CN113481309A - Method for high-sensitivity rapid quantitative detection of novel chloramphenicol resistance gene in environmental sample and application thereof - Google Patents
Method for high-sensitivity rapid quantitative detection of novel chloramphenicol resistance gene in environmental sample and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of environmental and gene detection, in particular to a method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample and application thereof. The method comprises the steps of extracting genomic DNA of an environmental sample; specific primers of target genes and amplification thereof; connecting the target gene fragment with a pEASY-T1 vector, transforming the target gene fragment into competent cells, selecting a single clone, and carrying out colony PCR on M13F/M13R by using a universal primer to verify a positive clone; carrying out gel recovery and purification on the PCR product of the positive clone, and then carrying out gradient dilution by 10 times to construct a Q-PCR standard curve; and (3) quantitatively detecting the novel chloramphenicol resistance gene in the environmental sample by referring to the constructed standard curve. Compared with the prior art, the method only needs to construct a standard curve without depending on the integrity of a database, can greatly shorten the detection time and cost, and can synchronously detect the non-culturable microorganisms and the novel chloramphenicol resistance genes in different environments, thereby improving the identification accuracy.
Description
Technical Field
The invention relates to the technical field of environmental and gene detection, in particular to a method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample and application thereof.
Background
Antibiotic Resistance Genes (ARGs) are functional genes which enable bacteria to generate Antibiotic resistance, can be widely spread in an environmental medium through various direct or indirect transmission and transfer ways, and cause the appearance of various resistant bacteria and even 'super bacteria' in the spreading process, thereby accelerating the transmission and enhancement of the Antibiotic resistance and seriously threatening the human health and the stability of an ecosystem. Resistance gene contamination has been listed as one of the three new pollution problems in the 21 st century by the united nations health organization. Therefore, the analysis and detection of the novel antibiotic resistance gene have important practical significance for the assessment and reduction treatment of the level of ARGs pollution in the environment.
Chloramphenicol has been widely used for disease control in livestock and aquaculture industries, but its use in veterinary clinical and food animals has been gradually banned due to its strong toxic side effects. Research shows that chloramphenicol residues in the environment have a significant correlation with the generation of chloramphenicol resistance, and ARGs can be transmitted among different bacteria by means of horizontal gene transfer, thereby causing the residues in the environment to be more durable. However, in the face of selection pressure in different environments, such as multiple antibiotic stress, microorganisms may derive some novel resistance genes based on existing resistance to cope with one or more selection pressures. The existing novel ARGs detection technology mainly depends on modes such as a pure culture technology, a high-throughput sequencing technology and the like. Although the pure culture technology can realize accurate qualitative analysis of the novel ARGs, the method has long verification period and complicated operation, but cannot realize high-throughput detection of non-culturable microorganisms and environmental samples; the high-throughput sequencing technology depends on the integrity of the existing database, cannot detect the occurrence condition of the novel resistance gene in time, and has long detection period and high price. Therefore, it is necessary to develop a simple, rapid and highly sensitive method for rapidly detecting a novel chloramphenicol resistance gene in different environmental samples.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample and an application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample, comprising the following steps:
(1) pretreating an environmental sample and extracting genome DNA;
(2) amplification of a target gene;
(3) constructing a standard plasmid;
(4) constructing a Q-PCR standard curve;
(5) quantitative detection of a novel chloramphenicol resistance gene in an environmental sample.
Further, the pretreatment of the environmental sample in the step (1) is as follows: centrifuging the collected environmental sample at 6000-.
Further, the amplification of the target gene in the step (2) is specifically: designing a specific Primer pair by adopting Primer Premier 5.0 software according to the nucleic acid sequence of the novel chloramphenicol resistance gene; separately from the environmental sample genomic DNA and ddH2And performing target gene PCR amplification by taking O as a template, detecting a PCR product by using 1% agarose gel electrophoresis, and then purifying, recovering and sequencing a target gene fragment by using a gel recovery kit.
Further, the construction of the standard plasmid in the step (3) is specifically as follows: connecting the target gene fragment purified in the step (2) with a vector, transforming the target gene fragment into competent cells, selecting single clones, and carrying out colony PCR (polymerase chain reaction) verification on positive clones by using a universal primer pair M13F/M13R.
Further, the construction of the Q-PCR standard curve in the step (4) is specifically as follows: performing gel recovery and purification on the PCR product of the positive clone in the step (3), determining the concentration of DNA in the recovered product, and calculating the copy number of the DNA in the recovered product; then, the DNA in the recovered product is diluted by 10 times of gradient, and the diluted products are respectively used as templates to carry out Q-PCR to construct a corresponding standard curve.
Further, the quantitative determination of the novel chloramphenicol resistance gene in the environmental sample in the step (5) specifically comprises: performing Q-PCR by using the genome DNA of the environmental sample as a template, wherein the system and the procedure are the same as those of the Q-PCR technology in the step (4); and (4) quantifying the copy number of the novel chloramphenicol resistance gene in the environmental sample according to the Q-PCR standard curve constructed in the step (4).
Further, when the nucleic acid sequence of the novel chloramphenicol resistance gene is SEQ ID NO: 1, comprising the following steps:
(1) pretreatment of environmental samples and extraction of genomic DNA: centrifuging the collected environmental sample at 8000rpm and 4 ℃ for 10 minutes, removing the supernatant, collecting the precipitate, and storing the precipitate at-80 ℃; extraction of genomic DNA Using FastDNATMSpin Kit (MP Biomedicals, USA) Kit, the specific steps refer to Kit instructions;
(2) amplification of the target gene: according to the nucleic acid sequence of the novel chloramphenicol resistance gene, a Primer Premier 5.0 software is adopted to design a specific Primer pair, the size of a target fragment is 394bp, and the Primer sequences are as follows:
P2-F：5’-ACAAGGGTTGGCGACATTCA-3’SEQ ID NO：2，
P2-R：5’-TGCTTGACTATCCTGGCGAC-3’SEQ ID NO：3；
separately from the environmental sample genomic DNA and ddH2Performing PCR amplification of target gene with O as template, wherein the total volume of PCR amplification reaction system is 50 μ l, specifically 2 μ l for 25 μ l 2 × SYBR Green Pro Taq HS Mix, 10 μ M P2-F and P2-R, 1 μ l DNA template, and ddH2O20 mu l; the reaction procedure is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature at 72 ℃ for 10min and 16 ℃; detecting PCR product by 1% agarose gel electrophoresis and using colloidal goldThe kit is collected to purify, recover and sequence the target gene fragment;
(3) construction of a Standard plasmid: ligating the target gene fragment purified in step (2) with pEASY-T1 vector at 37 ℃ for 5 minutes, and then adding 5. mu.l of the ligation product to 50. mu.l of competent cells (Escherichia coli DH 5. alpha.) in ice bath for 20 minutes; heat shock is carried out for 30 seconds at the temperature of 42 ℃, ice bath is carried out for 2 minutes, 250 mul LB liquid culture medium is added, and the mixture is cultured for 1 hour at the temperature of 37 ℃ and the rpm of 150; sucking 80 μ l of the culture, spreading on LB plate containing ampicillin, sealing with sealing film, and culturing at 37 deg.C for overnight; selecting single clones, and carrying out colony PCR verification on positive clones by using a universal primer pair M13F/M13R;
(4) construction of Q-PCR Standard Curve: performing gel recovery and purification on the PCR product of the positive clone in the step (3), determining the concentration of DNA in the recovered product, and calculating the copy number of the DNA in the recovered product; then, carrying out 10-fold gradient dilution on DNA in the recovered product, respectively carrying out Q-PCR by taking the diluted product as a template to construct a corresponding standard curve, wherein the Q-PCR reaction system is 50 mu l, specifically 25 mu l of 2 XSSYBR Green Pro Taq HS Mix, 10 mu M P2-F and P2-R are respectively 2 mu l, the DNA template is 1 mu l, and ddH is added2O20 mu l; the reaction procedure is as follows: 30s at 95 ℃; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles;
(5) quantitative detection of a novel chloramphenicol resistance gene in an environmental sample: performing Q-PCR by using the genome DNA of the environmental sample as a template, wherein the system and the procedure are the same as those of the Q-PCR technology in the step (4); and (4) quantifying the copy number of the novel chloramphenicol resistance gene in the environmental sample according to the Q-PCR standard curve constructed in the step (4).
In another aspect, the invention provides an application of any one of the methods for the high-sensitivity rapid quantitative detection of the novel chloramphenicol resistance gene in the environmental sample, which is used for the determination of the novel chloramphenicol resistance gene in the environmental sample.
Compared with the prior detection technology, the invention has the following remarkable characteristics and advantages:
compared with the current pure culture technical method, the detection time can be greatly shortened by utilizing the Q-PCR method, meanwhile, the non-culturable microorganisms and the novel chloramphenicol resistance genes in different environments can be synchronously detected, and the identification accuracy is improved;
compared with the existing high-throughput sequencing technology, the method only needs to construct a standard curve, does not need to depend on the integrity of a database, greatly reduces the detection cost and time, has higher sensitivity, and can realize the DNA concentration of 1.48 multiplied by 10-4ng/. mu.l of the sample was subjected to quantitative analysis of the novel chloramphenicol resistance gene.
Drawings
FIG. 1 is a graph at 3.19X 104-3.19×1010A Q-PCR dissolution curve was established in the copies/ng DNA range, representing the rate of decrease of fluorescence intensity at different temperature conditions, wherein the dissolution curve exhibited a single peak, indicating better primer specificity.
FIG. 2 is a graph at 3.19X 104-3.19×1010A Q-PCR standard curve was established in the copies/ng DNA range.
FIG. 3 is the copy number of the novel chloramphenicol resistance gene in different environmental samples.
Detailed Description
The following will describe in detail the implementation of the present invention and the analysis of the results with reference to examples in order to fully understand the characteristics and effects of the present invention. It should be noted that the described embodiments are illustrative, not limiting, and are not intended to limit the scope of the invention.
The nucleotide sequence is SEQ ID NO: 1 as examples:
example 1: extraction of genomic DNA from environmental samples
Respectively collecting sludge-water mixed liquid samples from a reactor and an aerobic tank of a sewage treatment plant, centrifuging the collected samples at 8000rpm and 4 ℃ for 10 minutes, removing supernatant, collecting precipitate, storing the precipitate at-80 ℃, and extracting genome DNA by adopting FastDNATMSpin Kit (MP Biomedicals, USA) Kit, and the specific steps refer to the Kit instructions. Genomic DNA integrity and concentration was checked by 1% agarose gel electrophoresis and a NanoDrop One ultramicro spectrophotometer (Thermo Fisher Scientific, USA).
Example 2: amplification of target Gene
According to the nucleic acid sequence (SEQ ID NO: 1) of the novel chloramphenicol resistance gene, a Primer Premier 5.0 software was used to design a specific Primer pair with a target fragment size of 394bp, and the Primer sequences were as follows:
P2-F：5’-ACAAGGGTTGGCGACATTCA-3’SEQ ID NO：2，
P2-R：5’-TGCTTGACTATCCTGGCGAC-3’SEQ ID NO：3；
the genomic DNA and ddH obtained in example 12Performing PCR amplification of target gene with O as template, wherein the total volume of PCR amplification reaction system is 50 μ l, specifically 2 μ l for 25 μ l 2 × SYBR Green Pro Taq HS Mix, 10 μ M P2-F and P2-R, 1 μ l DNA template, and ddH2O20 mu l; the reaction procedure is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature at 72 ℃ for 10min and 16 ℃. PCR products detected by 1% agarose gel electrophoresis and then recovered with gel kit (
DNA Gel Extraction Kit, NEB) the gene fragment of interest was purified, recovered and sequenced.
Example 3: construction of Standard plasmids
The target gene fragment purified in example 2 was ligated with pEASY-T1 vector (Beijing holotype gold organism) at 37 ℃ for 5 minutes, and then 5. mu.l of the ligation product was added to 50. mu.l of competent cells (Escherichia coli DH 5. alpha.) in ice bath for 20 minutes; heat shock is carried out for 30 seconds at the temperature of 42 ℃, ice bath is carried out for 2 minutes, 250 mul LB liquid culture medium is added, and the mixture is cultured for 1 hour at the temperature of 37 ℃ and the rpm of 150; sucking 80 μ l of the culture, spreading on LB plate containing ampicillin, sealing with sealing film, and culturing at 37 deg.C for overnight; single clones were picked and colony PCR verified for positive clones using the universal primer pair M13F/M13R.
Example 4: construction of Q-PCR Standard Curve
The PCR product of the positive clone in example 3 was gel recovered and purified, and the DNA concentration (148 ng/. mu.l) in the recovered product was measured using a NanoDrop One ultramicro spectrophotometer (Thermo Fisher Scientific, USA), and the copy number of DNA in the recovered product was calculated according to the corresponding equation. The DNA in the recovered product was then diluted in a 10-fold gradient, i.e., a DNA concentration gradient of 1.48X 10-4148 ng/. mu.l, corresponding to a concentration of the novel chloramphenicol resistance gene of 3.19X 104-3.19×1010And (3) taking the dilutions as templates to perform Q-PCR, and analyzing data by using software after the reaction is finished to construct a corresponding standard curve. The Q-PCR reaction system was 50. mu.l, specifically 25. mu.l of 2 XSSYBR Green Pro Taq HS Mix, 2. mu.l each of 10. mu. M P2-F and P2-R, 1. mu.l of DNA template, ddH2O20 mu l; the reaction procedure is as follows: 30s at 95 ℃; 95 ℃ 5s, 60 ℃ 30s, 40 cycles, while adding the dissolution profile. As shown in FIG. 1, at 3.19X 104-3.19×1010A Q-PCR dissolution curve is established in the copies/ng DNA range, and the result shows that the dissolution curve presents a single peak, so that the primer specificity is better, and the method can be used for quantitative detection of the novel chloramphenicol resistance gene. The primer pair is adopted at 3.19 multiplied by 104-3.19×1010A Q-PCR standard curve was established in the copies/ng DNA range, as shown in FIG. 2, the standard curve R2The value is 0.99, the PCR amplification efficiency is 89.4%, and the result shows that the standard curve can be used for quantitative analysis of the novel chloramphenicol resistance gene in a subsequent environmental sample.
Example 5: quantitative detection of novel chloramphenicol resistance gene in environmental sample
The Q-PCR technique of example 4 was used to quantitatively detect the novel chloramphenicol resistance gene in samples from different environments in a reaction system of 50. mu.l, specifically 25. mu.l of 2 XSSYBR Green Pro Taq HS Mix, 2. mu.l each of 10. mu. M P2-F and P2-R, 1. mu.l of DNA template, ddH2O20 mu l; the reaction procedure is as follows: 30s at 95 ℃; 95 ℃ 5s, 60 ℃ 30s, 40 cycles, while adding the dissolution profile. According to the standard curve constructed above, the copy number of the novel chloramphenicol resistance gene in the environmental sample was quantified. As shown in FIG. 3, the resistance genes of the novel chloramphenicol in the aerobic tanks of the reactor and the sewage treatment plant were 1.45X 10, respectively5copies/ng DNA and 0.65X 102copies/ng DNA。
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> method for high-sensitivity rapid quantitative detection of novel chloramphenicol resistance gene in environmental sample and application thereof
<130> CP121010582C
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Claims (8)
1. A method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample is characterized by comprising the following steps:
(1) pretreating an environmental sample and extracting genome DNA;
(2) amplification of a target gene;
(3) constructing a standard plasmid;
(4) constructing a Q-PCR standard curve;
(5) quantitative detection of a novel chloramphenicol resistance gene in an environmental sample.
2. The method for high-sensitivity rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to claim 1, characterized in that the environmental sample is pretreated in step (1) by: centrifuging the collected environmental sample at 6000-.
3. The method for high-sensitivity rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to claim 1, characterized in that the amplification of the target gene in step (2) is specifically: designing a specific Primer pair by adopting Primer Premier 5.0 software according to the nucleic acid sequence of the novel chloramphenicol resistance gene; separately from the environmental sample genomic DNA and ddH2And performing target gene PCR amplification by taking O as a template, detecting a PCR product by using 1% agarose gel electrophoresis, and then purifying, recovering and sequencing a target gene fragment by using a gel recovery kit.
4. The method for high-sensitivity rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to claim 3, characterized in that the standard plasmid in step (3) is specifically constructed as follows: connecting the target gene fragment purified in the step (2) with a vector, transforming the target gene fragment into competent cells, selecting single clones, and carrying out colony PCR (polymerase chain reaction) verification on positive clones by using a universal primer pair M13F/M13R.
5. The method for high-sensitivity rapid quantitative detection of a novel chloramphenicol resistance gene in an environmental sample according to claim 4, characterized in that the Q-PCR standard curve in step (4) is constructed by: performing gel recovery and purification on the PCR product of the positive clone in the step (3), determining the concentration of DNA in the recovered product, and calculating the copy number of the DNA in the recovered product; then, the DNA in the recovered product is diluted by 10 times of gradient, and the diluted products are respectively used as templates to carry out Q-PCR to construct a corresponding standard curve.
6. The method for high-sensitivity rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to claim 5, characterized in that the quantitative determination of the novel chloramphenicol resistance gene in the environmental sample in the step (5) is specifically: performing Q-PCR by using the genome DNA of the environmental sample as a template, wherein the system and the procedure are the same as those of the Q-PCR technology in the step (4); and (4) quantifying the copy number of the novel chloramphenicol resistance gene in the environmental sample according to the Q-PCR standard curve constructed in the step (4).
7. The method for high-sensitivity rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to claim 1, characterized in that, when the nucleotide sequence of the novel chloramphenicol resistance gene is SEQ ID NO: 1, comprising the following steps:
(1) pretreatment of environmental samples and extraction of genomic DNA: centrifuging the collected environmental sample at 8000rpm and 4 ℃ for 10 minutes, removing the supernatant, collecting the precipitate, and storing the precipitate at-80 ℃; extraction of genomic DNA Using FastDNATMSpin Kit (MP Biomedicals, USA) Kit, the specific steps refer to Kit instructions;
(2) amplification of the target gene: according to the nucleic acid sequence of the novel chloramphenicol resistance gene, a Primer Premier 5.0 software is adopted to design a specific Primer pair, the size of a target fragment is 394bp, and the Primer sequences are as follows:
P2-F: 5'-ACAAGGGTTGGCGACATTCA-3' SEQ ID NO: 2,
P2-R: 5'-TGCTTGACTATCCTGGCGAC-3' SEQ ID NO: 3;
separately from the environmental sample genomic DNA and ddH2Performing PCR amplification of target gene with O as template, wherein the total volume of PCR amplification reaction system is 50 μ l, specifically 2 μ l for 25 μ l 2 × SYBR Green Pro Taq HS Mix, 10 μ M P2-F and P2-R, 1 μ l DNA template, and ddH2O20 mu l; the reaction procedure is: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 40 s; keeping the temperature at 72 ℃ for 10min and 16 ℃; detecting the PCR product by 1% agarose gel electrophoresis, and then purifying, recovering and sequencing the target gene fragment by using a gel recovery kit;
(3) construction of a Standard plasmid: ligating the target gene fragment purified in step (2) with pEASY-T1 vector at 37 ℃ for 5 minutes, and then adding 5. mu.l of the ligation product to 50. mu.l of competent cells (Escherichia coli DH 5. alpha.) in ice bath for 20 minutes; heat shock is carried out for 30 seconds at the temperature of 42 ℃, ice bath is carried out for 2 minutes, 250 mul LB liquid culture medium is added, and the mixture is cultured for 1 hour at the temperature of 37 ℃ and the rpm of 150; sucking 80 μ l of the culture, spreading on LB plate containing ampicillin, sealing with sealing film, and culturing at 37 deg.C for overnight; selecting single clones, and carrying out colony PCR verification on positive clones by using a universal primer pair M13F/M13R;
(4) construction of Q-PCR Standard Curve: performing gel recovery and purification on the PCR product of the positive clone in the step (3), determining the concentration of DNA in the recovered product, and calculating the copy number of the DNA in the recovered product; then, carrying out 10-fold gradient dilution on DNA in the recovered product, respectively carrying out Q-PCR by taking the diluted product as a template to construct a corresponding standard curve, wherein the Q-PCR reaction system is 50 mu l, specifically 25 mu l of 2 XSSYBR Green Pro Taq HS Mix, 10 mu M P2-F and P2-R are respectively 2 mu l, the DNA template is 1 mu l, and ddH is added2O20 mu l; the reaction procedure is as follows: 30s at 95 ℃; 5s at 95 ℃, 30s at 60 ℃ and 40 cycles;
(5) quantitative detection of a novel chloramphenicol resistance gene in an environmental sample: performing Q-PCR by using the genome DNA of the environmental sample as a template, wherein the system and the procedure are the same as those of the Q-PCR technology in the step (4); and (4) quantifying the copy number of the novel chloramphenicol resistance gene in the environmental sample according to the Q-PCR standard curve constructed in the step (4).
8. Use of the method for the highly sensitive rapid quantitative determination of a novel chloramphenicol resistance gene in an environmental sample according to any of claims 1 to 7, which is characterized by being used for the determination of a novel chloramphenicol resistance gene in an environmental sample.
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