L Primer shown as SEQ ID No. 9 0.10μmol/L DNA template 2μL Make up water to 20~50μL

。

4. A method for detecting the MCR gene for non-disease diagnostic and therapeutic use, comprising the steps of:

(1) extracting DNA in a sample to be detected;

(2) respectively configuring PCR reaction systems with 5 sets of primer pairs according to claim 1;

(3) adding the DNA extracted in the step (1) into a PCR reaction system as a template, carrying out PCR amplification reaction and carrying out fluorescence PCR detection; when the actual melting curve Tm value of the PCR reaction system is consistent with the standard melting curve Tm value of any MCR gene, the sample to be detected has the MCR gene; wherein any of said MCR genes refers tomcr-1、mcr-3、mcr-8Andmcr-10any kind of gene.

5. The method for detecting MCR gene for non-disease diagnosis and treatment according to claim 4, wherein the step (1) comprises the following steps: taking 20 mu L of sample to be detected, placing the sample to be detected in a centrifuge tube with a diameter of 2.0mm FTA filter membrane sheet, drying at 56 ℃, adding 200 mu L of 10% SDS solution into the dried FTA filter membrane sheet, boiling for 10min, washing for 2 times by using FTA special buffer solution, then washing for two times by using TE buffer solution, drying at 56 ℃ and then using as a PCR reaction template.

6. The method for detecting MCR gene for non-disease diagnostic and therapeutic use according to claim 4, wherein the kit further comprises: 2 XPCR buffer, Taq enzyme, dNTP, MgCl₂Fluorescent dye EvaGreen and FTA test paper, 10% SDS solution and TE buffer solution.

7. The method for detecting the MCR gene for non-disease diagnosis and treatment according to claim 4 or 5, wherein the PCR reaction system is a 20-50 μ L fluorescent PCR reaction system, and the components and the content thereof are as follows:

PCR reaction system Components Final concentration PCR buffer solution 1× Mg^2＋Concentration of 2.0mmol/L dNTPs (containing dUTP) 0.15mmol/L Taq enzyme 2U 20 × EvaGreen fluorescent dye 1× Primer shown as SEQ ID No. 1 0.6μmol/L Primer shown as SEQ ID No. 2 0.6μmol/L Primer shown as SEQ ID No. 3 0.05μmol/L Primer shown as SEQ ID No. 4 0.05μmol/L Primer shown as SEQ ID No. 5 0.15μmol/L Primer shown as SEQ ID No. 6 0.15μmol/L Primer shown as SEQ ID No. 7 0.20μmol/L Primer shown as SEQ ID No. 8 0.10μmol/L Primer shown as SEQ ID No. 9 0.10μmol/L DNA template 2μL Make up water to 20~50μL

。

8. Use of the kit for detecting MCR gene according to any one of claims 1 to 3 for non-disease diagnosis and treatment in clinical or environmental monitoring.

9. A multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples, comprising: the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2, the primer pair shown in SEQ ID No. 7 and SEQ ID No. 8 and the primer pair shown in SEQ ID No. 7 and SEQ ID No. 9.

10. The multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples according to claim 9, further comprising 2 sets of primer pairs, the primer pairs shown in SEQ ID Nos. 3 and 4 and the primer pairs shown in SEQ ID Nos. 5 and 6.

Technical Field

The invention belongs to the field of medical treatment and public health and microbial detection, and particularly relates to a kit for detecting MCR genes, a detection method and application thereof.

Background

Polymyxin (polymyxin) is found in Paenibacillus polymyxa (B.), (B.)Paenibacilluspolymyxa) Five antibacterial polypeptides in the culture solution, such as A, B, C, D, E, are available, wherein polymyxin E (colistin) and polymyxin B (PMB) are two types of polymyxin which are most commonly used in clinic. Clinical use is severely restricted due to its neurotoxicity, but polymyxin is widely used in livestock breeding in asia, europe and north america.

Transferable drug resistance MCR (mobile colistin resistance) genes resistant to polymyxin have been found in clinical, aquaculture, animal food, and even healthy population samples in recent years. Frommcr-1Since the first report, it has been found in clinical and environmental samples many times, there are 10 classes of MCR genes worldwide ((C))mcr-1、mcr-2、mcr-3、mcr- 4、mcr-5、mcr-6、mcr-7、mcr-8、mcr-9、mcr-10) Are reported to be found, and each gene may contain multiple gene variation types, whereinmcr-1Up to 30 types of genetic variation have been found. Due to the wide use of polymyxin in the breeding industry, polymyxin drug resistance genes enter healthy human populations through the food chain from animal food to human populations. The transferability, high carrying rate and large-scale distribution of the MCR gene are causing great social attention and uneasiness. Polymyxin-resistant enterobacteriaceae bacteria continue to emerge and spread in medical clinics, and become a focus of attention of global health workers.

However, only those currently found in clinical specimensmcr-1、mcr-3、mcr-8Andmcr-10a sample present alone, andmcr-1andmcr-3simultaneous samples demonstrating partial MCR genotypesWithout the ability to spread across the population, the "superbacteria" carrying the MCR types that are prevalent in the clinic severely threaten the role of polymyxin as the "last line of defense" in clinical treatment. However, for the detection and early warning and specific typing of MCR gene in clinic, the traditional microbial culture method and drug sensitive test method cannot be popularized and used in large scale in clinic due to long time consumption, complicated operation process, high requirements on instruments and equipment, and the like. Therefore, it is urgent to find a method for efficiently identifying "superbacteria" carrying various MCR genes, and at the same time, it is clinically possible to rapidly identify and monitor the high-risk type of MCR which is currently prevalent in the clinic: (mcr-1、mcr-3、mcr-8Andmcr-10) The detection method of (2) is particularly important.

Disclosure of Invention

The invention aims to provide a kit for detecting the drug resistance MCR gene of the polymyxin mobilephonate.

It is another object of the present invention to provide a method for detecting the MCR gene for non-disease diagnostic and therapeutic uses.

The invention also aims to provide an application of the kit for detecting the MCR gene in non-disease diagnosis and treatment in clinical or environmental monitoring.

Still another object of the present invention is to provide a multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the kit for detecting the MCR gene comprises 5 primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2, primer pairs as shown in SEQ ID No. 3 and SEQ ID No. 4, primer pairs as shown in SEQ ID No. 5 and SEQ ID No. 6, primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8, and primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 9.

As a preferred embodiment of the kit for detecting an MCR gene provided by the present invention, the kit further comprises: 2 XPCR buffer, Taq enzyme, dNTP, MgCl₂Fluorescent dyes EvaGreen and FTA assaysPaper, 10% SDS solution, TE buffer.

As a preferred embodiment of the kit for detecting the MCR gene provided by the invention, the kit is a 20-50 mu L kit of a fluorescent PCR reaction system, and the components and the contents thereof are as follows:

	PCR reaction system
		Components	Final concentration
PCR buffer solution	1×
		Mg2＋Concentration of	2.0mmol/L
dNTPs (containing dUTP)	0.15mmol/L
		Taq enzyme	2U
20 × EvaGreen fluorescent dye	1×
		Primer shown as SEQ ID No. 1	0.6μmol/L
Primer shown as SEQ ID No. 2	0.6μmol/L
		Primer shown as SEQ ID No. 3	0.05μmol/L
Primer shown as SEQ ID No. 4	0.05μmol/L
		Primer shown as SEQ ID No. 5	0.15μmol/L
Primer shown as SEQ ID No. 6	0.15μmol/L
		Primer shown as SEQ ID No. 7	0.20μmol/L
Primer shown as SEQ ID No. 8	0.10μmol/L
		Primer shown as SEQ ID No. 9	0.10μmol/L
DNA template	2μL
		Make up water to	20~50μL

In a second aspect, a method for detecting the MCR gene for non-disease diagnostic and therapeutic use, comprising the steps of:

(1) extracting DNA in a sample to be detected;

(2) respectively preparing PCR reaction systems with the 5 groups of primer pairs;

(3) adding the DNA extracted in the step (1) into a PCR reaction system as a template, carrying out PCR amplification reaction and carrying out fluorescence PCR detection; when the actual melting curve Tm value of the PCR reaction system is consistent with the standard melting curve Tm value of any MCR gene, the sample to be detected has the MCR gene; wherein any of said MCR genes refers tomcr-1、mcr-3、mcr-8Andmcr-10any kind of gene.

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 following steps: taking 20 mu L of sample to be detected, placing the sample to be detected in a centrifuge tube with a diameter of 2.0mm FTA filter membrane sheet, drying at 56 ℃, adding 200 mu L of 10% SDS solution into the dried FTA filter membrane sheet, boiling for 10min, washing for 2 times by using FTA special buffer solution, then washing for two times by using TE buffer solution, drying at 56 ℃ and then using as a PCR reaction template.

As a preferred embodiment of the method for detecting MCR gene for non-disease diagnosis and therapeutic use provided by the present invention, the kit further comprises: 2 XPCR buffer, Taq enzyme, dNTP, MgCl2, fluorescent dye EvaGreen and FTA test paper, 10% SDS solution, TE buffer.

As a preferred embodiment of the method for detecting the MCR gene for non-disease diagnosis and treatment provided by the invention, the PCR reaction system is a 20-50 muL fluorescent PCR reaction system, and the components and the contents thereof are as follows:

	PCR reaction system
		Components	Final concentration
PCR buffer solution	1×
		Mg2＋Concentration of	2.0mmol/L
dNTPs (containing dUTP)	0.15mmol/L
		Taq enzyme	2U
20 × EvaGreen fluorescent dye	1×
		Primer shown as SEQ ID No. 1	0.6μmol/L
Primer shown as SEQ ID No. 2	0.6μmol/L
		Primer shown as SEQ ID No. 3	0.05μmol/L
Primer shown as SEQ ID No. 4	0.05μmol/L
		Primer shown as SEQ ID No. 5	0.15μmol/L
Primer shown as SEQ ID No. 6	0.15μmol/L
		Primer shown as SEQ ID No. 7	0.20μmol/L
Primer shown as SEQ ID No. 8	0.10μmol/L
		Primer shown as SEQ ID No. 9	0.10μmol/L
DNA template	2μL
		Make up water to	20~50μL

In a third aspect, the kit for detecting the MCR gene is applied to the detection of the MCR gene for clinical or environmental monitoring, non-disease diagnosis and treatment.

In a fourth aspect, a multiplex PCR primer set for detecting and identifying MCR genotyping in clinical samples, comprising: the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2, the primer pair shown in SEQ ID No. 7 and SEQ ID No. 8 and the primer pair 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, the multiplex PCR primer set further comprises 2 primer pairs, namely a primer pair shown in SEQ ID No. 3 and a primer pair shown in SEQ ID No. 4 and a primer pair shown in SEQ ID No. 5 and a primer pair shown in SEQ ID No. 6.

Compared with the prior art, the invention has the following advantages and effects:

(1) against the type of MCR currently prevalent in clinical samples: (mcr-1、mcr-3、mcr-8Andmcr-10) The prior art has no related reports, and when a conventional multiplex PCR detection scheme is adopted (namely, the detection scheme is designed respectively)mcr-1、mcr-3、mcr-8Andmcr-10different primers) to detect clinical samples, so that the amplification efficiency is reduced, an effective amplification curve and a dissolution curve cannot be obtained, products in a sample cannot be effectively detected, and the products cannot be distinguished (see comparative example 1). In the HRM method (high resolution melting curve analysis), the difference of single base is distinguished by the difference of Tm values of melting curves through the design of primers, so technicians can select amplified fragments with long distance when designing the primers, namely similar fragment sequences are designed to be avoided as much as possible in the detection and typing of multiplex PCR, so that the difference of the melting curves is large and is easy to distinguish. However, the present inventors have adopted the reverse design approach instead, and have utilized the difficulty in designing the original primermcr-8Andmcr-10the specific primer pair of (A) adopts a unified pre-primer sequence SEQ ID No. 7, and the following results are found in an unexpected way: will be provided withmcr-8Andmcr-10the specific primer pair adopts a unified pre-primer sequence SEQ IAfter the D No. 7, not only the differential typing of small differences is completed by an HRM method (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 as for mixingmcr-1Andmcr-3the clinical samples can be quickly and effectively detected and identified by adopting the multiplex PCR kit. Makes up the blank that no multiplex PCR kit for realizing the effective typing and detection of all MCR genes in clinical samples exists in the market at present, and particularly can effectively identify the typingmcr-8Andmcr-10blank for functional multiplex PCR kit.

(2) The kit is applied to clinical scenes, can effectively detect MCR genes, can effectively identify all MCR gene types popular in clinical samples, can quickly trace the source and know main popular types.

(3) The kit is particularly suitable for medical clinical monitoring scenes, can quickly, conveniently and effectively detect and early warn MCR genes in excrement samples, can monitor PCR products in real time, greatly reduces the consumption of manpower and material resources for monitoring movable polymyxin drug-resistant genes, and simplifies the spot check and monitoring of large batches of clinical samples in daily production life, so that the kit and the detection method thereof have greater application value in the medical clinical monitoring scenes, and can also be applied to the environment monitoring scenes for monitoring and early warning MCR types with high risk (MCR type (C) (in the environment monitoring scenes)mcr-1、mcr-3、mcr-8Andmcr-10）。

(4) the primers provided by the invention have no amplification signals for detection samples without mcr, which indicates that the primers have good specificity.

Drawings

FIG. 1 is a SeqMan sequence analysis chart for 46 MCR genes in NCBI database;

FIG. 2 shows the detection using the kit of the present inventionmcr-1(the specific primer pair is the primer shown in SEQ ID No. 3 and the primer shown in SEQ ID No. 4) and comprises three dissolution curves of positive templates, Tm: (mcr-1)=84.8℃；

FIG. 3 shows the detection using the kit of the present inventionmcr-3(it is specific toThe sex primer pair is a primer shown as SEQ ID No. 5 and a primer shown as SEQ ID No. 6), and comprises three positive template dissolution curves, Tm: (mcr-2)= 87.8℃；

FIG. 4 shows the detection using the kit of the present inventionmcr-8(the specific primer pair is the primer shown in SEQ ID No. 7 and the primer shown in SEQ ID No. 8) and comprises three dissolution curves of positive templates, Tm: (mcr-8)= 88.2℃；

FIG. 5 shows the detection using the kit of the present inventionmcr-10(the specific primer pair is the primer shown in SEQ ID No. 7 and the primer shown in SEQ ID No. 9) and comprises three dissolution curves of positive templates, Tm: (mcr-10)= 88.5℃；

FIG. 6 is a schematic view of a liquid crystal display devicemcr-1、mcr-3The mixed template sample is detected by the kit to obtain a dissolution curve;

FIG. 7 is a schematic view of a liquid crystal display devicemcr-1、mcr-3、mcr-8Andmcr-10the mixed template sample is detected by the kit to obtain a dissolution curve;

FIG. 8 shows the detection by single PCRmcr-8(the specific primer pair is the primer shown in SEQ ID No. 10 and the primer shown in SEQ ID No. 11) and comprises three positive template dissolution curves, Tm: (mcr-8)= 87.7℃；

FIG. 9 shows the detection by single PCRmcr-10(the specific primer pair is the primer shown in SEQ ID No. 12 and the primer shown in SEQ ID No. 13) and comprises three positive template dissolution curves, Tm: (mcr-10)= 75.1℃；

Wherein the ordinate of the dissolution profile is the Derivative of Derivative, in particular the Derivative of the change in fluorescence intensity over the change in temperature, i.e.d (fluorescence)/d (temperature); the abscissa is the Temperature (. degree.C.) which is the Temperature point for each of the solubilization processes. The dissolution temperature Tm is at the peak.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

Example 1

1. The primer sequence design in the kit of the invention is as follows: by analyzing MCR gene sequences in all known clinical samples respectively, sequence analysis is carried out on 59 genes by SeqMan (figure 1), a highly conserved segment is found out by analysis, the highly conserved segment without a secondary structure is selected by premier 3.0, a plurality of groups of primers are designed, the length of the primers is about 20 bases generally, and complementary sequences do not exist between the primers and in the primers. Primer list 1 is shown below:

TABLE 1 sequence List of primers and amplified fragments (both 5 '-3')

Target gene	Primer sequence 5'-3'	SEQ ID	Product Length (bp)
				Universal primer	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

Establishing and optimizing a reaction system: the target region template used in the establishment and optimization of the reaction system is obtained as follows: respectively takemcr-1、mcr-3、mcr-8Andmcr-10and (2) recovering the escherichia coli and the klebsiella pneumoniae strains of the carrying strains, culturing for 48 hours, taking 1mL of culture solution, washing with sterile water, redissolving into 1mL, respectively extracting genome nucleic acid by adopting a phenol-chloroform method or a kit, carrying out PCR amplification by using the primers, and taking the cells with Ct values of 24-27 as templates for optimizing a reaction system later. Wherein the initial reaction system is shown in Table 2:

TABLE 2 PCR reaction System designed initially

	PCR reaction system
		Components	Final concentration
PCR buffer solution	1×
		Mg2＋Concentration of	2.0mmol/L
dNTPs (containing dUTP)	0.15mmol/L
		Taq enzyme	2U
20 × EvaGreen fluorescent dye	1×
		Primer shown as SEQ ID No. 1	0.6μmol/L
Primer shown as SEQ ID No. 2	0.6μmol/L
		Primer shown as SEQ ID No. 3	0.05μmol/L
Primer shown as SEQ ID No. 4	0.05μmol/L
		Primer shown as SEQ ID No. 5	0.15μmol/L
Primer shown as SEQ ID No. 6	0.15μmol/L
		Primer shown as SEQ ID No. 7	0.20μmol/L
Primer shown as SEQ ID No. 8	0.10μmol/L
		Primer shown as SEQ ID No. 9	0.10μmol/L
Form panel	2μL
		Make up water to	40μL

2.1 optimization of primer concentration: in a reaction system, primer concentrations are respectively subjected to multiple-ratio continuous dilution from 0.1 mu mol/L to 0.8 mu mol/L for detection, and the optimal final primer concentration is determined to be reaction through analysis and comparison of test results: the concentration of the universal primers (primers shown in SEQ ID No:1 and SEQ ID No: 2) is 0.6 mu mol/L, and the concentration of the specific primers (primers shown in SEQ ID No:3 to SEQ ID No: 10) is 0.05-0.20 mu mol/L.

2.2 optimization of magnesium ion concentration: at the final concentration of the primersMgCl was added under the conditions of a degree of 0.2. mu. mol/L and other conditions in the reaction system as shown in Table 2₂The concentration of the magnesium ion 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 magnesium ion concentration in the reaction system of the kit through repeated experiments.

2.3 optimization of the amount of Taq DNA polymerase (Taq enzyme): on the premise that the final concentration of the primer is 0.2 mu mol/L, the final concentration of the magnesium ions is 2.5 mmol/L and other conditions in the reaction system are as shown in Table 2, 2U is selected as the dosage of the Taq enzyme in the reaction system of the kit by comparing the optimized experiment result of the dosage of the Taq enzyme (in Unit).

2.4 optimization of dNTPs (deoxyribonucleoside triphosphate) concentration: on the premise that the final concentration of the primer is 0.2 mu mol/L, the final concentration of the magnesium ion is 2.5 mmol/L and other conditions in the reaction system are as shown in the table 2, dNTPs with different concentrations are used for detection, and 0.2 mmol/L is selected as the usage amount of dNTPs in the reaction system of the kit after comprehensive evaluation.

As the primers and the target fragments related by the method are numerous, the error of the unsaturated dye SYBRGREEN is large, and the single-gene difference cannot be distinguished, the saturated dye EvaGreen is used as the fluorescent dye in the PCR process. The Evagreen has little interference on qPCR, no dye redistribution, saturation embedding in DNA double chains and far better distinguishing effect than SYBR Green. The primers and the fluorescent dye are used for establishing a reaction system, and finally the adopted fluorescent PCR reaction system is determined to be a 40 mu L system, and the required components and corresponding concentrations are shown in a table 3.

TABLE 3 optimized PCR reaction System

	PCR reaction system
		Components	Final concentration
PCR buffer solution	1×
		Mg2＋Concentration of	2.0mmol/L
dNTPs (containing d)UTP）	0.15mmol/L
		Taq enzyme	2U
20 × EvaGreen fluorescent dye	1×
		Primer shown as SEQ ID No. 1	0.6μmol/L
Primer shown as SEQ ID No. 2	0.6μmol/L
		Primer shown as SEQ ID No. 3	0.05μmol/L
Primer shown as SEQ ID No. 4	0.05μmol/L
		Primer shown as SEQ ID No. 5	0.15μmol/L
Primer shown as SEQ ID No. 6	0.15μmol/L
		Primer shown as SEQ ID No. 7	0.20μmol/L
Primer shown as SEQ ID No. 8	0.10μmol/L
		Primer shown as SEQ ID No. 9	0.10μmol/L
Form panel	2μL
		Make up water to	40μL

Note: when the reaction volumes of the fluorescent PCR are different, the reagents are adjusted proportionally.

3. The detection method of the kit specifically comprises the following steps:

(1) extracting DNA in a sample to be detected;

because the known mobile 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 detected, such as a feces sample, placing the sample to be detected in a centrifugal tube with a diameter of 2.0mm FTA filter membrane sheet, then drying the sample at 56 ℃, adding 200 microliters of 10% SDS solution into the dried FTA filter membrane sheet, boiling the dried FTA filter membrane sheet for 10min, washing the FTA filter membrane sheet for 2 times by using FTA special buffer solution, then washing the FTA filter membrane sheet for two times by using TE buffer solution, and drying the FTA filter membrane sheet at 56 ℃ to be used as a PCR reaction.

(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 the table 3, carrying out PCR amplification reaction and carrying out fluorescence PCR detection, carrying out high-resolution dissolution analysis on PCR amplification products in the PCR detection, and carrying out the following procedures: 95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15s, 60 ℃ for 15s, the dissolution rate is 0.2 ℃/s; when the actual melting curve Tm value of any one of the PCR-A reaction system and the PCR-B reaction system is consistent with the standard melting curve Tm value of any kind of MCR genes, the sample to be detected has the MCR genes.

Wherein, the PCR amplification reaction program conditions are as follows: 2 min at 95 ℃ for 1 cycle; 5 sec at 95 ℃ and 40 sec at 60 ℃ for 40 cycles.

In this example, a real-time fluorescence quantitative PCR instrument of Applied Biosystems ABI 7500 manufactured by Seimer Feishel technologies, Ltd is used for the fluorescence PCR detection, but the detection is not limited thereto. Selecting a detection channel of the instrument: in the fluorescent PCR reaction, the fluorescence signal of the reaction tube of the apparatus is collected, and the specific method of the selected fluorescence detection channel is different from apparatus to apparatus, and the instruction of the apparatus is referred.

Example 2

The primer pairs shown in table 1 in example 1 were selected, and the genomic DNA of bacteria in samples of various sources was extracted from a culture solution of 33 bacteria in total, which was a culture solution of a bacterium to be tested and other non-target strains, by using a phenol-chloroform method or a kit. Wherein the standard strain is a strain purchased or preserved in a food safety and detection laboratory of southern China university; the isolate is from excrement of various culture farms, farmer markets and healthy people in Shenzhen city, adopts Shen,et al.(2018) the method of (1) identifies the presence or absence of the MCR gene in the strains, and the strains are identified by 16S sequencing or mass spectrometry.

In a 40. mu.L fluorescent PCR reaction system (prepared as shown in Table 3), 2. mu.L of the genomic DNA of the above-extracted different strains was added, and fluorescent PCR detection was performed according to the PCR reaction conditions in example 1.

The results of the experiment are shown in table 4 below.

TABLE 4 fluorescent quantitation qPCR results for different strains

Bacterial strains	Sourceb	qPCR	Shen, et al.(2018)c
				Target strain (n =14)
Klebsiella spKlebsiella(n=3)
				Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis	Fecal isolate 1	＋a	＋
Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis	Fecal isolate 2	＋	＋
				Klebsiella pneumoniaeKlebsiellapneumoniassp. rhinoscleromatis	Fecal isolate 3	＋	＋
Escherichia coliEscherichiacoli(n=11)		＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 1	＋	＋
Escherichia coliEscherichiacoli	Fecal isolate 2	＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 3	＋	＋
Escherichia coliEscherichiacoli	Fecal isolate 4	＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 5	＋	＋
Escherichia coliEscherichiacoli	Fecal isolate 6	＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 7	＋	＋
Escherichia coliEscherichiacoli	Fecal isolate 8	＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 9	＋	＋
Escherichia coliEscherichiacoli	Fecal isolate 10	＋	＋
				Escherichia coliEscherichiacoli	Fecal isolate 11	＋	＋
Non-target strain (n =19)
				Enterobacter spEnterbacter(n=4)
Enterobacter cloacaeEnterbacter cloacae	CICC No.21539	-	-
				Enterobacter sakazakiiEnterbactersakazakii	ATCC No.29544	-	-
Enterobacter aerogenesEnterbacteraerogenes	CICC No.10293	-	-
				Enterobacter aerogenesEnterobacteraerogenes	ATCCNo.13408	-	-
Escherichia coliEscherichiacoli(n=12)
				Escherichia coliE. coli O157:H7	CICC No.21530	-	-
Escherichia coliE. coli O157:H7	SZCIQ No.13813	-	-
				Escherichia coliE. coli O157:H7	ADCPC No.931	-	-
Escherichia coliEscherichiacoli	ATCC No.9637	-	-
				Escherichia coliEscherichiacoli	SZCIQNo.eco3	-	-
Escherichia coliEscherichiacoli	SZCIQNo.eco5	-	-
				Escherichia coliEscherichiacoli	SZCIQNo.jm109	-	-
Escherichia coliEscherichiacoli	CMCC No.44104	-	-
				Escherichia coliEscherichiacoli	CMCC No.44105	-	-
Escherichia coliEscherichiacoli	CMCC No.44106	-	-
				Escherichia coliEscherichiacoli	CMCC No.44111	-	-
Escherichia coliEscherichiacoli	CGMCC No.1.129	-	-
				Klebsiella spKlebsiella(n=2)
Klebsiella pneumoniaeKlebsiellapneumonia	CMCC No.46102	-	-
				Klebsiella pneumoniaeKlebsiellapneumonia	CICC No.10781	-	-
SalmonellaSalmonella（n=1）		-	-
				Salmonella dublinSalmonella dublin	GZCDCNo.dbl1a	-	-

a. +, positive; -, negative

ATCC, American type culture Collection, university of Denver, Manassas, Mevirginia, zip code 10801; CMCC, China medical microbial strain preservation center, Hua Tuo Lu No. 31 institute of biological medicine industry base in Daxing district, Beijing, zip code 102629; CGMCC, China general microbiological culture Collection center, No. 3 of Xilu No. 1 of Beijing, Chaoyang, North Cheng, zip code 100101; CICC, industrial microbial strain preservation management center, No. 6 building of No. 24 institute of Zhonglu Jiuxiangqiao of the rising district, Beijing city, zip code: 100015, respectively; SZCIQ, Shenzhen entry and exit inspection and quarantine bureau, Guangdong province Shenzhen, Futian district of Shenzhen 1011 number, postcode 518000; ADCPC, Chongqing animal disease control and prevention center, Chongqing city Yuzhongjiang river two way No. 8, postcode: 400042. wherein the strains from ATCC, CMCC, CGMCC and CICC are all available. Strains from SZCIQ and ADCPC were separately donated by these agencies; these strains have been disclosed in the literature "Xing-long Xiao, Li Zhang, Hui Wu et. al, Simultaneous Detection of Salmonella, Listeriophytogens, and Staphyloccusaureusby Multiplex Real-Time PCR analysis Using High-Resolution Messing [ J ]. Food Analytical Methods, 2014,7(10): 1960-.

c. MCR detection methods reference "Yingbo, s., Hongwei, z., Jiao, x., yongqiong, w., Qijing, z., & Walsh, t.r., et al, (2018). antitropogenic and environmental factors associated with high interaction of MCR-1 carriage in human access chip.

Upon detection, if the culture solution to be tested contains MCR variants (MCR gene variants, that ismcr-1、mcr-3、mcr-8Andmcr-10) Any one or more of them shows a positive amplification curve, and the Tm value of the dissolution curve is equal tomcr-1、mcr-3、mcr-8Andmcr-10the Tm values of any standard dissolution curve match, whereinmcr-1、mcr-3、mcr-8Andmcr-10the standard dissolution curves of (A) are shown in FIGS. 2-5; if the culture solution to be detected does not contain MCR variants, no amplification signal exists, and the primer pair has good sensitivity and specificity. The strains in the table 4 are detected, and the result shows that the strains containing the target genes are detected, but the non-target strains are negative without amplification curves, which indicates that the method has good specificity.

In order to determine the MCR type in the sample to be tested, the sample to be tested needs to be identified through a melting curve. Using the Tm values as the interpretation standards, the peak values of the standard dissolution curves corresponding to different types of MCR genes shown in FIGS. 2 to 5 were found to correspond to drug-resistant genes when the measured Tm values were 84.8, 87.8, 88.2 and 88.5mcr-1、mcr-3、mcr-8Andmcr-10it is demonstrated that the method has good resolution.

For the Presence of Co-occurrences in clinical samplesmcr-1、mcr-3In the case of (2), the mixed DNA template was further examined, as shown in FIG. 6mcr-1Andmcr-3in the presence of both, T can be obtained in the PCR reaction system_mBimodal dissolution curves with values of 84.8 ℃ and 87.8 ℃ and T_m(mcr-1) And T_m(mcr-3) And (5) the consistency is achieved.

In summary, the detection method of the kit of the invention can not only effectively detect MCR (MCR) found in all clinical samples in the sample to be detectedmcr-1、mcr-3、mcr-8Andmcr-10) When present in a clinical samplemcr-1Andmcr-3in addition, the MCR gene type can be effectively detected and identified.

Further, the kit of the present invention may be used for verificationmcr-1、mcr-3、mcr-8Andmcr-10any combination ofThe MCR gene is used for effective detection and early warning, and the inventor artificially carries outmcr-1、mcr-3、mcr-8Andmcr-10mixing to form 4 mcr gene type mixed DNA templates, obtaining a bimodal dissolution curve with Tm values of 84.8 ℃ and 88.5 ℃ by the PCR reaction system (shown in figure 7), wherein the melting curve is consistent with Tm (mcr-1) and Tm (mcr-10), so that when the DNA templates are mixed, the Tm values are equal to the Tm values of the DNA templatesmcr-1、mcr-3、mcr-8Andmcr-10when the MCR gene types exist simultaneously, the kit can detect the functions of a plurality of MCR gene types simultaneously, and plays a role in effective detection and early warning.

Example 3

In this example, 112 fresh stool samples of healthy people collected by the Huada Zhi stool collection set are suspended in a preservation solution and stored at 4 ℃ for no more than one week.

The primer set shown in Table 1 in example 1 was selected, total DNA of fecal bacteria extracted by the boiling method described in example 1 and the DNA extracted by the Shen method described in example 2 were used,et al(2018) using the extracted DNA template as a different DNA template for PCR detection comparison. The results show that, when the percentage of Shen,et al(2018) in which the method detects 12.5% (14/112) of positive rate, and the method detects 13.4% (15/112) of positive rate. The invention shows that the DNA extracted from the sample to be detected by the boiling method is used as the template and has good detectable rate.

It should be noted that: FIGS. 2 to 7 show 2 to 3 parallel experiments, and although the melting curves in each of the graphs do not overlap, the Tm automatically calculated by the software of the PCR detection apparatus is the same for each melting curve, so that the slight difference in the form of the 3 melting curves obtained by performing 3 parallel experiments on the same DNA sample is a normal phenomenon in the art.

Comparative example 1

This comparative example differs from example 1 in that: table 1 is modified as shown in table 5 below, specifically,

TABLE 5 sequence List of primers and amplified fragments (both 5 '-3')

Target gene	Primer sequence 5'-3'	SEQ ID	Product Length (bp)
				Universal primer	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 shown in Table 5mcr-1、mcr-3、mcr-8Andmcr-10when the combination of the respective specific primers and the universal primer is used for single PCR detection, the target fragment can be specifically identified in a single PCR system, whereinmcr-8Andmcr-10performing single PCR detection to obtain Tm values of 87.7 and 75.1, respectively, andmcr-8andmcr-10the Tm values of the standard dissolution curves agree with (mcr-8Andmcr-10the standard dissolution curves are shown in FIGS. 8 and 9), the corresponding drug resistance genes aremcr-8Andmcr-10. To further facilitate typing by Tm valuesmcr-8Andmcr-10the primer set of (1), wherein amplified fragments are selected to be distant from each other. However, when the primer pair combinations shown in table 5 are applied to a multiplex PCR system, it is found that the mutual interference exists between the primer pairs, so that the amplification efficiency is reduced, an effective amplification curve and a dissolution curve cannot be obtained, products in a sample cannot be effectively detected, and the products cannot be differentiated and typed.

Although there is a multiplex PCR technique for detection and early warning of various types of MCR genes in the prior art, primers for detecting various types of MCR genes are different and amplified fragments are selected to be distant from each other, so that the detection or typing can be performed by the Tm value of HRM (high resolution melting curve analysis) method. But against the type of MCR currently prevalent in the clinic: (mcr-1、mcr-3、mcr-8Andmcr-10) Detecting and identifying the type of the cells, and screening outmcr-1、mcr-3、mcr-8Andmcr-10the respective corresponding specific primer pairs (single PCR detection can well detect and identify MCR genes), and when multiple PCR detection is carried out after combination, the mutual interference among the primer pairs is found, so that the amplification efficiency is reduced, an effective amplification curve and a dissolution curve cannot be obtained, products in a sample cannot be effectively detected, and the products cannot be distinguished and typed.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.