CN117417989B - Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes - Google Patents

Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes Download PDF

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CN117417989B
CN117417989B CN202311533955.9A CN202311533955A CN117417989B CN 117417989 B CN117417989 B CN 117417989B CN 202311533955 A CN202311533955 A CN 202311533955A CN 117417989 B CN117417989 B CN 117417989B
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CN117417989A (en
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张宁
杨威
韩昱
胡晓悦
郑丽丽
赵博伟
王珏
王晓芳
王芸萌
于海航
潘保革
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HEBEI INSTITUTE OF ANIMAL HUSBANDRY AND VETERINARY
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Abstract

The invention relates to the technical field of biology, in particular to a multiplex fluorescence quantitative PCR kit and a multiplex fluorescence quantitative PCR method for detecting drug-resistant genes. The kit comprises a nucleic acid amplification reagent, a reference substance and a standard substance, wherein the nucleic acid amplification reagent contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 6 and a probe with a nucleotide sequence shown as SEQ ID NO. 7-SEQ ID NO. 9. Compared with the traditional bacterial drug resistance detection, the detection kit and the detection method do not need to separate and culture strains, so that the detection speed is increased, the labor cost is reduced, and the detection kit and the detection method have the advantages of simplicity, convenience, rapidness, high sensitivity, high specificity and the like.

Description

Multiplex fluorescence quantitative PCR kit and method for detecting drug-resistant genes
Technical Field
The invention relates to the technical field of biology, in particular to a multiplex fluorescence quantitative PCR kit and a multiplex fluorescence quantitative PCR method for detecting drug-resistant genes.
Background
Carbapenem is the first drug to treat MDR enterobacteriaceae, and oxazolidinone drugs, linezolid and polymyxin, are the last lines of defense to treat infections caused by resistant gram-positive and gram-negative bacteria, respectively. The drug-resistant genes blaNDM, mcr-1 and cfr respectively mediate the bacteria to produce drug resistance to carbapenem antibiotics, polymyxin and oxazolidinone drugs-linezolid, and the bacteria carrying one of the drug-resistant genes is called super drug-resistant bacteria, so that the appearance and the transmission of the super drug-resistant bacteria seriously threaten the health and public health safety of human beings. At present, no detection method for simultaneously detecting three drug-resistant genes of blaNDM, mcr-1 and cfr exists. Therefore, it is an urgent need to establish a detection technology that is faster, simpler, more stable, more efficient and accurate, and lower in cost, and that ensures public health safety.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides a multiplex fluorescence quantitative PCR kit and a method for detecting drug-resistant genes blaNDM, mcr-1 and cfr.
The invention provides a multiplex fluorescence quantitative PCR kit for detecting drug-resistant genes, which comprises a nucleic acid amplification reagent, a reference substance and a standard substance, wherein the nucleic acid amplification reagent contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 6 and a probe with a nucleotide sequence shown as SEQ ID NO. 7-SEQ ID NO. 9. Alternatively, FAM is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 7, BHQ-1 is marked at the 3' end, HEX is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 8, -BHQ-2 is marked at the 3' end, cy5 is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 9, and-BHQ-2 is marked at the 3' end.
The invention provides a multiplex fluorescence quantitative PCR method for detecting drug-resistant genes, which comprises the following steps:
S1, extracting bacterial genome DNA in a sample to be detected to obtain template DNA in the sample to be detected;
s2, preparing a fluorescent quantitative PCR reaction system;
S3, fluorescence quantitative PCR amplification: 95 ℃ for 15min;95℃for 10sec and 60℃for 30sec 45 total times, and collecting fluorescent signals of the template DNA in step 2 of each cycle; and the positive result is judged that Ct is less than or equal to 30.
S4, preparing a standard curve by adopting a reference sample to obtain the concentration of the drug-resistant gene in the sample to be detected.
Optionally, the fluorescent quantitative PCR reaction system contains 2 XM 5 GoldStar TaqMan Mixture mu L, and primers shown in SEQ ID NO 1-SEQ ID NO 6 with concentration of 10 mu mol/L are respectively 0.3-0.5 mu L, 0.1-0.3 mu L and 0.1-0.3 mu L; the probes shown in SEQ ID NO 7-SEQ ID NO 9 with the concentration of 10 mu mol/L are respectively 0.3-0.5 mu L, 0.3-0.5 mu L and 1 mu L of template, and the ultrapure water is complemented to 20 mu L.
The invention provides a primer and a probe for detecting drug resistance genes, the nucleotide sequence of the primer is shown in SEQ ID NO. 1-SEQ ID NO. 6, and the nucleotide sequence of the probe is shown in SEQ ID NO. 7-SEQ ID NO. 9. Alternatively, FAM is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 7, BHQ-1 is marked at the 3' end, HEX is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 8, -BHQ-2 is marked at the 3' end, cy5 is marked at the 5 'end of the nucleotide sequence shown in SEQ ID NO. 9, and-BHQ-2 is marked at the 3' end.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
compared with the traditional bacterial drug resistance detection, the detection kit and the detection method do not need to separate and culture strains, so that the detection speed is increased, the labor cost is reduced, and the detection kit and the detection method have the advantages of simplicity, convenience, rapidness, high sensitivity, high specificity and the like.
The kit and the method for detecting the drug resistance gene can monitor the reaction process in real time by adopting a fluorescent quantitative PCR technology, do not need electrophoresis and tube closing operation, prevent pollution, and identify the drug resistance type of the strain according to Ct value by detecting a fluorescent signal curve released in the dissolution process in a reaction system; can meet the clinical requirement of rapidly and accurately detecting drug-resistant genes.
Drawings
FIG. 1 shows the results of plasmid synthesis assays;
FIG. 2 shows amplification curves of a triple Tap Man fluorescent quantitative PCR experiment of an mcr-1 gene plasmid;
FIG. 3 shows amplification curves of a triple Tap Man fluorescent quantitative PCR experiment of a blaNDM-1 gene plasmid;
FIG. 4 is a cfr gene plasmid triple Tap Man fluorescent quantitative PCR experimental amplification curve;
FIG. 5 is a single annealing temperature amplification plot; FIG. 5A is a drug resistance gene mcr-1; FIG. 5B drug resistance gene blaNDM; FIG. 5C is a drug resistant gene cfr;
FIG. 6 shows the results of triple annealing temperature amplification; FIG. 6-1A, FIG. 6-2A, FIG. 6-3A are drug-resistant gene mcr-1; FIG. 6-1B, FIG. 6-2B, FIG. 6-3B drug resistance gene blaNDM; FIGS. 6-1C, 6-2C, and 6-3C are drug resistant genes cfr;
FIG. 7A is a PCR primer and probe amplification curve for drug-resistant gene mcr-1 triple TaqMan fluorescence quantification; FIG. 7B is a PCR primer and probe amplification curve of drug-resistant gene blaNDM triple TaqMan fluorescence quantification; FIG. 7C is a graph of amplification of the drug resistant gene cfr triple TaqMan fluorescent quantitative PCR primer and probe; FIG. 7D is a triplet TaqMan fluorescent quantitative PCR primer and probe optimization result;
FIG. 8 is a triple TaqMan fluorescent quantitative PCR specific amplification curve: wherein: a is a standard mixture; b is mcr-1 standard; c is blaNDM standard; d is cfr standard; e is Escherichia coli containing mcr-1 and blaNDM; f is Escherichia coli containing mcr-1; g is Escherichia coli containing blaNDM; h is staphylococcus aureus containing cfr; i is E.coli 25922; j is staphylococcus aureus 25923; k sterile and enzyme-free deionized water;
FIG. 9 shows the results of a triple TaqMan fluorescent quantitative PCR sensitivity assay;
Fig. 10 shows a partial clinical test result.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
The embodiment of the invention provides a multiplex fluorescence quantitative PCR kit for detecting drug-resistant genes, which comprises a nucleic acid amplification reagent, a reference substance and a reference substance. The nucleic acid amplification reagent contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 6 and a probe with a nucleotide sequence shown as SEQ ID NO. 7-SEQ ID NO. 9.
In the embodiment of the invention, 3 pairs of specific primers and 3 TaqMan probes are designed by referring to mcr-1 gene blaNDM-1 gene and cfr gene sequences registered in GenBank and aiming at a conserved region thereof through screening experiments, as shown in Table 1. The sequences of the primers and probes are specifically shown in Table 1.
TABLE 1
The designed 3 pairs of specific primers were used to determine by PCR and sequencing alignment, and biosystems were commissioned for synthesis.
As a preferred technical scheme of the multiplex fluorescence quantitative PCR kit, the volumes of the primer and the probe in the nucleic acid amplification reagent are shown in Table 2:
TABLE 2
Numbering device Concentration of Volume range Preferred volume
SEQ ID NO:1 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:2 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:3 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:4 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:5 10μmol/L 0.1～0.3μL 0.2μL
SEQ ID NO:6 10μmol/L 0.1～0.3μL 0.2μL
SEQ ID NO:7 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:8 10μmol/L 0.3～0.5μL 0.4μL
SEQ ID NO:9 10μmol/L 0.3～0.5μL 0.4μL
As a preferable technical scheme of the multiplex fluorescence quantitative PCR kit, the control substances comprise a positive control substance and a negative control substance; the positive reference substance contains a mixture of 3 mutant plasmids with nucleotide sequences shown in SEQ ID NO. 10-SEQ ID NO. 12 in equal proportion; the negative control is process water.
As a preferred technical scheme of the multiplex fluorescence quantitative PCR kit, the standard substance comprises a mixture of mutant plasmids with gradient concentration; the concentration of the mixture of mutant plasmids was in turn ：102copies/μL、103copies、104copies/μL、105copies/μL、106copies、107copies/μL、108copies/μL、109copies.
As a preferred technical scheme of the multiplex fluorescence quantitative PCR kit, the nucleic acid amplification reagent also contains 2×M5 GoldStar TaqMan Mixture.
The invention also provides a multiplex fluorescence quantitative PCR method for detecting the drug-resistant genes, which comprises the following steps:
S1, extracting bacterial genome DNA in a sample to be detected to obtain template DNA in the sample to be detected;
s2, preparing a fluorescent quantitative PCR reaction system;
S3, fluorescence quantitative PCR amplification: 95 ℃ for 15min;95℃for 10sec and 60℃for 30sec 45 total times, and collecting fluorescent signals of the template DNA in step 2 of each cycle; and the positive result is judged that Ct is less than or equal to 30.
S4, preparing a standard curve by adopting a reference substance to obtain the concentration of the drug-resistant gene in the sample to be detected.
As a preferable technical scheme of the multiplex fluorescence quantitative PCR method for detecting the drug-resistant genes, the fluorescence quantitative PCR reaction system contains 2 XM 5 GoldStar TaqMan Mixture mu L, and primers shown in SEQ ID NO 1-SEQ ID NO 6 with concentration of 10 mu mol/L are respectively 0.3-0.5 mu L, 0.1-0.3 mu L and 0.1-0.3 mu L; the probes shown in SEQ ID NO 7-SEQ ID NO 9 with the concentration of 10 mu mol/L are respectively 0.3-0.5 mu L, 0.3-0.5 mu L and 1 mu L of template, and the ultrapure water is complemented to 20 mu L.
Preferably, the fluorescent quantitative PCR reaction system contains 2 XM 5 GoldStar TaqMan Mixture mu L and primers shown in SEQ ID NO. 1-SEQ ID NO. 6 with concentration of 10 mu mol/L, wherein the primers are respectively 0.4 mu L, 0.2 mu L and 0.2 mu L; the probes shown in SEQ ID NO. 7-SEQ ID NO. 9 with the concentration of 10 mu mol/L are respectively 0.4 mu L, 0.4 mu L and 1mu L of template, and the ultrapure water is complemented to 20 mu L.
The invention also provides a primer and a probe for detecting the drug-resistant gene, which are characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 6, and the nucleotide sequence of the probe is shown as SEQ ID NO. 7-SEQ ID NO. 9. Preferably, the nucleotide sequence shown in SEQ ID NO. 7 is marked with FAM at the 5 'end, BHQ-1 at the 3' end, HEX at the 5 'end and-BHQ-2 at the 3' end of the nucleotide sequence shown in SEQ ID NO. 8, cy5 at the 5 'end and-BHQ-2 at the 3' end of the nucleotide sequence shown in SEQ ID NO. 9.
Examples
The reagents used in the examples are all commercially available.
Materials:
Bacterial strains and samples: escherichia coli quality control bacteria (ATCC 25922) were purchased from Qingdao sea Bo biotechnology Co., ltd; escherichia coli, salmonella, staphylococcus aureus, etc. are all isolated from pig manure, oral fluid and stored in the laboratory of livestock and veterinary research institute of Hebei province in 2021-2023. Environmental samples, pharyngeal swabs and anal swabs are all collected in farms in the Hebei province area.
Molecular biological agents: TIANcombi DNA Lyse & Det PCR Ki nucleic acid extraction kit, beijing Tiangen Biochemical technology Co., ltd; 2 XM 5 GoldStar TaqMan Mixture probe premix, beijing polymeric Biotechnology Co., ltd; primers and probes, anhui general Biotechnology Co., ltd.
The main instrument is as follows: rogowski 96.
Example 1
A multiplex fluorescent quantitative PCR kit for detecting drug resistance genes, the composition of which is shown in table 3:
table 3:
the using method of the kit comprises the following steps:
s1, extracting bacterial genome DNA in a sample to be detected, and referring to the instruction of a commercial extraction kit;
S2, preparing a fluorescent quantitative PCR reaction system; as shown in table 4:
TABLE 4 Table 4
S3, fluorescence quantitative PCR amplification: 95 ℃ for 15min;95℃for 10sec and 60℃for 30sec 45 times, and collecting a fluorescent signal of the template DNA in step 2 of each cycle; the positive result is determined that Ct is less than or equal to 30, when the Ct value is more than 30, the randomness of the reaction is increased, the repeatability test error is increased, and the influence of nonspecific products can be accompanied;
S4, preparing a standard curve by adopting a standard substance to obtain the concentration of the drug-resistant gene in the sample to be detected.
The preparation method of the standard curve comprises the following steps:
The standard substance group is used as a template, a reaction system is prepared according to the method shown in table 4, and the reaction amplification is carried out according to the fluorescent quantitative PCR method shown in S3, so that a standard curve is drawn.
Example 2
The construction method of the plasmids p-mcr-1, p-blaNDM-1 and p-cfr is as follows:
3 pairs of designed specific primer pairs mcr-F, mcr-R were used; NDM-F, NDM-R, cfr-F, cfr-R primer pair, and performing PCR amplification. The primer sequences are shown in Table 5 below:
TABLE 5
Primer name Nucleotide sequence Sequence numbering
mcr-F tgtcggtatgctcgttggc SEQ ID NO:13
mcr-R gatgaatgcggtgcggtct SEQ ID NO:14
NDM-F tggaccgatgaccagaccg SEQ ID NO:15
NDM-R gggaacgccgcaccaaacg SEQ ID NO:16
cfr-F tgaagtataaagcaggttgggagtca SEQ ID NO:17
cfr-R accatataattgaccacaagcagc SEQ ID NO:18
Target fragments of 486bp, 409bp and 746bp were obtained and synthesized by Jiangsu Samson Biotechnology Co., ltd. With PUC57 empty vector plasmid, and the identification result is shown in FIG. 1. The synthesized plasmids were designated as p-mcr-1, p-blaNDM-1 and p-cfr and used as standards. Plasmid DNA concentration was determined and copy number was performed.
The calculation formula is as follows: 6.02X10 23 plasmid concentration/(660 plasmid length) =copy number.
The nucleotide sequences of the plasmids are shown in Table 6:
TABLE 6
The concentration was determined to be 10.7X10 10 copies/. Mu. L, p-blaNDM-1 for p-mcr-1 and 11.32X10 10 copies/. Mu. L, p-cfr for 7.52X10 10 copies/. Mu.L.
Example 3
This example is used to illustrate the linear relationship of the standard curve:
the reference (recombinant standard plasmids p-mcr-1, p-blaNDM-1 and p-cfr were mixed in equal proportions and serially diluted 10-fold to the order of (10 2～109) copies/. Mu.L) were used as templates, and the reaction amplification was performed by the triple fluorescence quantitative PCR method of S3 in example 1 and a standard curve was drawn.
The experimental results are shown in fig. 2, 3 and 4. FIG. 2 shows amplification curves of a triple Tap Man fluorescent quantitative PCR experiment of an mcr-1 gene plasmid; FIG. 3 shows amplification curves of a triple Tap Man fluorescent quantitative PCR experiment of a blaNDM-1 gene plasmid; FIG. 4 is an amplification curve of cfr gene plasmid triple Tap Man fluorescent quantitative PCR experiment.
The results show that the standard curves for 8 concentration gradients have good linear relationships with correlation coefficients of p-mcr-1, p-blaNDM-1 and p-cfr being 0.9922, 0.9973 and 0.9953, respectively.
Example 4
This example illustrates the optimization process of annealing temperature:
The reaction system shown in Table 3 in example 1 was prepared, and the annealing temperature was optimized by single fluorescence quantitative PCR under 20. Mu.L system, and the annealing temperatures were set to 54℃at 55℃at 56℃at 57℃at 58℃at 59℃at 60℃at 61℃at 62℃at 63℃under the following conditions: 95 ℃ for 15min;95 ℃ 10sec, 54-63 ℃ 30sec, 40 cycles total. Fluorescence signals were collected in the second step of each cycle.
The experimental results are shown in fig. 5 and 6. The results demonstrate that the temperature is optimal using the temperature of example 1 of the present invention.
Example 5:
This example illustrates the optimization procedure of primer and probe concentrations:
The reaction system shown in Table 3 in example 1 was prepared, and four gradients of 0.2. Mu.L, 0.4. Mu.L, 0.6. Mu.L, and 0.8. Mu.L were used to perform fluorescent quantitative PCR amplification of three genes at a concentration of 10. Mu. Mol/L, with 3 replicates for each set and a negative control with ultrapure water as a template to determine the optimal primer concentration. The probe volumes were set to four gradients of 0.2. Mu.L, 0.4. Mu.L, 0.6. Mu.L, 0.8. Mu.L at a concentration of 10. Mu. Mol/L for fluorescent quantitative PCR amplification of three genes, 3 replicates per group and a negative control with ultrapure water as template, and the optimal probe concentrations were determined.
The optimized results of the triple TaqMan fluorescent quantitative PCR primer and the probe are shown in figure 7, wherein figure A is the amplification curve of the drug-resistant gene mcr-1 triple TaqMan fluorescent quantitative PCR primer and the probe; FIG. B shows the amplification curve of the drug-resistant gene blaNDM triple TaqMan fluorescent quantitative PCR primer and the probe; and C, a drug resistant gene cfr triple TaqMan fluorescent quantitative PCR primer and a probe amplification curve.
Finally, determining the volume of the drug-resistant gene mcr-1 and the drug-resistant gene blaNDM primer to be 0.8 mu L at the concentration of 10 mu mol/L and the volume of the drug-resistant gene cfr primer to be 0.4 mu L at the concentration of 10 mu mol/L; the volumes of the drug-resistant gene mcr-1, the drug-resistant gene blaNDM and the drug-resistant gene cfr probes are all 0.4 mu L at the concentration of 10 mu mol/L.
Example 6
This example is used to illustrate specificity and sensitivity assays:
1. specificity experiments
Using standard plasmids p-mcr-1, p-bla NDM-1 and p-cfr as positive controls, using sterile enzyme-free deionized water as negative controls, and containing drug-resistant genes mcr-1 and blaNDM; coli containing drug-resistant gene mcr-1 and drug-resistant gene blaNDM; staphylococcus aureus containing a drug resistant gene cfr; DNA extraction of E.coli quality control bacteria (ATCC 25922) and Staphylococcus aureus quality control bacteria (ATCC 25923) was used as templates. And (3) performing triple TaqMan fluorescent quantitative PCR detection by using an optimized system, and verifying the specificity of the method. The triple TaqMan fluorescent quantitative PCR specific amplification curve is shown in FIG. 8, and the statistical results are shown in Table 7.
TABLE 7
2. Sensitivity test
P-mcr-1, p-bla NDM-1 and p-cfr were subjected to 10-fold gradient dilutions at concentrations of p-mcr-1 (10.7X10 2～10.79×109)copies/μL、p-blaNDM-1 (11.32X10 2～11.32×109) copies/. Mu.L and p-cfr (7.52X10 2～7.52×109) copies/. Mu.L, respectively, as triple fluorescence quantitative PCR amplification templates.
Experiments were performed using the kit and method of example 1, and the results are shown in fig. 9. The lowest order of magnitude of detection for sensitivity experiments on recombinant standard plasmids p-mcr-1, p-blaNDM-1 and p-cfr was 10 2) copies/. Mu.L in the order of magnitude of (10 2～109) copies/. Mu.L.
Example 7
Results of the repeatability experiments in this example:
3 concentration gradients (10 6～108) are selected from the recombinant plasmid diluted by 10 times and used as templates for repeated detection, and the variation coefficients in batches and between batches are compared.
Triple fluorescence quantitative PCR experiments were performed with recombinant plasmids p-mcr-1, p-bla NDM-1 and p-cfr, respectively, using (10.79×104～10.79×106)copies/μL、(11.32×104～11.32×106)copies/μL、(7.52×104～7.52×106)copies/μL as template. The intra-group variation coefficient and the inter-group variation number were 0.01% to 0.04%, and the reproducibility was good, as shown in table 8.
TABLE 8
Example 8
This example is used to illustrate primer design and screening:
Searching MCR-1, blaNDM-1 and cfr drug resistance gene sequences according to NCBI, designing MCR-1 (1-10), blaNDM-1 (11-20) and cfr (21-30) primers by PRIMER PREMIER, and screening out hairpin-free structures, dimers, cross dimers and mismatches through simulation; and the sequence and screening results of the upstream and downstream primers with appropriate reaction temperature are shown in Table 9, and fluorescence modification is performed. The cfr (21-30) primer had a hairpin structure and was found not to form very severe hairpins according to query PRIMER PREMIER, which had not much effect on the experiment.
TABLE 9
Example 9
The present example is used to illustrate the technical effects obtained by clinical testing:
selecting 800 parts of samples collected in pig farms in partial regions of Hebei province according to the following The TIANcombi DNA Lyse & DET PCR KIT instruction manual extracts total DNA and stores it at-20 ℃.
800 Bacterial samples were tested and co-tested for 66 strains of bacteria containing the drug resistance gene, among which 32 strains of E.coli, 17 strains of Salmonella, 15 strains of Klebsiella pneumoniae and 2 strains of Staphylococcus aureus containing the drug resistance gene were tested.
The established triple fluorescence quantitative detection results are as follows: 32 parts of a sample containing a drug-resistant gene mcr-1, 40 parts of a sample containing a drug-resistant gene blaNDM and 2 parts of a sample containing a drug-resistant gene cfr; 8 samples containing both drug-resistant gene mcr-1 and blaNDM. The results detected by the conventional PCR detection method are as follows: 21 parts of sample containing drug-resistant gene mcr-1, 34 parts of sample containing drug-resistant gene blaNDM and 2 parts of sample containing drug-resistant gene cfr. The difference sample of the conventional PCR detection result is further detected to be a missing detection sample. The partial detection results are shown in fig. 10, wherein: 1. 2, 4, 5 and 6 contain drug-resistant genes bla NDM; 3 contains drug-resistant genes mcr-1 and bla NDM at the same time; 7 contains a drug-resistant gene cfr.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. The multiplex fluorescence quantitative PCR kit for detecting the drug-resistant genes is characterized by comprising a nucleic acid amplification reagent, a reference substance and a standard substance, wherein the nucleic acid amplification reagent contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 6 and a probe with a nucleotide sequence shown as SEQ ID NO. 7-SEQ ID NO. 9.
2. The kit according to claim 1, wherein the nucleotide sequence shown in SEQ ID NO. 7 is labeled with FAM at the 5 'end, BHQ-1 at the 3' end, HEX at the 5 'end, -BHQ-2 at the 3' end, cy5 at the 5 'end and-BHQ-2 at the 3' end, and the nucleotide sequence shown in SEQ ID NO. 9.
3. The kit according to claim 1, wherein, in the nucleic acid amplification reagent,
The volume of the primer shown in SEQ ID NO. 1-SEQ ID NO. 6 with the concentration of 10 mu mol/L is 0.3-0.5 mu L, 0.1-0.3 mu L and 0.1-0.3 mu L respectively; the volume of the probe shown in SEQ ID NO 7-SEQ ID NO 9 with the concentration of 10 mu mol/L is 0.3-0.5 mu L, 0.3-0.5 mu L and 0.3-0.5 mu L respectively.
4. The kit according to claim 3, wherein, in the nucleic acid amplification reagent,
The volume of the primer shown in SEQ ID NO. 1-SEQ ID NO. 6 with the concentration of 10 mu mol/L is 0.4 mu L, 0.2 mu L and 0.2 mu L respectively; the volumes of the probes represented by SEQ ID NO:7 to SEQ ID NO:9, which contained the probes at a concentration of 10. Mu. Mol/L, were 0.4. Mu.L, respectively.
5. The kit of claim 1, wherein the controls comprise a positive control and a negative control;
the positive control contains a mixture of 3 mutant plasmids with nucleotide sequences shown in SEQ ID NO. 10-SEQ ID NO. 12 in equal proportion;
The negative reference substance is process water.
6. The kit of claim 1, wherein the standard comprises a mixture of mutant plasmids at a gradient concentration;
The concentration of the mixture of mutant plasmids was in turn ：102copies/μL、103copies、104copies/μL、105copies/μL、106copies、107copies/μL、108copies/μL、109copies.
7. The kit of claim 1, wherein the nucleic acid amplification reagent further comprises 2×m5 GoldStar TaqMan Mixture.
8. A primer and a probe for detecting drug-resistant genes are characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 6, and the nucleotide sequence of the probe is shown as SEQ ID NO. 7-SEQ ID NO. 9.
9. The primer and probe of claim 8, wherein the nucleotide sequence of SEQ ID NO. 7 is labeled with FAM at the 5 'end, BHQ-1 at the 3' end, HEX at the 5 'end, -BHQ-2 at the 3' end, cy5 at the 5 'end and-BHQ-2 at the 3' end of the nucleotide sequence of SEQ ID NO. 8.
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