阅读说明：本技术 一种环境中抗生素抗性基因的快速定量试剂盒及其检测方法 (Rapid quantitative kit for antibiotic resistance gene in environment and detection method thereof ) 是由 谢冰 王盼亮 奚慧 乔子茹 杜飞 于 2019-08-28 设计创作，主要内容包括：本发明公开了一种环境中抗生素抗性基因的定量检测试剂盒及其定量检测方法。该试剂盒包括以下成分：Solution I、Solution II、Solution III、ddH<Sub>2</Sub>O和ROX。所述检测方法主要步骤为：1)以环境样品的DNA为模板,将其与Solution I和Solution II加入八联排管中,并根据qPCR仪型号确定是否加入ROX,最后添加ddH<Sub>2</Sub>O；2)将标准质粒Solution III(与Solution I对应)按10倍梯度稀释,随后将不同梯度的标准质粒Solution III分别与Solution I和Solution II加入八联排管中,ddH<Sub>2</Sub>O和ROX加入方法与步骤1)相同；3)将步骤1)和2)中的样品同时进行荧光定量PCR扩增,最终得出抗性基因标准曲线和基因丰度。本发明的试剂盒可以快速高效地定量分析环境中抗生素抗性基因的丰度,不仅特异性好,精确度高,而且能大大缩短实验周期,提高实验操作效率。(The invention discloses a quantitative detection kit and a quantitative detection method for antibiotic resistance genes in an environment. The kit comprises the following components: solution I, Solution II, Solution III, ddH 2 O and ROX. The detection method mainly comprises the following steps: 1) adding DNA of an environment sample, Solution I and Solution II into an eight-connected calandria by taking the DNA of the environment sample as a template, determining whether ROX is added or not according to the model of a qPCR instrument, and finally adding ddH 2 O; 2) diluting standard plasmid Solution III (corresponding to Solution I) according to 10-fold gradient, adding standard plasmid Solution III with different gradients, Solution I and Solution II into eight-connected pipes, ddH 2 The adding method of O and ROX is the same as that of the step 1); 3) and (3) carrying out fluorescence quantitative PCR amplification on the samples in the steps 1) and 2) simultaneously to finally obtain a resistance gene standard curve and gene abundance. The kit can rapidly and efficiently quantitatively analyze the antibiotics in the environmentThe abundance of the resistance gene has good specificity and high accuracy, and can greatly shorten the experimental period and improve the experimental operation efficiency.)

1. A kit for rapidly and quantitatively detecting antibiotic resistance genes in an environment is characterized by comprising Solution I10 x, Solution II 2 x, Solution III standard plasmids, ROX and ddH₂O。

2. The kit of claim 1, wherein said Solution I is composed of a forward primer and a reverse primer for a single gene of interest; the concentration of the forward primer and the reverse primer in Solution I is 4 mu M.

3. The kit according to claim 2, wherein the forward and reverse primers are selected from several classes of antibiotic resistance genes with high abundance in the environment, and the classes of the resistance genes set by Solution I include: sulfonamides sul1 and sul2, tetracyclines tetM and tetQ, beta-lactams bla_CTX-MAnd bla_TEMAnd the macrolides ermA and ermB.

4. The kit of claim 1, wherein said Solution II is selected from the group consisting of HotStart DNA Polymerase, SYBR Green I, dNTPs, Mg²⁺And buffer system and ddH₂And (C) O.

5. The kit according to claim 4, wherein the concentration of the HotStart DNA Polymerase is 0.2U/. mu.l; the concentration of SYBR Green I is 0.5 ×; the Mg²⁺Was 4 mM.

6. The kit of claim 4, wherein the dNTPs comprise dATPs, dTTPs, dGTPs, and dCTPs; the molar weight ratio of the dATPs, dTTPs, dGTPs and dCTPs is 1:1:1:1, and the molar concentration of the dATPs, dTTPs, dGTPs and dCTPs is 40 mM.

7. The kit of claim 1, wherein said Solution III is a plasmid standard corresponding to said Solution I, and wherein said plasmid standard is a standard plasmid for the corresponding gene constructed from pMD18-T or pMD19-T or pMD 20-T.

8. The kit according to claim 7, wherein the standard plasmid is used in accordance with the requirementsDiluting with 10 times of gradient for more than 5 gradients, making Ct and standard plasmid copy number into standard curve according to quantitative result, and requiring R²≥0.99。

9. The kit of claim 1, wherein the addition amount of ROX is determined according to the model number of the qPCR apparatus.

10. A method for rapid detection of antibiotic resistance genes is characterized by comprising the following specific steps:

(1) selecting appropriate DNA extraction kit according to the type of the environment sample to be detected, and after DNA extraction, using an ultramicro spectrophotometer to measure the concentration and A of the DNA_260/280Carrying out measurement;

(2) diluting the DNA sample qualified for detection to a certain concentration, and then adding Solution I10X, ROX and ddH in sequence₂O and Solution II 2 x to eight rows of tubes, setting a qPCR reaction system to be 20 mu L or 10 mu L, and setting more than 3 parallels for each reaction;

(3) mixing Solution III diluted by 5 gradient concentrations with ROX and ddH₂O and Solution II 2 x are added into the eight-connected-row pipe according to the step (2);

(4) putting the sample prepared in the step (3) into a qPCR instrument, and setting a reaction program as follows: pre-denaturation: 94 ℃ for 5 min; setting 40 cycles and denaturation: 30s at 94 ℃; annealing: the annealing temperature is set according to the Solution I primer for 30 s; extension: 72 ℃ for 30 s; ③ 72 ℃ for 10 min; dissolution curve: the dissolution curve can be set or set at 65-95 ℃ according to an instrument system, and the dissolution curve is increased by 0.5 ℃ every 5 seconds;

(5) meanwhile, setting negative control according to the step (2);

(6) log copy number of standard plasmid after reaction₁₀Making a standard curve of the target gene according to the value and the corresponding Ct value;

(7) and substituting the Ct value of the sample to be detected into the standard curve, calculating the copy number of the quantitative resistance gene of the sample to be detected, and calculating the copy number of the resistance gene in the sample.

Technical Field

The invention belongs to the technical field of environment and molecular biology, and relates to a rapid quantitative kit for antibiotic resistance genes in an environment and a detection method thereof.

Background

Since the discovery of antibiotics by Fleming in microbiologists in the second thirty th of the twentieth century, it has been widely used in the fields of medical care and health and in livestock and poultry farming because of its ability to treat and prevent human and animal diseases. However, with the heavy use or abuse of antibiotics, they remain in the environment in large quantities. And the residual antibiotics in the environmental system induce the generation and the transmission of antibiotic resistance genes and the generation of drug-resistant bacteria. Antibiotic resistance genes are considered to be a novel, persistent environmental pollutant with potential environmental and human health risks. A great deal of research shows that antibiotic resistance genes are various in types and widely distributed in artificial environments including farms and surrounding environments, sewage treatment plants and sewage discharge receiving water bodies thereof, river sediments, landscape water bodies, drinking water bodies, soil, refuse landfills and the like, and even researchers find that antibiotic resistance genes and resistant bacteria exist in the air. These drug-resistant bacteria can be transferred into the human body by direct or indirect routes. There is increasing evidence that antibiotic-resistant bacteria and resistance genes in the environment are closely associated with clinical resistance. The number of cases of serious illness or death due to antibiotic resistant bacterial infections is increasing every year. Statistically, two million people die annually in the united states from infections with drug-resistant bacteria, with approximately 23000 dying from infections and 25000 dying from infections with multiple drug-resistant bacteria annually in europe. Therefore, the quantitative detection and risk assessment of the antibiotic resistance genes in the environment are very important, and especially the health risk assessment of the antibiotic resistance genes in the environment is of great significance.

The quantitative detection method of antibiotic resistance gene in environment mainly includes Polymerase Chain Reaction (PCR), DNA sequencing technology and DNA molecular hybridization technology. The PCR technique is the most commonly used technique for rapid amplification of specific genes in vitro, and the technical principle is similar to the DNA replication in cells. And the method has the advantages of simple operation, high sensitivity, quick reaction and low requirement on the purity of the sample, and can detect the pg-grade DNA quantity. Real-time fluorescent quantitative PCR (qPCR) is used as a supplement of a PCR technology and becomes a powerful tool for quantitative analysis of resistance genes in the environment, and the qPCR technology has strong stability and good repeatability and realizes quantitative research of the resistance genes. At present, the qPCR technology is widely applied to research on abundance of resistance genes in environments such as rivers, lakes, sewage treatment systems, soil, breeding industry and the like.

The antibiotic resistance gene quantitative PCR reaction comprises the selection of primers, the establishment of standard products, the establishment of a reaction system and the optimization of conditions. However, there are often inconveniences in the quantitative analysis of antibiotic resistance genes. For example, in a conventional qPCR amplification system configuration, reagents such as forward and reverse primers, fluorescent dye, enzyme, dNTP, etc. are usually required to be stored separately, and when amplification is to be performed, samples are added to the same PCR tube separately. Therefore, in addition to the disadvantages of long time consumption and complex operation in the operation process, the reagent is polluted by taking the reagent for many times, and the subsequent use is influenced. Meanwhile, the preparation of the standard plasmid is a more complicated process.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a quantitative kit for rapidly detecting antibiotic resistance genes and a detection method thereof, and the kit can rapidly, efficiently and accurately carry out quantitative research on the resistance genes in different environments.

The invention aims to solve the problem of complicated operation steps in the quantitative analysis process of antibiotic resistance genes in the environment, and provides a quantitative kit and a detection method thereof, which can quickly, efficiently and accurately determine the antibiotic resistance genes in the environment.

The invention provides a kit for rapidly and quantitatively detecting antibiotic resistance genes in an environment, which contains Solution I (10X), Solution II (2X), Solution III (standard plasmid), ROX and ddH₂O。

Wherein, the Solution I consists of a forward primer and a reverse primer of a single target gene.

The concentration of the forward primer and the reverse primer in Solution I is preferably 4 mu M.

The forward primer and the reverse primer are selected from several types of antibiotic resistance genes with high abundance in each environment, and the types of the resistance genes mainly set by Solution I include but not only: sulfonamides (sul1 and sul2), tetracyclines (tetM and tetQ), beta-lactams (bla_CTX-MAnd bla_TEM) And macrolides (ermA and ermB).

The kit can customize corresponding Solution I and Solution III of related target resistance genes according to customer requirements.

Wherein the Solution II is selected from the group consisting of HotStart DNA Polymerase, SYBR Green I, dNTPs, Mg²⁺And optimized buffer system and ddH₂And (C) O.

The concentration of the HotStart DNA Polymerase is preferably 0.2U/. mu.l.

The concentration of SYBR Green I is preferably 0.5X.

The dNTPs comprise dATPs, dTTPs, dGTPs and dCTPs, the molar ratio of each dNTPs is preferably 1:1:1:1, and the optimal molar concentration of each dNTPs is 40 mM.

The Mg²⁺The concentration of (B) is preferably 4 mM.

Wherein, the Solution III is a plasmid standard product corresponding to the Solution I, and is a standard plasmid of a corresponding gene constructed by T plasmid vectors such as pMD18-T, pMD19-T, pMD20-T and the like.

When the standard plasmid is used, the standard plasmid needs to be diluted by more than 5 gradients according to 10-fold gradients, Ct and the copy number of the standard plasmid are made into a standard curve according to a quantitative result, and R is required²≥0.99。

Wherein, whether the ROX is added or not is determined according to the model of the qPCR instrument.

Wherein the ddH is added₂O (double distilled water) to the amount of the reaction system。

The invention also provides a method for rapidly detecting the antibiotic resistance gene, which comprises the following specific contents and steps:

(1) selecting appropriate DNA extraction kit according to the type of the environment sample to be detected, and after DNA extraction, using an ultramicro spectrophotometer to measure the concentration and A of the DNA_260/280Carrying out measurement;

(2) diluting a DNA sample qualified for detection to a certain concentration (the concentration of a suggested template is 50-100 ng), and then sequentially adding Solution I (10 x), ROX (the adding amount is determined according to the model of the instrument) and ddH₂O and Solution II (2X) to eight-row pipes, the qPCR reaction system is recommended to be set to be 20 mu L or 10 mu L, and each reaction is set to be more than 3 parallels;

(3) 5 gradient concentrations of Solution III (standard plasmid) diluted with ROX (the amount of addition is determined according to the type of the instrument), ddH₂O and Solution II (2X) are added into the eight-row pipe according to the step (2);

(4) putting the sample prepared in the step (3) into a qPCR instrument, and setting a reaction program as follows: pre-denaturation: 94 ℃ for 5 min; setting 40 cycles and denaturation: 30s at 94 ℃; annealing: annealing temperature (set according to Solution I primer), 30 s; extension: 72 ℃ for 30 s; ③ 72 ℃ for 10 min; dissolution curve: can be set according to the instrument system (or the dissolution curve is set at 65-95 ℃ and the temperature is increased by 0.5 ℃ every 5 seconds);

(5) meanwhile, setting negative control according to the step (2);

(6) log copy number of standard plasmid after reaction₁₀Making a standard curve of the target gene according to the value and the corresponding Ct value (cycle threshold);

(7) and substituting the Ct value of the sample to be detected into the standard curve, calculating the copy number of the quantitative resistance gene of the sample to be detected, and calculating the copy number of the resistance gene in the sample.

The invention has the beneficial effects that: the kit provided by the invention can solve the problem of complicated operation steps in the quantitative analysis process of the antibiotic resistance genes in the environment. The invention also provides a detection method capable of quickly, efficiently and accurately detecting the antibiotic resistance genes in the environment, and the kit and the detection method can quickly carry out quantitative research on various types of resistance genes in the environment.

Drawings

FIG. 1 abundance of sulfonamide resistance genes in Suzhou river.

Figure 2 abundance of tetracycline resistance genes in the suzhou river.

FIG. 3 abundance of beta-lactam resistance genes in Suzhou river.

FIG. 4 abundance of macrolide resistance genes in the Suzhou river.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.