CN103981263B - The quantitative detecting method of antibiotics resistance gene in soil - Google Patents

Abstract

The invention discloses a method for quantitatively detecting antibiotic resistance genes in soil, comprising the following steps: 1) selecting pigs that often take antibiotics, using pig feces DNA as a template, and using PCR to amplify target resistance gene fragments; 2) 1. Cut the gel to recover and purify the amplified target resistance gene, connect the resistance gene to the T carrier, then transform the competent Escherichia coli cells, and select positive clones after smearing the LB plate; 3), use agarose gel electrophoresis Detect the quality of the expanded culture solution, select the successfully cloned bacteria solution to extract the plasmid, detect the concentration of the plasmid, convert the copy number of the resistance gene carried by the plasmid, and dilute it by 10 times as a dilution gradient for use as a resistance gene standard; 4) Genomic DNA of the soil is extracted, and the soil sample DNA and the resistance gene standard are subjected to quantitative PCR amplification at the same time, so as to finally obtain the copy number of the resistance gene of the soil sample.

Description

Quantitative detection method of antibiotic resistance gene in soil

technical field

The invention belongs to the technical field of detection of soil environmental pollutants, and in particular relates to a quantitative detection method of antibiotic resistance genes in soil.

Background technique

In 1928, Alexander Fleming discovered penicillin. In the 1940s, penicillin was put into large-scale production, and then streptomycin, tetracycline, vancomycin, methicillin and other antibiotics were discovered one after another. my country is a big producer and consumer of antibiotics. In 2007, 210,000 tons of antibiotics were produced. In addition to exporting about 30,000 tons, 180,000 tons were used domestically (among them, aquaculture accounted for 97,000 tons). At present, antibiotics have been widely used in the prevention and treatment of human and animal diseases, and are also used as growth promoters to increase the growth rate of livestock and poultry. It has become a habit to use a large amount of antibiotics in modern farms. In the case of using one antibiotic to produce drug resistance, on the one hand, the farms increase the dosage of antibiotics, and on the other hand, they are constantly looking for new antibiotics. At the same time, they will also use Compound antibiotic preparations. Agricultural soil is the main destination of veterinary antibiotics. Most antibiotics are excreted in the form of parent compounds with feces and urine. More than 80% of livestock and poultry manures are directly applied to farmland without comprehensive treatment, resulting in antibiotics in the soil. Accumulation of resistance genes (ARGs) and tolerant bacteria. There are many kinds of microorganisms in the soil, and the number is huge. High-density microorganisms are more likely to promote the horizontal transfer of genes. ARGs can be integrated into some mobile genetic elements, such as plasmids, transposons, integrons, etc., through direct or indirect contact ( Such as food chain) and other ways into the human body, increase the drug resistance of the human body, and then pose long-term potential harm to human health and the entire ecosystem. Antibiotic therapy is the main or even the only therapy for infectious diseases, and the increase and spread of drug resistance of pathogenic bacteria has become a huge problem facing the global disease treatment.

Pruden et al. proposed as early as 2006 that ARGs were regarded as a new type of environmental pollutants. There are many types of antibiotic resistance genes, and the number is huge. For example, there are three categories and more than 40 types of genes encoding tetracycline resistance. Establishing a quantitative detection method for ARGs in soil is useful for further systematic monitoring and evaluation of antibiotic resistance genes in the soil environment. Its ecological risk is very necessary. At present, there is no commercial resistance gene standard product, and ordinary PCR can only detect the presence or absence of the gene qualitatively, and the sensitivity is not high.

Contents of the invention

The technical problem to be solved by the invention is to provide a quantitative detection method for antibiotic resistance genes in soil.

In order to solve the above-mentioned technical problems, the invention provides a kind of quantitative detection method of antibiotic resistance gene in soil, comprises the following steps:

1) Select pigs that often take antibiotics, and the types of antibiotics taken should include the antibiotics corresponding to the target resistance gene;

Extract the genomic DNA of the pig manure of the above-mentioned pigs, and use the pig manure DNA as a template to amplify the target resistance gene fragment by PCR;

Remarks: If the target resistance gene cannot be obtained, re-select pig manure and repeat the above step 1) until the target resistance gene can be obtained;

2), cutting the gel to recover and purify the amplified target resistance gene, connect the resistance gene to the T carrier, then transform competent Escherichia coli cells, select positive clones after smearing LB plates, and cultivate them with LB culture medium (enlarge to cultivate);

3) Use agarose gel electrophoresis to detect the quality of the expanded culture solution, select the successfully cloned bacteria solution to extract the plasmid, detect the plasmid concentration, convert the copy number of the resistance gene carried by the plasmid, and dilute it by 10 times as a dilution gradient Standby, as a resistance gene standard;

4), extract the genomic DNA of the soil, perform quantitative PCR amplification on the soil sample DNA and the resistance gene standard at the same time, generate a standard curve according to the copy number of the resistance gene standard and its amplified CT value, and then obtain the soil The copy number of the resistance gene of the sample.

As the improvement of the quantitative detection method of antibiotic resistance gene in soil of the present invention:

The target resistance gene is 100-350bp in length;

The target resistance genes are: penicillin resistance gene blaTEM gene, tetracycline resistance gene tetC gene, tetracycline resistance gene tetM gene.

As the further improvement of the quantitative detection method of antibiotic resistance gene in the soil of the present invention:

In the step 3), the formula for converting the plasmid concentration into the absolute template copy number carried by each μL plasmid solution is: Copies/μL=[x/(a+b)×660]×10 ^-9 ×6.02×10 ²³ , where x is the plasmid concentration (ng/μL); a is the vector length (bp); b is the target resistance gene length (bp).

As a further improvement of the quantitative detection method of the antibiotic resistance gene in the soil of the present invention: in the step 1),

The reaction system for PCR amplification is:

2×EasyTaqPCRSuperMix10μL;

DNA template (concentration is 18-20ng/μL, for example, 19.1ng/μL) 1μL;

1 μL each of the front and back primers (10 μM) of the target resistance gene;

The remainder is double distilled water;

The reaction conditions are:

Pre-denaturation at 94°C for 4 minutes,

Extend at 72°C for 7 min.

Remarks: For different target genes, set the annealing gradient according to the Tm value of the primers, and optimize to the corresponding annealing temperature.

As the further improvement of the quantitative detection method of antibiotic resistance gene in the soil of the present invention:

When the target resistance gene is the penicillin resistance gene blaTEM gene, the sequences of the front and back primers are AAGCCATACCAAACGACGAG and CCGCCTCCATCCAGTCTAT respectively; the annealing temperature is 66°C;

When the target resistance gene is the tetracycline resistance gene tetC gene, the sequences of the front and back primers are GCGGGATATCGTCCATTCCG and GCGTAGAGGATCCACAGGACG respectively; the annealing temperature is 68°C;

When the target resistance gene is the tetracycline resistance gene tetM gene, the sequences of the front and rear primers are ACAGAAAGCTTTATTAAC and TGGCGTGTCTATGATGTTCAC respectively; the annealing temperature is 46°C.

Remarks: When the target resistance gene is the penicillin resistance gene blaTEM gene, its front and rear primer sequences are specially created by the present invention (that is, this primer sequence has not been reported); the blaTEM gene fragments reported so far are greater than 350bp, the length It is not suitable for quantitative PCR amplification. We selected 30 known blaTEM gene sequences (GenBank database) for comparison and found that the blaTEM gene is well conserved. The primers were redesigned according to the conserved sequence, and the generated target fragment was 129bp. The length is suitable for quantitative PCR amplification, and this pair of primers can be considered as universal primers for detecting blaTEM gene.

As the further improvement of the quantitative detection method of antibiotic resistance gene in the soil of the present invention:

In the step ⁴ ), the R2 value for generating the standard curve should be greater than 0.98, the closer to 1, the higher the reliability of the result; the range of amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal.

In the present invention:

Step 1) is to take pig manure from a farm where antibiotics have been used for a long time to extract standard DNA. The details are as follows: Pig manure was collected in farms where antibiotics were used for a long time, and the genomic DNA of pig manure was extracted with a soil genome extraction kit (FastDNA spinkit for soil); using pig manure DNA as a template, a common PCR program was used to amplify the target resistance gene. The length should be between 100-350bp.

In step 2) and step 3), electrophoresis detection is performed after PCR amplification, and the correct target gene fragment is recovered by gel cutting, purified and amplified, inserted into the T carrier, and then commercialized competent Escherichia coli cells are used for transform. After coating the LB plate, select positive clones (single colonies that meet the color of white instead of blue are positive clones), and culture them in small quantities with LB culture medium. Take 1 μL of bacterial liquid for bacterial liquid PCR (primer is universal carrier primer M13, pre-denaturation at 94°C for 10 minutes, annealing temperature at 55°C); verify whether the target gene is cloned successfully (bacterial liquid PCR product is detected by agarose gel electrophoresis, when its length When the length of the load body is the length of the target resistance gene, the cloning is determined to be successful).

Take 2-4mL of the successfully cloned bacteria solution to extract the plasmid, sequence the plasmid, and compare the inserted gene fragment to be correct, then use NanoDrop to detect the plasmid concentration, convert the copy number of the resistance gene carried by the plasmid, and convert the plasmid concentration into the amount carried by each μL plasmid solution The formula for the absolute template copy number is: Copies/μL=[x/(a+b)×660]×10 ^-9 ×6.02×10 ²³ , where x is the plasmid concentration (ng/μL); a is the vector length ( bp); b is the length of the target resistance gene (bp). And according to the 10-fold dilution serial dilution for use as the resistance gene standard.

In step 4), the genomic DNA of the soil sample to be tested is extracted with the soil genome extraction kit (FastDNA spinkit for soil), and the soil sample DNA and the standard are simultaneously subjected to quantitative PCR amplification. The quantitative PCR reaction system is a 20 μL reaction system, specifically including 10 μL2 ×SYBRqPCRSuperMix, 1 μL template (concentration diluted to 1-5 ng/μL) and 0.4 μL each of the front and back primers (10 μM) of the target gene; the balance is twice redistilled water.

The reaction conditions are: pre-denaturation at 94°C for 30s; 94°C for 5s, annealing for 15s (the annealing temperature of the three different target resistance genes is the same as the optimized annealing temperature set in step 1), 72°C for 10s, 40 Cycle, the final melting curve program is 95°C for 5s, 60°C for 1min, and the fluorescence signal is continuously detected while the temperature is raised to 95°C. ^The R2 value of the generated standard curve should be greater than 0.98, the closer to 1, the higher the reliability of the result. The amplification efficiency E should be in the range of 0.8-1.2, the closer to 1, the more ideal. A standard curve is generated from the copy number of the known plasmid and its amplified CT value, and then the copy number of the resistance gene of the soil sample is obtained.

The present invention is characterized in that the pig manure DNA sample is used to accurately and quickly prepare the resistance gene standard product. Compared with pig manure, most of the soil is not polluted by antibiotics or is less polluted, and the copy number of the resistance gene in the soil is relatively low. It is difficult to amplify the target resistance gene through qualitative PCR with low sensitivity, let alone prepare standard products. Regarding the extraction of total genes from pig manure, we tested and compared manual extraction and other kit extraction methods, and found that the soil genome extraction kit (FastDNA spinkit for soil) can be used to extract pig manure genes, and it is very convenient and accurate. Plasmid standard products can be stored at -20°C, and 10-20 times can be used for one preparation; the resistance gene standard product and soil sample DNA are simultaneously subjected to quantitative PCR amplification. The present invention is easy to operate, except for the preparation of the standard product, the soil sample can obtain the detection result within 3 hours; the detection result has high sensitivity, and the quantity is also more intuitive; it does not depend on the cultivation of microorganisms, and can detect non-culturable microorganisms ( More than 99.9% of the microorganisms cannot be cultivated) The resistance gene carried by it makes the final quantification result more comprehensive and credible.

The invention clarifies the quantitative detection methods of the penicillin resistance gene blaTEM gene, the tetracycline resistance gene tetC gene and the tetracycline resistance gene tetM gene, and can also be used as a reference for the quantitative detection of other resistance genes.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is blaTEM gene quantitative PCR standard curve;

Fig. 2 is the tetC gene quantitative PCR standard curve;

Fig. 3 is the tetM gene quantitative PCR standard curve;

Figure 4 is a quantitative comparison of tetracycline resistance genes in different soils.

detailed description

The following examples will further illustrate the feasibility and application effect of the method of the present invention.

Example 1, Quantitative detection of penicillin resistance gene blaTEM in different soils

The β-lactamase encoded by blaTEM can invalidate penicillin. Get fresh forest soil, pasture soil, and tea garden soil to measure the soil water content. Each soil sample and pig manure sample weigh 0.25g to extract DNA, and the volume is 100 μ L [the pig manure The feces produced by pigs taking antibiotics for a long time; the DNA of the above soil samples and pig manure samples were extracted using the Soil Genome Extraction Kit (FastDNAspinkitforsoil)].

30 known blaTEM gene sequences (GenBank database) were selected for comparison, and it was found that the blaTEM gene was well conserved. The primers were redesigned according to the conserved sequences, and the generated target fragment was 129 bp. This pair of primers can be considered as universal primers for detecting blaTEM gene.

Use pig feces sample DNA as a template for PCR. The common PCR reaction system is a 20 μL reaction system, which specifically includes 10 μL 2×EasyTaqPCRSuperMix, 1 μL template (19.1 ng/μL concentration) and 1 μL each of blaTEM front and back primers (10 μM), and the balance For the double redistilled water. The reaction conditions were: pre-denaturation at 94°C for 4 minutes; 35 cycles of annealing at 94°C for 45 s, annealing at 66°C for 45 s, and 1 min at 72°C; and finally extension at 72°C for 7 minutes.

2), cutting the gel to recover and purify the amplified blaTEM gene fragment (129bp), connect the fragment to the T vector, and then transform competent Escherichia coli cells, and select positive clones after smearing LB plates (satisfied that the color is white instead of The blue single colony is a positive clone), and expanded culture with LB medium (37°C, 200 rpm overnight). Take 1 μL of bacterial liquid for bacterial liquid PCR to verify that the target gene is successfully cloned. The above PCR verification system is: 20 μL reaction system, specifically including 10 μL of 2×EasyTaqPCRSuperMix, 1 μL of bacterial liquid (concentration: 19.1ng/μL) as DNA template and universal carrier primer M13 (10 μM) 1 μL each of the front and rear primers, and the balance is redistilled water twice; the reaction conditions are: pre-denaturation at 94°C for 10 min, 94°C for 45 s, annealing at 55°C for 45 s, 72°C for 1 min, 35 cycles, and finally extension at 72°C for 7 min ; When the bacteria liquid PCR product is detected by agarose gel electrophoresis, when its length is the length of the length loading body of the target resistance gene, then it is judged as the amplified bacterial liquid of successful cloning);

Take 3mL of the positive bacterial liquid to extract the plasmid, send the plasmid to the sequencing company for sequencing, and compare the inserted gene fragments on the NCBI website according to the returned sequencing results, and use NanoDrop to detect that the plasmid concentration is 62.9ng/μL, according to the conversion formula Copies/μL =[x/(a+b)×660]×10 ^-9 ×6.02×10 ²³ , where x is the plasmid concentration of 62.9ng/μL; a is the length of T vector 3829bp; b is the length of blaTEM gene 129bp. The copy number of the blaTEM gene carried by the plasmid was calculated to be 1.43×10 ¹⁰ , and diluted to a copy number of 1.43×10 ⁸ to 1.43×10 ³ according to a 10-fold dilution gradient as a standard.

Simultaneously carry out quantitative PCR amplification of soil sample DNA and standards, specifically including 10 μL 2×SYBRqPCRSuperMix, 1 μL template (based on the above-mentioned standard with a copy number of 1.43×10 ⁸ to 1.43×10 ³ , and soil sample DNA (concentration diluted to 1 -5ng/μL) as a template) and 0.4 μL of primers (10 μM) before and after blaTEM; the reaction conditions were: pre-denaturation at 94 °C for 30 s; The program was 95°C for 5s, 60°C for 1min, and the fluorescent signal was detected continuously while the temperature was raised to 95°C.

The standard curve is y=-3.6666x+41.812 (FIG. 1), where x is the log ₁₀ gene blaTEM copy number, and y is the critical cycle number, which is the CT reading of the quantitative PCR instrument. According to the K value calculation of the standard curve, the amplification efficiency e is 87.38% (the formula is e=10 ^-1/k -1), R ² =0.9937, and the copy number of the blaTEM gene of the soil sample is obtained, and the blaTEM gene per gram of dry soil Copy number of blaTEM = blaTEM gene copy number obtained from the marking line × 100 μL × template dilution factor / [0.25 × (1-water content)], therefore, forest soil 1.58 × 10 ⁶ copies/g, pasture soil 1.08 × 10 ⁶ Copies/g, tea garden soil 1.57×10 ⁶ copies/g. It shows that the blaTEM gene is contained in the three different types of soils, and there is little difference in the quantity.

Verification test 1. The forest soil, pasture soil, and tea garden soil in Example 1 were quantitatively detected according to the probe method. The front and rear primer sequences were CACTATTCTCAGAATGACTTGGT, and the CATGACAGTAAGAGAATTATGCA probe was CCAGTCACAGAAAAGCATCTTACGG. The results obtained were: forest soil 1.31×10 ⁶ copies/ g. Pasture soil 0.98×10 ⁶ copies/g, tea garden soil 1.29×10 ⁶ copies/g.

Comparative Example 1-1, the annealing temperature in Example 1 was increased from "66°C" to "70°C"; the rest were exactly the same as in Example ¹ ; the obtained standard curve was meaningless, in which the R2 value was 0.815, and the amplification efficiency E is 22.6 (the R2 value of generating the standard curve should be greater than 0.98, the closer to ¹ , the higher the reliability of the result. The range of amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal.) Therefore, the final The resulting copy number of the blaTEM gene per gram of dry soil is not reliable.

Comparative Example 1-2, the annealing temperature in the quantitative PCR program of Example 1 was lowered from "66°C" to "55°C"; the rest were exactly the same as in Example ¹ ; the obtained standard curve was meaningless, and the R2 value was 0.994, The amplification efficiency E is ^2.10 (the R2 value of the generated standard curve should be greater than 0.98, the closer to 1, the higher the reliability of the result. The range of the amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal. ) so the resulting number of copies of the blaTEM gene per gram of dry soil is not reliable.

Example 2, Quantitative comparison of tetracycline resistance genes tetC and tetM in different soils

tetC encodes an efflux pump protein, which can excrete tetracycline out of the cell, thereby reducing the intracellular drug concentration to play a protective role. tetM encodes a ribosome protection protein, which causes a change in ribosome configuration, preventing tetracycline from binding to ribosomes and failing.

Get fresh forest soil, pasture soil, tea garden soil and measure soil water content, each soil sample and pig manure sample weigh 0.25g to extract DNA, and volume is 100 μ L [the described pig manure is the excrement that the pig place that takes antibiotic for a long time; The above-mentioned soil DNA samples and pig manure samples were extracted using the Soil Genome Extraction Kit (FastDNAspinkitforsoil)].

The primer sequences before and after tetC are GCGGGATATCGTCCATTCCG, GCGTAGAGGATCCACAGGACG, respectively, and the target fragment length is 207bp.

The front and back primer sequences of tetM are ACAGAAAGCTTTATTAAC, TGGCGTGTCTATGATGTTCAC, and the target fragment length is 171bp.

Use pig manure sample DNA as a template to carry out PCR respectively. The common PCR reaction system is a 20 μL reaction system, which specifically includes 10 μL 2×EasyTaqPCRSuperMix, 1 μL template (concentration: 19.1ng/μL) and a primer before and after a target resistance gene (10 μM) 1 μL each, and the rest was twice double-distilled water. The reaction conditions were: pre-denaturation at 94°C for 4min; 35 cycles at 94°C for 45s, annealing for 45s, and 72°C for 1min; and finally extension at 72°C for 7min. The tetC annealing temperature is 68°C, and the tetM annealing temperature is 46°C.

Cut the gel to recover and purify the amplified target resistance gene fragment (tetC is 207bp; tetM is 171bp), connect the fragment to the T vector, then transform competent E. A single colony that is white instead of blue is a positive clone), and expanded culture with LB medium (37°C, 200 rpm overnight). Take 1 μL of bacterial liquid for bacterial liquid PCR to verify the successful cloning of the target gene (the above PCR verification system is: 20 μL reaction system, specifically including 10 μL of 2×EasyTaqPCRSuperMix, 1 μL of bacterial liquid as a DNA template, and 1 μL of universal carrier primer M13 (10 μM) before and after primers , and the balance is twice redistilled water; the reaction conditions are: 94°C pre-denaturation for 10 minutes, 94°C for 45s, 55°C for 45s, 72°C for 1min, 35 cycles, and finally 72°C extension for 7min; Agarose gel electrophoresis detection, when its length is the length of the length of the target resistance gene load body, then it is judged as the amplified bacterial solution of successful cloning);

Take 3mL of the positive bacterial liquid to extract the plasmid, send the plasmid to the sequencing company for sequencing, and compare the inserted gene fragments on the GenBank database according to the returned sequencing results, then use NanoDrop to detect the plasmid concentration, according to the conversion formula Copies/μL=[x/( a+b)×660]×10 ^-9 ×6.02×10 ²³ , where x is the plasmid concentration in ng/μL; a is the length of the T vector in bp; b is the length of the target resistance gene in bp. Calculate the copy number of the target resistance gene carried by the plasmid, and dilute it by 10 times as a dilution series as a standard. The copy number of the tetC gene was obtained to be 1.68×10 ¹⁰ , and the copy number of the tetM gene was 7.66×10 ⁹ , and the copy number was 1.68×10 ⁸ to 1.68×10 ³ as a standard by diluting 10 times as a dilution gradient. According to the 10-fold dilution gradient, the copy number is 7.66×10 ⁷ ~7.66×10 ² as the standard.

Quantitative PCR amplification was performed on the soil sample DNA and the standard at the same time. The tetC gene and the tetM gene had to be amplified separately due to different annealing temperatures, specifically including 10 μL 2×SYBRqPCRSuperMix, 1 μL template (the standard product of the above tetC gene or tetM gene, and the soil Sample DNA was used as a template, diluted to 1-5ng/μL) and 0.4 μL each of primers (10 μM) before and after the target resistance gene; the reaction conditions were: pre-denaturation at 94°C for 30s; 94°C for 5s, annealing for 15s, 72°C for 10s, 40°C cycle, the tetC annealing temperature was 68°C, and the tetM annealing temperature was 46°C. The final melting curve program was 95°C for 5s, 60°C for 1min, and the fluorescence signal was detected continuously while the temperature was raised to 95°C.

The standard curve of the tetC gene is y=-3.3972x+40.705 (Fig. 2), where x is the log ₁₀ gene tetC copy number, and y is the critical cycle number, which is the CT reading of the quantitative PCR instrument. According to the K value calculation of the standard curve, the amplification efficiency e is 96.95% (the formula is e=10 ^-1/k -1), R ² =0.9954, and the copy number of the tetC gene of the soil sample is obtained, and the tetC gene per gram of dry soil Copy number of the tetC gene = copy number of tetC gene obtained from the marking line × 100 μL × template dilution factor / [0.25 × (1-water content)], forest soil 7.49 × 10 ³ copies/g, pasture soil 1.63 × 10 ⁶ copies/ g. Tea garden soil: 2.93×10 ³ copies/g. It indicated that the tetC gene was contained in different types of soil, but the quantity varied greatly, and the pasture soil was highly polluted by the resistance gene.

The standard curve of the tetM gene is y=-3.3903x+42.193 (Fig. 3), where x is the log ₁₀ gene tetM copy number, and y is the critical cycle number, which is the CT reading of the quantitative PCR instrument. According to the K value calculation of the standard curve, the amplification efficiency e is 97.22% (the formula is e=10 ^-1/k -1), R ² =0.9975, and the copy number of the tetM gene of the soil sample is obtained, per gram of dry soil tet, M gene copy number = tetM gene copy number obtained from the marking line × 100 μL × template dilution factor / [0.25 × (1-water content)], forest soil 4.64 × 10 ⁴ copies/g, pasture soil 6.59 × 10 ⁶ Copies/g, tea garden soil 3.85×10 ⁴ copies/g. It indicated that the tetM gene was contained in different types of soil, but the quantity varied greatly, and the pasture soil was highly polluted by the resistance gene.

Comparing the two tetracycline resistance genes in the soil (Figure 4), it was found that the content of the two resistance genes in the pasture soil was significantly higher than that in the other two soils, indicating that the pasture soil was more polluted. At the same time, the content of tetM gene in various soils was higher than that of tetC gene, which indicated that tetM gene was more common than tetC gene.

Comparative example 2-1, the two kinds of annealing temperatures in the quantitative PCR program of embodiment 2 are all increased by 5 degrees; the rest are completely the same as embodiment 2;

^The R2 value of the standard curve corresponding to the tetracycline resistance gene tetC was 0.907, and the amplification efficiency E was 7.650;

^The R2 value of the standard curve corresponding to the tetracycline resistance gene tetM is 0.538, and the amplification efficiency E is 180;

The resulting standard curve is meaningless (the R2 value of the generated standard curve should be greater than 0.98, the closer to ¹ , the higher the reliability of the result. The range of amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal. ) Therefore, the final copy number of the target resistance gene per gram of dry soil is unreliable.

Comparative example 2-2, the two kinds of annealing temperatures in embodiment 2 are all lowered by 5°C; the rest are completely the same as embodiment 2;

^The R2 value of the standard curve corresponding to the tetracycline resistance gene tetC was 0.445, and the amplification efficiency E was 463;

^The R2 value of the standard curve corresponding to the tetracycline resistance gene tetM is 0.525, and the amplification efficiency E is 269;

The resulting standard curve is meaningless (the R2 value of the generated standard curve should be greater than 0.98, the closer to ¹ , the higher the reliability of the result. The range of amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal. ) Therefore, the final copy number of the target resistance gene per gram of dry soil is unreliable.

<110> Zhejiang University

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Claims (3)

1. the quantitative detection method of antibiotic resistance gene in soil is characterized in that comprising the following steps: 1) Select pigs that often take antibiotics; Extract the genomic DNA of the pig manure of the above-mentioned pigs, and use the pig manure DNA as a template to amplify the target resistance gene fragment by PCR; The target resistance gene has a length of 100-350bp; The target resistance genes are: penicillin resistance gene blaTEM gene, tetracycline resistance gene tetC gene, tetracycline resistance gene tetM gene; The reaction system for PCR amplification is: 2×EasyTaqPCRSuperMix10μL; DNA template, the concentration is 18～20ng/μL1μL; The front and back primers of the target resistance gene, 10 μM each 1 μL; The remainder is double distilled water; The reaction conditions are: Pre-denaturation at 94°C for 4 minutes, Extend at 72°C for 7 minutes; When the target resistance gene is the penicillin resistance gene blaTEM gene, the sequences of the front and back primers are AAGCCATACCAAACGACGAG and CCGCCTCCATCCAGTCTAT respectively; the annealing temperature is 66°C; When the target resistance gene is the tetracycline resistance gene tetC gene, the sequences of the front and back primers are GCGGGATATCGTCCATTCCG and GCGTAGAGGATCCACAGGACG respectively; the annealing temperature is 68°C; When the target resistance gene is the tetracycline resistance gene tetM gene, the sequences of the primers before and after are ACAGAAAGCTTTATATAAC, TGGCGTGTCTATGATGTTCAC; the annealing temperature is 46°C; 2), cutting the gel to recover and purify the amplified target resistance gene, connect the resistance gene to the T vector, then transform competent Escherichia coli cells, select positive clones after coating with LB plates, and cultivate them with LB culture medium; 3) Use agarose gel electrophoresis to detect the quality of the expanded culture solution, select the successfully cloned bacteria solution to extract the plasmid, detect the plasmid concentration, convert the copy number of the resistance gene carried by the plasmid, and dilute it by 10 times as a dilution gradient Standby, as a resistance gene standard; 4), extract the genomic DNA of the soil, perform quantitative PCR amplification on the soil sample DNA and the resistance gene standard at the same time, generate a standard curve according to the copy number of the resistance gene standard and its amplified CT value, and then obtain the soil The copy number of the resistance gene of the sample. 2. the quantitative detection method of antibiotic resistance gene in soil according to claim 1, is characterized in that: In the step 3), the formula for converting the plasmid concentration into the absolute template copy number carried by each μL plasmid solution is: Copies/μL=[x/(a+b)×660]×10 ^-9 ×6.02×10 ²³ , where x is the plasmid concentration in ng/μL; a is the length of the vector in bp; b is the length of the target resistance gene in bp. 3. the quantitative detection method of antibiotic resistance gene in soil according to claim 2, it is characterized in that: in described step ⁴ ), the R value that generates standard curve should be greater than 0.98, the closer to 1, the result reliability The higher; the range of amplification efficiency E should be 0.8-1.2, the closer to 1, the more ideal.