CN113234806A - Rapid detection method for fluoroquinolone antibiotic resistance based on single base mutation and application thereof - Google Patents
Rapid detection method for fluoroquinolone antibiotic resistance based on single base mutation and application thereof Download PDFInfo
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Abstract
The invention provides a rapid detection method of fluoroquinolone antibiotic resistance based on single base mutation and application thereof, wherein full-length amplification primers are designed for fluoroquinolone antibiotic target gene gyrA, the nucleotide sequence of an upstream primer of an optimal specific primer is SEQ ID NO.1, the nucleotide sequence of a downstream primer is SEQ ID NO.2, the primer pair can obtain the full-length fluoroquinolone antibiotic target gene gyrA of bacteria to be detected through conventional PCR, and then is compared with the gyrA gene sequence of fluoroquinolone antibiotic sensitive bacteria to judge whether base mutation exists.
Description
Technical Field
The invention relates to the technical field of environmental and biological detection, in particular to a rapid detection method of fluoroquinolone antibiotic resistance based on single base mutation and application thereof.
Background
In recent years, the rapid development of the large-scale livestock and poultry breeding industry leads to the increasing use amount of antibiotics, the antibiotics are often used for the purposes of preventing and treating animal diseases in livestock and poultry breeding, promoting the growth of livestock and poultry and the like, however, because some antibiotics are not completely absorbed or metabolized in animals, and are finally discharged through urine and feces in the form of parent compounds or metabolites, the content of the antibiotics in the environment is continuously increased, and some antibiotics such as fluoroquinolone antibiotics can be stably maintained in the environment for several months. Studies have reported that trace amounts of antibiotics are sufficient to exert selective pressure on bacteria, eventually leading to the development of bacterial resistance, which poses a serious threat to public health safety. Therefore, it is necessary to accurately and rapidly detect whether a pathogenic microorganism commonly found in the environment has antibiotic resistance.
The fluoroquinolone antibiotics are chemically synthesized broad-spectrum antibacterial drugs, and mainly inhibit the activity of DNA gyrase by specifically combining DNA gyrase A subunits (gyrase A and gyrA) related to bacterial DNA replication, interfere bacterial DNA to form supercoils, and prevent the normal function of the DNA so as to play a bactericidal role.
The main molecular mechanisms of the bacteria for generating the resistance of the fluoroquinolone antibiotics are the following: one is plasmid-mediated resistance gene, such as qnr family gene and qepA gene, which can be rapidly detected by fluorescent quantitative PCR technology; the other is to reduce the accumulation of intracellular fluoroquinolone antibiotics, and related genes involved in the molecular mechanism are numerous and have no specificity; finally, single base mutation of the target gene DNA gyrase A subunit causes missense mutation of amino acid codes of the DNA gyrase A subunit, and the corresponding protein configuration is changed, so that the binding capacity of the DNA gyrase A subunit and fluoroquinolone antibiotics is weakened, and further fluoroquinolone antibiotic resistance is generated.
The research based on the single base mutation mechanism is mainly researched through whole genome sequencing, and the defects of long experimental detection period, high consumption and the like exist.
Disclosure of Invention
The invention mainly aims to provide a primer pair for amplifying a fluoroquinolone antibiotic target gene, aims to specifically obtain a gyrA gene full-length sequence of bacteria, analyzes a base mutation site of the gyrA gene, and is favorable for quickly judging whether a strain has fluoroquinolone antibiotic resistance.
In order to achieve the above object, the present invention provides a primer pair for amplifying a fluoroquinolone antibiotic target gene, comprising:
the nucleotide sequence of the upstream primer is SEQ ID NO. 1;
the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target genes, wherein the nucleotide sequence of the upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any base; the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target gene, wherein the nucleotide sequence of the upstream primer is SEQ ID NO. 1; the nucleotide sequence of the downstream primer comprises a nucleotide sequence which is different from the nucleotide sequence of SEQ ID NO.2 by any one base.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target genes, wherein the nucleotide sequence of the upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any base;
the nucleotide sequence of the downstream primer comprises a nucleotide sequence which is different from the nucleotide sequence of SEQ ID NO.2 by any one base.
The invention also provides a kit for amplifying the fluoroquinolone antibiotic target genes, which comprises any one of the primer pairs for amplifying the fluoroquinolone antibiotic target genes.
Optionally, the kit further comprises a DNA polymerase and an amplification buffer.
The invention also provides a rapid detection method of the fluoroquinolone antibiotic resistance based on the single base mutation of the target gene, which comprises the following steps:
carrying out PCR amplification on the genome of the bacteria to be detected by using any one of the primer pair for amplifying the fluoroquinolone antibiotic target gene or the kit for amplifying the fluoroquinolone antibiotic target gene to obtain a target strip;
sequencing the target strip, comparing the target strip with a target gene sequence of fluoroquinolone antibiotic sensitive bacteria, and judging whether a mutant base exists;
if the mutant base exists, the bacteria to be detected are fluoroquinolone antibiotic resistant bacteria;
if the mutant base does not exist, the bacteria to be detected are fluoroquinolone antibiotic sensitive bacteria.
The technical scheme of the invention designs a full-length amplification primer for a fluoroquinolone antibiotic target gene gyrA gene, the nucleotide sequence of an upstream primer of the optimal specific primer is SEQ ID NO.1, the nucleotide sequence of a downstream primer is SEQ ID NO.2, the primer pair can obtain the full length of the fluoroquinolone antibiotic target gene gyrA of bacteria to be detected through conventional PCR, and then the full length of the fluoroquinolone antibiotic target gene gyrA of the bacteria to be detected is compared with the sequence of the gyrA gene of fluoroquinolone antibiotic sensitive bacteria to judge whether base mutation exists.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a diagram showing the electrophoresis result of the primer pair for amplifying fluoroquinolone antibiotic target genes on the target gene product of an amplification sensitive strain according to the present invention;
FIG. 2 shows the comparison of the target gene sequence of the sensitive strain and the target gene sequence of the model strain;
FIG. 3 is a diagram showing the electrophoresis results of the primer pair for amplifying fluoroquinolone antibiotic target genes of the present invention on the target gene products of the amplification sensitive strains and the mutant strains;
FIG. 4 shows the comparison of the target gene sequences of the sensitive strain and the mutant strain with the target gene sequence of the model strain;
FIG. 5 is a graph showing the results of experiments on the minimum inhibitory concentrations of enrofloxacin and ciprofloxacin against sensitive strains and mutant strains.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a primer pair for amplifying fluoroquinolone antibiotic target genes, which comprises the following components:
the nucleotide sequence of the upstream primer is SEQ ID NO. 1;
the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
The fluoroquinolone antibiotics specifically bind with DNA gyrase A subunits (gyrase A, gyrA) related to bacterial DNA replication, inhibit the activity of the DNA gyrase, interfere bacterial DNA to form supercoils, and prevent the normal function of the DNA, thereby playing a bactericidal role.
The single base mutation of the target gene DNA gyrase A subunit causes missense mutation of the amino acid code of the DNA gyrase A subunit, and the corresponding protein configuration is changed, so that the binding capacity of the DNA gyrase A subunit and fluoroquinolone antibiotics is weakened, and further fluoroquinolone antibiotic resistance is generated.
The full-length sequence of the gyrA gene is shown as SEQ ID NO.3, and an amplification primer is designed based on the full-length sequence of the gyrA gene, so that the nucleotide sequence of an upstream primer is SEQ ID NO.1 (gyrA-F: 5'-ATGAGCGACCTTGCGAGA-3'), and the nucleotide sequence of a downstream primer is SEQ ID NO.2 (gyrA-R: 5'-TTATTCTTCTTCTGGCTCGTCG-3'). The primer pair can be amplified to obtain a gyrA gene full-length sequence, and the gyrA gene full-length sequence of the bacteria to be detected can be compared with a gyrA gene full-length sequence of fluoroquinolone antibiotic sensitive bacteria to find whether the gyrA gene full-length sequence of the bacteria to be detected has mutation or not, so that whether the bacteria to be detected are fluoroquinolone antibiotic resistant bacteria or not is judged.
The technical scheme of the invention designs a full-length amplification primer for a fluoroquinolone antibiotic target gene gyrA gene, the nucleotide sequence of an upstream primer of the optimal specific primer is SEQ ID NO.1, the nucleotide sequence of a downstream primer is SEQ ID NO.2, the primer pair can obtain the full length of the fluoroquinolone antibiotic target gene gyrA of bacteria to be detected through conventional PCR, and then the full length of the fluoroquinolone antibiotic target gene gyrA of the bacteria to be detected is compared with the sequence of the gyrA gene of fluoroquinolone antibiotic sensitive bacteria to judge whether base mutation exists.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target genes, wherein the nucleotide sequence of the upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any base; the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target gene, wherein the nucleotide sequence of the upstream primer is SEQ ID NO. 1; the nucleotide sequence of the downstream primer comprises a nucleotide sequence which is different from the nucleotide sequence of SEQ ID NO.2 by any one base.
The invention also provides a primer pair for amplifying the fluoroquinolone antibiotic target genes, wherein the nucleotide sequence of the upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any base; the nucleotide sequence of the downstream primer comprises a nucleotide sequence which is different from the nucleotide sequence of SEQ ID NO.2 by any one base.
The nucleotide sequence of the upstream primer of the primer pair for amplifying the fluoroquinolone antibiotic target gene is SEQ ID NO.1, the nucleotide sequence of the downstream primer is SEQ ID NO.2, and the optimal primer sequence of the primer pair has good PCR amplification effect and no impurity band. It is understood that, in the PCR amplification, the primers and the templates are combined one by one through the base complementary pairing principle, wherein the nucleotide sequence of the primers is mutated by one base and cannot be combined with the corresponding template position, and the amplification effect is not influenced.
The invention also provides a kit for amplifying the fluoroquinolone antibiotic target genes, which comprises any one of the primer pairs for amplifying the fluoroquinolone antibiotic target genes.
Optionally, the kit further comprises a DNA polymerase and an amplification buffer.
The invention also provides a rapid detection method of the fluoroquinolone antibiotic resistance based on the single base mutation of the target gene, which comprises the following steps:
carrying out PCR amplification on the genome of the bacteria to be detected by using any one of the primer pair for amplifying the fluoroquinolone antibiotic target gene or the kit for amplifying the fluoroquinolone antibiotic target gene to obtain a target strip;
sequencing the target strip, comparing the target strip with a target gene sequence of fluoroquinolone antibiotic sensitive bacteria, and judging whether a mutant base exists;
if the mutant base exists, the bacteria to be detected are fluoroquinolone antibiotic resistant bacteria;
if the mutant base does not exist, the bacteria to be detected are fluoroquinolone antibiotic sensitive bacteria.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 design of primer set for amplification of fluoroquinolone antibiotic target genes
Taking a gyrA full-length sequence of a fluoroquinolone antibiotic sensitive bacterium Escherichia coli K12 substr MG1655(GenBank NC-000913.3) published by NCBI as reference, designing a specific Primer pair by using Primer Premier 5.0 software, and obtaining an upstream Primer nucleotide sequence as follows: gyrA-F: 5'-ATGAGCGACCTTGCGAGA-3' (SEQ ID NO.1), and the downstream primer nucleotide sequence is: gyrA-R: 5'-TTATTCTTCTTCTGGCTCGTCG-3' (SEQ ID NO. 2).
Example 2 verification of specificity of primer set obtained in example 1
Taking the genome DNA of a fluoroquinolone antibiotic sensitive type model strain Escherichia coli K12 as a template, and carrying out conventional PCR amplification on the full length of a gyrA gene of the fluoroquinolone antibiotic target gene by using an upstream primer and a downstream primer which are designed and obtained in example 1 and used for amplifying the fluoroquinolone antibiotic target gene, wherein the total volume of a PCR amplification reaction system is 50 mu l, and the specific steps are as follows:
the PCR amplification reaction program is as follows: 95 ℃ for 5 min; at 95 ℃ for 1 min; 30s at 55 ℃; 72 ℃ for 3 min; 30 cycles; 72 ℃ for 10 min; keeping the temperature at 16 ℃.
After the PCR was completed, 5. mu.l of the PCR product was subjected to agarose gel electrophoresis (1% w/v), and the condition of the band on the gel was observed by using a gel imaging system, and compared with the size of the gyrA fragment of the fluoroquinolone antibiotic sensitive type strain in NCBI.
As shown in FIG. 1, lanes 1 and 2 represent an Escherichia coli K12 group and a negative control group using water as a template, respectively, wherein a target band in lane 1 is amplified, the size of the target band is 2628bp, the size of the target band is consistent with the full-length size of a gyrA fragment of Escherichia coli K12 strain, the target band is better in unicity and has no non-specific amplification result, and the negative control group in lane 2 has no target band and primer dimer.
Subsequently, the PCR products were sent to the huadamo corporation for sequencing, and sequencing results of PCR amplification products of genomic DNA of Escherichia coli K12 were compared and analyzed with the gyrA gene of Escherichia coli K12 substr MG1655 model strain in NCBI using DNAMAN software, and the results are shown in fig. 2, which indicates that the PCR products have 100% sequence similarity with the gyrA gene of Escherichia coli K12 substr MG1655 model strain. This indicates that the primer pair has high specificity and can specifically amplify the full-length sequence of gyrA.
Example 3 method for rapidly detecting single base mutation of gyrA gene
(1) Obtaining the genome DNA of a mutant strain to be detected Escherichia coli EM (strain EM) and a mutant strain to be detected Escherichia coli CM (strain CM).
(2) And (2) amplifying and sequencing the full lengths of the gyrA genes of the strains to be detected EM and CM by using the specific primer pair obtained in the example 1 according to the PCR amplification reaction system and the PCR amplification reaction program in the example 2, comparing and analyzing the full lengths of the gyrA genes of the EM and CM and the gyrA gene of the model strain Escherichia coli K12 substr MG1655 by using DNAMAN software to obtain the information of the base mutation sites on the gyrA gene of the strain to be detected, and screening the fluoroquinolone antibiotic resistant strains according to the information of the base mutation sites.
The amplification results are shown in fig. 3, lanes 1 to 3 represent PCR amplification products of gyrA genes of Escherichia coli K12, mutant strain EM, and mutant strain CM, respectively, and it can be seen from fig. 3 that lanes 1 to 3 obtained better results by amplifying gyrA genes using the specific primer pair of example 1, no non-specific band and primer dimer appeared, and the size of the band also corresponds to the full length size of gyrA genes. While lane 4 is a negative control group in which no target band appears, indicating that the gyrA genes of the three strains were successfully obtained.
And performing sequence comparison by using DNAMAN software according to a sequencing result to obtain base mutation site information on gyrA genes of the strain EM and the strain CM. The sequence comparison result is shown in fig. 4, the first row of sequence in fig. 4 is the gyrA gene sequence of model strain Escherichia coli K12 substart MG1655 in NCBI, the second row is the gyrA gene sequencing result of sensitive strain Escherichia coli K12, the third row is the gyrA gene sequencing result of mutant strain CM, the fourth row is the gyrA gene sequencing result of mutant strain EM, according to the comparison result in fig. 4, the sensitive strain does not have base mutation, single base mutation (a > G) occurs at the 260 th base of mutant strain EM, the 87 th amino acid is mutated from aspartic acid to glycine, and the 248 th base of gyrA gene of mutant strain CM has single base mutation (C > T), the 83 th amino acid is mutated from serine to leucine. Therefore, the method can detect the single base mutation of the fluoroquinolone antibiotic target gene gyrA gene, thereby judging whether the bacteria have fluoroquinolone antibiotic resistance.
Example 4 phenotypic validation of fluoroquinolones antibiotic resistance of bacteria to be detected
The resistance of sensitive strain Escherichia coli K12, mutant strain EM and mutant strain CM to fluoroquinolone antibiotics was measured by 2-fold dilution method, and as shown in FIG. 5, the minimum inhibitory concentrations of enrofloxacin (labeled: ENR) and ciprofloxacin (labeled: CIP) to sensitive strain Escherichia coli K12 were 0.06mg/L and 0.016mg/L, respectively, and the minimum inhibitory concentrations of enrofloxacin (labeled: ENR) and ciprofloxacin (labeled: CIP) to mutant strain EM were 0.24mg/L and 0.128mg/L, respectively, and the minimum inhibitory concentrations of CM to mutant strain were 0.48mg/L and 0.256mg/L, respectively.
Compared with a sensitive strain Escherichia coli K12, the minimum inhibitory concentrations of enrofloxacin and ciprofloxacin to the mutant strain EM and the mutant strain CM are remarkably increased, and the mutant strain EM and the mutant strain CM are proved to be fluoroquinolone antibiotic resistant strains, which are consistent with the base mutation result of the gyrA gene in example 3, so that the fluoroquinolone antibiotic resistant strains can be rapidly detected through single base mutation site information on the gyrA gene.
The above description is only an alternative embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> rapid detection method for fluoroquinolone antibiotic resistance based on single base mutation and application thereof
<130> CP121010523C
<160> 3
<170> PatentIn version 3.3
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ctgcgtttgc agaaactgac cggtcttgag cacgaaaaac tgctcgacga atacaaagag 1440
ctgctggatc agatcgcgga actgttgcgt attcttggta gcgccgatcg tctgatggaa 1500
gtgatccgtg aagagctgga gctggttcgt gaacagttcg gtgacaaacg tcgtactgaa 1560
atcaccgcca acagcgcaga catcaacctg gaagatctga tcacccagga agatgtggtc 1620
gtgacgctct ctcaccaggg ctacgttaag tatcagccgc tttctgaata cgaagcgcag 1680
cgtcgtggcg ggaaaggtaa atctgccgca cgtattaaag aagaagactt tatcgaccga 1740
ctgctggtgg cgaacactca cgaccatatt ctgtgcttct ccagccgtgg tcgcgtctat 1800
tcgatgaaag tttatcagtt gccggaagcc actcgtggcg cgcgcggtcg tccgatcgtc 1860
aacctgctgc cgctggagca ggacgaacgt atcactgcga tcctgccagt gaccgagttt 1920
gaagaaggcg tgaaagtctt catggcgacc gctaacggta ccgtgaagaa aactgtcctc 1980
accgagttca accgtctgcg taccgccggt aaagtggcga tcaaactggt tgacggcgat 2040
gagctgatcg gcgttgacct gaccagcggc gaagacgaag taatgctgtt ctccgctgaa 2100
ggtaaagtgg tgcgctttaa agagtcttct gtccgtgcga tgggctgcaa caccaccggt 2160
gttcgcggta ttcgcttagg tgaaggcgat aaagtcgtct ctctgatcgt gcctcgtggc 2220
gatggcgcaa tcctcaccgc aacgcaaaac ggttacggta aacgtaccgc agtggcggaa 2280
tacccaacca agtcgcgtgc gacgaaaggg gttatctcca tcaaggttac cgaacgtaac 2340
ggtttagttg ttggcgcggt acaggtagat gactgcgacc agatcatgat gatcaccgat 2400
gccggtacgc tggtacgtac tcgcgtttcg gaaatcagca tcgtgggccg taacacccag 2460
ggcgtgatcc tcatccgtac tgcggaagat gaaaacgtag tgggtctgca acgtgttgct 2520
gaaccggttg acgaggaaga tctggatacc atcgacggca gtgccgcgga aggggacgat 2580
gaaatcgctc cggaagtgga cgttgacgac gagccagaag aagaataa 2628
Claims (7)
1. A primer pair for amplifying a fluoroquinolone antibiotic target gene is characterized by comprising:
the nucleotide sequence of the upstream primer is SEQ ID NO. 1;
the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
2. A primer pair for amplifying fluoroquinolone antibiotic target genes is characterized in that the nucleotide sequence of an upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any one base;
the nucleotide sequence of the downstream primer is SEQ ID NO. 2.
3. A primer pair for amplifying fluoroquinolone antibiotic target genes is characterized in that the nucleotide sequence of an upstream primer is SEQ ID NO. 1;
the nucleotide sequence of the primer comprises a nucleotide sequence which is different from SEQ ID NO.2 by any one base.
4. A primer pair for amplifying fluoroquinolone antibiotic target genes is characterized in that the nucleotide sequence of an upstream primer comprises a nucleotide sequence which is different from SEQ ID NO.1 into any one base;
the nucleotide sequence of the downstream primer comprises a nucleotide sequence which is different from the nucleotide sequence of SEQ ID NO.2 by any one base.
5. A kit for amplifying a fluoroquinolone antibiotic target gene, comprising the primer set for amplifying a fluoroquinolone antibiotic target gene according to any one of claims 1 to 4.
6. The kit for amplifying the fluoroquinolone antibiotic target gene as set forth in claim 5, further comprising DNA polymerase and an amplification buffer.
7. A rapid detection method of fluoroquinolone antibiotic resistance based on target gene single base mutation is characterized by comprising the following steps:
performing PCR amplification on the genome of a bacterium to be detected by using the primer pair for amplifying the fluoroquinolone antibiotic target gene according to any one of claims 1 to 4 or the kit for amplifying the fluoroquinolone antibiotic target gene according to claim 5 or 6 to obtain a target strip;
sequencing the target strip, comparing the target strip with a target gene sequence of fluoroquinolone antibiotic sensitive bacteria, and judging whether a mutant base exists;
if the mutant base exists, the bacteria to be detected are fluoroquinolone antibiotic resistant bacteria;
if the mutant base does not exist, the bacteria to be detected are fluoroquinolone antibiotic sensitive bacteria.
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Application publication date: 20210810 |