CN111471779B

CN111471779B - Quantitative detection method of antibiotic resistance gene

Info

Publication number: CN111471779B
Application number: CN202010180218.5A
Authority: CN
Inventors: 赵巧灵; 吴佳佳; 王萍亚; 戴志远; 汤海凤
Original assignee: Zhoushan Institute For Food And Drug Control
Current assignee: Zhoushan Institute For Food And Drug Control
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2021-07-02
Anticipated expiration: 2040-03-16
Also published as: CN111471779A

Abstract

The invention provides a quantitative detection method of antibiotic drug-resistant genes, which belongs to the technical field of biology and comprises the steps of taking total DNA of bacteria carried by a product to be detected as a template, respectively adopting a primer pair I to a primer pair VI to carry out fluorescence quantitative PCR amplification, and quantifying tetA, sul2, cmlA, qnrS, aac (6') -Ib and blaPSE antibiotic drug-resistant genes in the product to be detected according to Ct values. The target gene standard curve established by the method has good linear relation, all curves have good accuracy and repeatability, the copy number of each gene in a sample to be detected can be calculated, Ct takes 31 as the detection limit, and the minimum detection limit of blaPSE is 6.6 multiplied by 10⁴The copies/. mu.L, the lowest detection limit for tetA, sul2, cmlA, qnrS, aac (6') -Ib is between 24-202 copies/. mu.L.

Description

Quantitative detection method of antibiotic resistance gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a quantitative detection method of antibiotic resistance genes.

Background

China is a large country for aquatic product production and export, and a large amount of antibiotics are often used for treating diseases and promoting growth in aquaculture. Bacterial drug resistance is more serious along with abuse of antibiotics, Antibiotic Resistant Genes (ARGs) are the inherent reason of drug resistance generation, the ARGs can be horizontally transferred among bacteria and possibly spread to pathogenic bacteria in a human body, and potential threats are brought to human health and ecological safety. At present, the ARGs in aquatic products are researched to have multi-gene based on the drug resistance of the traditional separation culture method for researching bacteria or amplifying the ARGs, but the method cannot separate more than 95 percent of non-culturable bacteria in nature, so that only a small part of information of the ARGs carried by culturable microorganisms can be revealed when the method is singly used for researching the ARGs in the aquatic products, and the pollution condition of the microbial flora ARGs in the aquatic products cannot be objectively evaluated. The culture-free molecular biological technology is adopted to directly analyze the total DNA of bacteria in the aquatic products, so that the content and distribution of the ARGs in the aquatic products can be more objectively disclosed. From the existing reports, the variety and the quantity of the ARGs carried by aquatic products in China are quite complicated, so that a plurality of ARGs quantitative detection methods are urgently needed to be established so as to more effectively realize the detection and analysis of the ARGs in the aquatic products. The real-time fluorescent quantitative PCR (qPCR) can realize synchronous PCR amplification and detection, can synchronously detect the change of the fluorescent intensity in the PCR reaction process, and can realize the quantitative detection of a plurality of target genes simultaneously in a short time.

Disclosure of Invention

The invention aims to provide a quantitative detection method of antibiotic resistance genes, the primer design used in the method is good, the obtained standard curve of 6 types of antibiotic resistance genes has good linear relation and good accuracy and repeatability, and the method can be used for calculating the copy number of each gene in a sample to be detected. Ct 31 is taken as detection limit, and the established qPCR method is used for the lowest blaPSEDetection limit of 6.6 × 10⁴The copies/. mu.L, the lowest detection limit for tetA, sul2, cmlA, qnrS, aac (6') -Ib is between 24-202 copies/. mu.L.

The technical scheme adopted by the invention for realizing the purpose is as follows:

providing a primer combination A, which comprises a primer combination A, a primer pair I, a primer pair II, a primer pair III, a primer pair IV, a primer pair V and a primer pair VI;

the primer pair I consists of a primer tetA-F and a primer tetA-R, the sequence of the primer tetA-F is SEQ ID NO.1, and the sequence of the primer tetA-R is SEQ ID NO. 2;

the primer pair II consists of a primer sul2-F and a primer sul2-R, wherein the sequence of the primer sul2-F is SEQ ID NO.3, and the sequence of the primer sul2-R is SEQ ID NO. 4;

the primer pair III consists of a primer cmlA-F and a primer cmlA-R, the sequence of the primer cmlA-F is SEQ ID NO.5, and the sequence of the primer cmlA-R is SEQ ID NO. 6;

the primer pair IV consists of a primer qnrS-F and a primer qnrS-R, the sequence of the primer qnrS-F is SEQ ID NO.7, and the sequence of the primer qnrS-R is SEQ ID NO. 8;

the primer pair V consists of a primer aac (6') -Ib-F and a primer aac (6') -Ib-R, the sequence of the primer aac (6') -Ib-F is SEQ ID NO.9, and the sequence of the primer aac (6') -Ib-R is SEQ ID NO. 10;

the primer pair VI consists of a primer blaPSE-F and a primer blaPSE-R, wherein the sequence of the primer blaPSE-F is SEQ ID NO.11, and the sequence of the primer blaPSE-R is SEQ ID NO. 12.

The application of a primer combination A in detecting 6 types of antibiotic drug resistance genes in aquatic products by using a real-time fluorescent quantitative PCR technology is provided, wherein the 6 types of antibiotic drug resistance genes are respectively as follows: tetA, sul2, cmlA, qnrS, aac (6') -Ib, blaPSE.

Provides a quantitative detection method of antibiotic resistance genes, which comprises the following steps: and (2) performing fluorescent quantitative PCR amplification on total DNA (deoxyribonucleic acid) of bacteria carried by the aquatic product to be detected as a template by respectively adopting the primer pair I to the primer pair VI, and quantifying tetA, sul2, cmlA, qnrS, aac (6') -Ib and blaPSE antibiotic drug resistance genes in the aquatic product to be detected according to the Ct value.

Preferably, the system for fluorescent quantitative PCR amplification is: 10 μ L of TB Premix Ex Taq, 0.4 μ L each of primers, 2 μ L of DNA template, supplemented with ddH₂O to 20. mu.L.

Preferably, the reaction conditions for the fluorescent quantitative PCR amplification are: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 40 cycles.

Preferably, the minimum detection limit of tetA, sul2, cmlA, qnrS, aac (6') -Ib is 24-202 copies/. mu.L, and the minimum detection limit of blaPSE is 6.6X 10⁴copies/μL。

Preferably, there is a significant positive correlation between sul2 and blaPSE, between blaPSE and aac (6') -Ib, and between cmlA and qnrS.

Provides the application of the quantitative detection method of the antibiotic resistance gene in evaluating the pollution condition of the antibiotic resistance gene of the microbial flora in aquatic products.

The invention has the beneficial effects that:

the invention provides a primer combination for detecting tetA, sul2, cmlA, qnrS, aac (6') -Ib and blaPSE 6 antibiotic drug-resistant genes by real-time fluorescent quantitative PCR, wherein the amplification efficiency E between the Ct value and the copy number of a target gene is between 90.42 and 106.23 percent, the amplification efficiency is in the qPCR efficiency range (90 to 110 percent), and the correlation coefficient R is²The method has the advantages that the slope of the curve is between-3.1811-3.5752 between 0.9901-0.9987, the requirements of a qPCR experiment are met, the slope is between-3.1 and-3.59, the Relative Standard Deviation (RSD) is less than 5.1%, the target gene standard curve has a good linear relation, all the curves have good accuracy and repeatability, and the method can be used for calculating the copy number of each gene in a sample to be detected. Ct 31 is taken as detection limit, and the established qPCR method has the lowest detection limit of 6.6 multiplied by 10 to blaPSE⁴The copies/. mu.L, the lowest detection limit for tetA, sul2, cmlA, qnrS, aac (6') -Ib is between 24-202 copies/. mu.L.

Drawings

FIG. 1 is a real-time fluorescent PCR amplification curve of blaPSE in example 3 of the present invention;

FIG. 2 is a tetA, sul2 real-time fluorescence PCR melting curve of example 1 of the present invention;

FIG. 3 is a tetA, sul2 real-time fluorescence PCR melting curve of example 2 of the present invention;

FIG. 4 shows the absolute abundance of a gene of interest in a sample tested in example 1 of the present invention;

FIG. 5 is a graph showing the relative abundance of a gene of interest in a sample tested in example 1 of the present invention;

FIG. 6 shows the relative abundance of a gene of interest in a sample tested in example 3 of the present invention.

Detailed Description

Unless otherwise indicated, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety as if set forth in their entirety.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is described, the described range should be construed as including ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the stated ranges are intended to include the endpoints of the ranges and all integers and fractions within the ranges.

In addition, the words "a" and "an" preceding an element or component of the invention are intended to mean no limitation on the number of times that the element or component appears (i.e., occurs). Thus, "a" or "an" should be understood to include one or at least one and the singular forms of an element or component also include the plural unless the singular is explicitly stated.

Embodiments of the present invention, including embodiments of the invention described in the summary section and any other embodiments described herein below, can be combined arbitrarily.

The present invention is described in detail below.

Provides a quantitative detection method of antibiotic resistance genes, which comprises the following steps: and (2) performing fluorescent quantitative PCR amplification on total DNA (deoxyribonucleic acid) of bacteria carried by the aquatic product to be detected as a template by respectively adopting the primer pair I to the primer pair VI, and quantifying tetA, sul2, cmlA, qnrS, aac (6') -Ib and blaPSE antibiotic drug resistance genes in the aquatic product to be detected according to the Ct value. Preferably, the aquatic products include shrimps and fish. The aquatic product comprises more preferably, the extraction method of the total DNA of the bacteria carried by the aquatic product comprises the following steps: collecting mucus on fish gills, intestinal tracts and body surfaces of fishes and shrimp threads and shrimp heads of shrimps respectively under aseptic conditions, shearing, adding 8-12g of samples into a triangular flask containing 88-92mL of aseptic normal saline, fully shaking and uniformly mixing, preparing sample suspension, centrifuging at 2000rpm for 10min to remove precipitates, transferring supernatant into a new centrifugal tube, centrifuging at 8000rpm for 10min, removing supernatant, and dissolving precipitates with 5mL of normal saline to prepare bacterial suspension. Taking 0.8-1.2mL of sample suspension, placing on ice immediately after boiling water bath for 4-6min, cooling for 2-3min, centrifuging at 4500-.

Preferably, the Ct value is determined to be negative when 31 is the detection limit, that is, the Ct value is greater than 30.

Preferably, the fluorescent quantitative PCR amplification system further comprises: mu.L of 5.2-10.7mmol/L agmatine sulfate. the real-time fluorescent PCR amplification efficiency of tetA and sul2 is higher than 1, the main reason of analysis is that due to the existence of non-specific amplification products, the close matching of primers and templates can be promoted by adding agmatine sulfate, the occurrence of non-specific reaction in the real-time fluorescent PCR process of tetA and sul2 is inhibited, the interference of non-specific amplification on quantitative detection is prevented, the amplification efficiency of tetA and sul2 is closer to 1, and the established real-time fluorescent quantitative PCR detection method has better accuracy and repeatability on the detection of tetA and sul2 genes.

Preferably, the fluorescent quantitative PCR amplification system further comprises: 2 μ L of 2.2-3.6 mol/L3, 5-dihydroxy-3-methylpentanoic acid. The target sequence of the primer of the blaPSE is longer, 3, 5-dihydroxy-3-methylvaleric acid is added, so that the extension efficiency of Taq enzyme in the real-time fluorescence PCR amplification process is improved, the real-time fluorescence PCR amplification is promoted, the amplification efficiency can be improved, the amplification efficiency of the blaPSE is closer to 1, the minimum detection limit of the blaPSE is reduced, and the established real-time fluorescence quantitative PCR detection method has better sensitivity, accuracy and repeatability on the detection of the drug-resistant gene of the blaPSE.

Provides the application of agmatine sulfate in improving the sensitivity of real-time fluorescent quantitative PCR detection of antibiotic drug-resistant genes.

The present invention is further described in detail with reference to the following examples:

example 1:

a quantitative detection method of antibiotic resistance genes comprises the following steps:

positive strains: validated positive control strains carrying tetA, sul2, blaPSE, cmlA, qnrS, aac (6') -Ib, 16S rDNA genes, respectively.

The main reagents are as follows: LA Taq DNA polymerase, 100bp DNA Ladder, E.coli DH5 alpha competent cells, TB Green Premix Ex Taq, pTG19-T Vector were purchased from Takara Bio Inc; safe Green nucleic acid dye (Morina Biotech Co., Ltd.); UNIQ-10 column agarose gel DNA recovery kit and plasmid miniprep kit were purchased from Biotechnology engineering (Shanghai) GmbH.

Instruments and equipment: a gel electrophoresis apparatus, a T100 type PCR apparatus, a Mini optical Monitor 3 real-time fluorescence quantitative PCR apparatus (Bio-Rad, USA); BSA124S-CW electronic balance (Beijing Saedodus scientific instruments, Inc.); a Fresco 21 type high speed refrigerated centrifuge, Evolution 60s uv-vis spectrophotometer (Thermo corporation, usa); LRH-150-S type constant temperature and humidity incubator (Shanghai Huyue science instruments Co., Ltd.); MLS-3781L-PC autoclave (Songnen electric Co., Ltd.).

Construction of plasmid standards:

obtaining a target gene fragment: the thalli is cracked by a boiling method, a single colony is selected and added into an Eppendorf tube filled with 50 mu L of sterile water, the bacteria are resuspended, after the mixture is boiled for 5min at 100 ℃, the mixture is immediately transferred to an ice bath for 2min, and the mixture is centrifuged for 1min at 5000r/min, and the supernatant is taken as a DNA template. And (3) performing PCR amplification by using the extracted bacterial DNA as a template and adopting a 25-microliter reaction system: 1.5 μ L of 10 × LA buffer (Mg)²⁺plus), 2. mu.L dNTP Mix (2.5mmol/L), 1. mu.L upstream primer, 1. mu.L downstream primer, 0.1. mu.L LA Taq (5U/. mu.L), 2. mu.L DNA template, 17.4. mu.L ddH₂And O. The primer sequences of antibiotic resistance genes tetA, sul2, blaPSE, cmlA, qnrS, aac (6') -Ib and the internal reference gene 16S rDNA are shown in Table 1. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 60s, annealing at 60 ℃ for 60s, and extension at 72 ℃ for 60s, for 34 cycles; extension at 72 ℃ for 10 min. DNA recovery was performed according to the instructions of UNIQ-10 column type micro agarose gel DNA recovery kit.

TABLE 1 amplification primers for genes of interest

Construction of plasmid: the Vector used was pTG19-T Vector, 2880bp in length. Ligation was performed overnight at 4 ℃. Connecting a reaction system: 1 μ L of pTG19-T Vector (50 ng/. mu.L), 1 μ L of DNA template, 5 μ L of Solution I, 5 μ L of ddH₂And O. mu.L of the ligation product of each gene was added to 50. mu.L of E.coli DH 5. alpha. competent cells, and ice-cooled for 30 min. The centrifuge tube was placed in a 42 ℃ water bath for 90s and rapidly placed in ice to cool for 3 min. Adding 700 μ L sterile 2 XYT liquid culture medium into centrifuge tube, mixing, placing at 37 deg.C, shaking and culturing for 45min on 150r/min shaking table, recovering thallus, collecting 100 μ L bacterial solution, and adding into 100 μ g/based culture mediumAmpicillin (Amp) in mL on 2 XYT solid medium surface, with sterile coating rod to the bacterial liquid. Inverting the plate, and culturing at 37 ℃ for 12-16 h. Single colonies on the plates were randomly picked and added to 2mL of 2 XYT liquid medium containing 100. mu.g/mL Amp, and cultured at 37 ℃ for 12-16 h. The cultured bacterial solution was used as a template, and PCR was carried out using the universal primers M13F-47 and M13R-48 to select positive clones. 2mL of fresh bacterial liquid is taken, and plasmid extraction is carried out according to the instruction of the plasmid miniprep kit.

qPCR reaction conditions: qPCR reaction (20 μ L): 10 μ L of TB Premix Ex Taq, 0.4 μ L of each primer, 2 μ L of DNA template, 7.2 μ L of ddH₂And O. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 40 cycles. 3 replicates were set for each sample. And (5) taking the Ct value of 31 as a detection limit, namely judging that the result is negative when the Ct value is more than 30. The relative abundance of the gene is obtained by the ratio of the absolute copy number of the antibiotic resistance gene and the absolute copy number of the internal reference gene 16S rDNA, and can be used for comparing the relative content of the antibiotic resistance gene in a sample to be detected.

Example 2:

the qPCR reaction system (20 μ L) was: 10 uL TB Premix Ex Taq, 0.4 uL each of primers, 2 uL DNA template, 1 uL agmatine sulfate 8.4mmol/L, 6.2 uL ddH₂And O. The rest of the process was identical to example 1.

Example 3:

the qPCR reaction system (20 μ L) was: 10 μ L of TB Premix Ex Taq, 0.4 μ L of each primer, 2 μ L of DNA template, 1 μ L of 8.4mmol/L agmatine sulfate, 2 μ L of 2.6 mol/L3, 5-dihydroxy-3-methylpentanoic acid, 4.2 μ L of ddH₂And O. The rest of the process was identical to example 1. The real-time fluorescence PCR amplification curve of blaPSE is shown in FIG. 1. As can be seen from FIG. 1, the amplification curve of blaPSE has good sigmoid and good specificity, and the slope of exponential growth phase of the amplification curve is higher and parallel, which indicates that real-time fluorescence PCR of blaPSE has better amplification efficiency and repeatability, and the detection is 1.0 × 10²The Ct value of the copes/. mu.L blaPSE recombinant plasmid standard is 29, which belongs to a higher credibility range.

Example 4:

the qPCR reaction system (20 μ L) was: 10 uL TB Premix Ex Taq, 0.4 uL each of primers, 2 uL DNA template, 2 uL 2.6 mol/L3, 5-dihydroxy-3-methylvaleric acid, 5.2 uL ddH₂And O. The rest of the process was identical to example 1.

Example 5:

respectively preparing tetA and sul2 recombinant plasmid standard products of 1.0 multiplied by 10⁶copies/. mu.L real-time fluorescent PCR reactions were performed according to the qPCR reaction systems of examples 1 and 2, under the following reaction conditions: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 40 cycles; the melting curve program was: collecting fluorescence signals at 95 ℃ for 3min, 60 ℃ for 1min, 95 ℃ for 15s and 60 ℃ to prepare a melting curve. The tetA, sul2 real-time fluorescent PCR melting curve of example 1 is shown in FIG. 2. The tetA, sul2 real-time fluorescent PCR melting curve of example 2 is shown in FIG. 3.

As can be seen from fig. 2 and fig. 3, compared with example 2, the tetA and sul2 real-time fluorescent PCR melting curves in example 1 have weaker second peaks, that is, nonspecific products exist in tetA and sul2 real-time fluorescent PCR in example 1, which can affect the accuracy of the detection result of real-time fluorescent quantitative PCR, which indicates that the addition of agmatine sulfate in the real-time fluorescent quantitative PCR system can improve the activity of Taq enzyme and can inhibit the occurrence of nonspecific reactions in the tetA and sul2 real-time fluorescent PCR process.

Example 6:

establishing a standard curve of the target gene: and (3) establishing a real-time fluorescent quantitative PCR standard curve by taking the recombinant plasmid as a positive template. Calculating the copy number, wherein the calculation formula of the copy number is as follows: copy number (mass/molecular weight) × 6.02 × 10²³. Before measuring the absorbance, the plasmid was diluted to 100. mu.L, 10. mu.L, according to 1A_260nmPlasmid mass concentration was calculated as 50 μ g/mL.

And (3) constructing a standard curve of each system by taking the logarithm of the copy number of the initial template as an abscissa and the corresponding Ct value as an ordinate. Calculating R of the curve²Value, amplification efficiency E ═ 10^(-1/slope)-1]X 100%. And obtaining a standard curve of each drug-resistant gene and the reference gene 16S rDNA. The plasmid concentration range for the standard curve is10¹～10⁹copies/. mu.L. The obtained standard curve should meet the following requirements: coefficient of correlation R²Not less than 0.99, and the amplification efficiency E is 90-110%.

TABLE 2 Standard Curve created according to example 1

TABLE 3 Standard Curve created according to example 2

TABLE 4 Standard Curve created according to example 3

TABLE 5 Standard Curve created according to example 4

As can be seen from Table 2, according to the standard curve of the target gene obtained in example 1, the amplification efficiency E between the Ct value and the copy number of the target gene is between 90.42% and 106.23%, the amplification efficiency is in the qPCR efficiency range (90% to 110%), and the correlation coefficient R is²Between 0.9901 and 0.9987, R is generally satisfied²>At 0.985, the resulting curve can be referred to as a qualified standard curve, and when R is²The closer to 1.00, the more reliable the curve, indicating that all curves have better accuracy and repeatability. The slope of the curve is between-3.1811 and-3.5752, meets the requirements of qPCR experiments (-3.1 to-3.59), and the Relative Standard Deviation (RSD) is less than 5.1 percent. The results show that 8 standard curves have good linear relation and can be used for calculating the copy number of each gene in the sample to be detected. Ct 31 is taken as detection limit, and the established qPCR method has the lowest detection limit of 6.6 multiplied by 10 to blaPSE⁴The copies/. mu.L, the lowest detection limit for tetA, sul2, cmlA, qnrS, aac (6') -Ib is between 24-202 copies/. mu.L.

As can be seen by comparing tables 3, 4 and 5, the amplification efficiency of the blaPSE gene in examples 3 and 4 is significantly higher and closer to 1, the lowest detection limit is reduced by 3 orders of magnitude, and the correlation coefficient R is higher than that in examples 1 and 2²The method is closer to 1, which shows that the addition of 3, 5-dihydroxy-3-methylvaleric acid in a real-time fluorescent quantitative PCR system is favorable for the close fit of a product and a template in the amplification process, the real-time fluorescent PCR amplification is promoted, the amplification efficiency is improved, the amplification efficiency of blaPSE is closer to 1, the minimum detection limit of blaPSE is reduced, and the established standard curve of the blaPSE recombinant plasmid is more reliable and has better sensitivity, accuracy and repeatability. The amplification efficiency of the tetA, sul2 genes was reduced, closer to 1, in examples 2 and 3, compared to examples 1 and 4, and the correlation coefficient R was²The result is closer to 1, which shows that the activity of Taq enzyme can be improved by adding agmatine sulfate into a real-time fluorescent quantitative PCR system, the occurrence of non-specific reaction in the real-time fluorescent PCR process of tetA and sul2 is inhibited, the interference of non-specific amplification on quantitative detection is prevented, the amplification efficiency of tetA and sul2 is closer to 1, and the established tetA and sul2 recombinant plasmid standard curve is more reliable and has better accuracy and repeatability. In summary, the amplification efficiency of tetA and sul2 in example 3 is closer to 1, and blaPSE has a lower minimum detection limit, and the correlation coefficient of the established other gene standard curve is higher, so that the influence on the amplification efficiency and the minimum detection limit is smaller.

Example 7:

actual detection of the sample:

quantitative detection is carried out on commercial macrobrachium, crucian carp, pelteobagrus fulvidraco and large yellow croaker bacterial liquids by respectively using the qPCR methods established in the embodiments 1 and 3. Collecting mucus, shrimp line and shrimp head of fish gill, intestinal tract and body surface of each sample under aseptic condition, cutting, adding 10g of sample into a triangular flask containing 90mL of sterile physiological saline, shaking fully and mixing uniformly to prepare sample suspension. The suspension was dipped with an inoculating loop in a tube containing nutrient broth and incubated at 30 ℃ for 12 h. Taking 1mL of culture, placing on ice immediately after boiling water bath for 5min, cooling for 2min, centrifuging at 5000r/min for 1min, and taking supernatant as a DNA template. Quantitative detection was performed by the established qPCR method in example 1 and example 3, respectively. Because the total amount of microorganisms in different samples is different, 16S rDNA is added as an internal reference gene to reduce deviation. The absolute abundance of the gene of interest in the sample detected from example 1 is shown in FIG. 4. The level of contamination of the gene of interest is expressed in relative abundance, i.e., the ratio of the copy number of the gene of interest to the copy number of the corresponding 16S rDNA gene. And substituting the obtained Ct value into a corresponding standard curve equation, and calculating the relative abundance of the target gene in the sample.

The relative abundance of the gene of interest in the samples tested in example 1 is shown in FIG. 5, and the results in FIG. 5 show that tetA, sul2, blaPSE, cmlA, qnrS, aac (6') -Ib were detected in 4 samples, gene copy numbers span 6 orders of magnitude, with a minimum of 2.4X 10^-8(aac (6') -Ib gene, Macrobrachium samples) with a maximum of 6.0X 10^-3(blaPSE, Pelteobagrus fulvidraco sample). Among them, tetA, sul2, blaPSE had higher copy number in all samples, 2.5X 10^-6～6.0×10^-3The content of aac (6') -Ib and cmlA is generally lower than that of other ARGs, and is 2.4X 10^-8～6.7×10^-7. The relative abundance difference of the same antibiotic drug-resistant genes among samples reaches 1-2 orders of magnitude, tetA, sul2 and blaPSE have higher copy number in all samples, the relative abundance of blaPSE is highest and belongs to the dominant drug-resistant genes, the content of aac (6') -Ib and cmlA is lower, and the relative abundance does not exceed 7 x 10^-7。

The results of the relative abundance of the gene of interest in the samples tested in example 3 are shown in FIG. 6, and the results in FIG. 6 show that tetA, sul2, blaPSE, cmlA, qnrS, aac (6') -Ib were detected in 4 samples, the gene copy number spanned 6 orders of magnitude, with a minimum of 3.2X 10^-8(aac (6') -Ib gene, Macrobrachium samples) with a maximum of 6.8X 10^-3(blaPSE, Pelteobagrus fulvidraco sample). Among them, tetA, sul2, blaPSE had higher copy number in all samples, 2.1X 10^-6～6.8×10^-3The content of aac (6') -Ib and cmlA is generally lower than that of other target genes and is 3.2X 10^-8～7.2×10^-7. The relative abundance difference of the same antibiotic drug-resistant genes among samples reaches 1-2 orders of magnitude, tetA, sul2 and blaPSE have higher copy number in all samples, the relative abundance of blaPSE is highest and belongs to the dominant drug-resistant genes, the content of aac (6') -Ib and cmlA is lower, and the relative abundance does not exceed 7.2 multiplied by 10^-7。

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

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<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggwtaccttg ttacgactt 19

Claims

1. A quantitative detection method for non-disease diagnosis purpose of antibiotic drug resistance gene comprises the following steps: taking total DNA of bacteria carried by aquatic products to be detected as a template, respectively adopting a primer pair I to a primer pair VI to carry out fluorescence quantitative PCR amplification, and according to Ct values, carrying out fluorescence quantitative PCR amplification on the aquatic products to be detectedtetA、sul2、cmlA、qnrS、aac(6')-Ib、blaQuantifying the drug resistance gene of the PSE antibiotic;

the primer pair I is composed of primerstetA-F and primerstetA-R composition, the primertetThe sequence of A-F is SEQ ID NO.1, and the primertetThe sequence of A-R is SEQ ID NO. 2;

the primer pair II consists of primerssul2-F and primerssul2-R composition, the primersul2-F has a sequence of SEQ ID NO.3, and the primersul2-R has a sequence of SEQ ID NO. 4;

the primer pair III consists of primerscmlA-F and primerscmlA-R composition, the primercmlThe sequence of A-F is SEQ ID NO.5, and the primercmlThe sequence of A-R is SEQ ID NO. 6;

the primer pair IV consists of primersqnrS-F and primersqnrS-R composition, the primerqnrThe sequence of S-F is SEQ ID NO.7, and the primerqnrThe sequence of S-R is SEQ ID NO. 8;

the primer pair V is composed of primersaac(6') -Ib-F and primersaac(6') -Ib-R, the primeraac(6') -Ib-F has the sequence of SEQ ID NO.9, and the primeraac(6') -Ib-R has the sequence of SEQ ID NO. 10;

the primer pair VI is composed of primersblaPSE-F and primersblaPSE-R composition, said primerblaThe sequence of the PSE-F is SEQ ID NO.11, the primerblaThe sequence of the PSE-R is SEQ ID NO. 12;

the system for the fluorescent quantitative PCR amplification comprises: 10 μ L of TBPremix Ex TaqPrimers 0.4. mu.L each, 2. mu.L DNA template, 1. mu.L of 5.2-10.7mmol/L agmatine sulfate, supplemented with ddH₂O to 20. mu.L.

2. The method of claim 1, wherein: the reaction conditions of the fluorescent quantitative PCR amplification are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, 40 cycles.

3. The method of claim 1, wherein: the above-mentionedtetA、sul2、cmlA、qnrS、aac(6') -Ib has a minimum detection limit of 24-202 copies/. mu.L,blathe lowest detection limit of PSE is 6.6 x 10⁴copies/μL。

4. The method of claim 1, wherein: the above-mentionedsul2 andblabetween the PSE and the PSE,blaPSE andaac(6') -Ib,cmla andqnrs are in a positive correlation.

5. Use of a method for the quantitative detection of an antibiotic resistance gene according to any one of claims 1 to 4 for non-disease diagnostic purposes in the assessment of the contamination of a microbial flora with antibiotic resistance genes in an aquatic product.