CN112322707A

CN112322707A - Detection method of resistance gene intl1 in offshore sea area

Info

Publication number: CN112322707A
Application number: CN202011317374.8A
Authority: CN
Inventors: 汪光义; 郭嘉懿; 何艺科; 李佳倩
Original assignee: Tianjin University
Current assignee: CHINA OCEAN MINERAL RESOURCES RESEARCH AND DEVELOPMENT ASSOCIATION; Tianjin University
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-02-05

Abstract

The invention discloses a method for detecting an anti-sex gene intl1 in offshore areas, which comprises the steps of extracting DNA in an environmental sample; amplifying a target fragment; constructing a standard plasmid template; purifying the DNA fragment, connecting with a PGM-18 carrier at constant temperature, taking 5ul of the connecting product after the connection is finished, adding the connecting product into a 1.5ml centrifuge tube containing 50ul of competent cells (H5 alpha), uniformly mixing, and carrying out ice bath for 30 min; the mixture was heat shocked at 42 ℃ for 60s and then iced for 2 min. Adding 800ul LB liquid culture medium, shaking and culturing at 37 deg.C and 170rpm for 1 h; extracting plasmids by using a plasmid small extraction kit according to the instruction provided by a merchant, and sending the extracted plasmids to a company for sequencing; establishing a Q-PCR quantitative standard curve; and (3) determining the resistance gene intl1 of the environmental sample, and converting the Ct value into the copy number according to a quantitative standard curve, thereby quantifying the concentration of the intl1 gene in the environmental sample.

Description

Detection method of resistance gene intl1 in offshore sea area

Technical Field

The invention relates to the technical field of common gene detection, in particular to a method for detecting an anti-gene intl1 in offshore areas.

Background

Currently, in the treatment of bacterial infections for human, animal and agricultural use, the extensive use of antibiotics causes the local bacterial community to exert antibiotic-selective pressure to make the bacteria resistant, producing resistance genes (ARG). Since the first reported discovery, ARGs have been extensively detected in natural environments such as surface rivers, groundwater, and sediments, where aquatic ecosystems are considered one of the ideal environments for acquiring and disseminating ARGs. The inti1 gene is a major transmissible vector, and compared with other mobile genetic elements, it is particularly suitable for transferring and transmitting antibiotic resistance, which can be transferred between bacterial cells and species in water, sediments and soil, and even to human commensal pathogens and bacteria, posing a threat to human health, so it is necessary to select a suitable and efficient method for accurate detection of the intl1 gene.

Disclosure of Invention

The present invention aims to overcome the following technical problems:

1. solves the problems of long time consumption and high price of the existing method for detecting the resistance gene tetG based on a sequencing method.

2. Solves the problem that the intl1 gene carried by the unculturable microorganisms in the seawater can not be accurately quantified when the resistance gene is detected based on a selective culture method.

The technical scheme of the invention is a detection method of a resistance gene intl1 in an offshore sea area, which comprises the following steps:

1) extraction of DNA from environmental samples

The seawater sample was retained on a 0.22 μm pore size microfiltration membrane using a filtration system and the DNA on the membrane was extracted using the E.N.Z.A.TM.Water DNA Kit (Omega, USA), the specific procedures of which are described in the Kit instructions.

2) Amplification of fragments of interest

Referring to relevant documents, primers designed aiming at specific genes of tetracycline resistance gene tetG are obtained, the size of a target fragment is about 146p, and the information of the primers is as follows:

an upstream primer: 5'-GGCTTCGTGATGCCTGCTT-3'

A downstream primer: 5'-CATTCCTGGCCGTGGTTCT-3'

A blank and an environment sample are used for carrying out a PCR experiment, and the system is as follows: 2 XPCR Master Mix 12.5. mu. L, ddH₂O9.5. mu.L, forward primer (10. mu.M) 1. mu.L, and reverse primer (10. mu.M) 1. mu. L, DNA 1. mu.L. The PCR procedure used: pre-denaturation at 95 ℃5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles; final extension at 72 ℃ for 5 min. And (3) carrying out 1% agarose gel electrophoresis detection on the PCR product, cutting and recovering the presented target band, and sequencing.

3) Construction of Standard plasmid templates

(1) Purifying the DNA fragment, connecting with a PGM-18 carrier at constant temperature, taking 5ul of the connecting product after the connection is finished, adding the connecting product into a 1.5ml centrifuge tube containing 50ul of competent cells (H5 alpha), uniformly mixing, and carrying out ice bath for 30 min;

(2) the mixture was heat shocked at 42 ℃ for 60s and then iced for 2 min. Adding 800ul LB liquid culture medium, shaking and culturing at 37 deg.C and 170rpm for 1 h;

(3) sucking 100 μ L of the solution, spreading the solution on an LB plate containing ampicillin, sealing the plate with a sealing film, and performing inverted culture at 37 ℃ overnight;

(4) picking single colony to shake culture in LB liquid selective culture medium containing ampicillin (50 mug/ml) for 12 h;

(5) extracting the plasmid by using a plasmid small extraction kit according to the instruction provided by the merchant, and sending the extracted plasmid to the company for sequencing.

4) Establishing a Q-PCR quantitative standard curve

After extraction is finished, use

The concentration of the plasmid was measured with an ND-1000UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA) nucleic acid content meter, and the exact copy number of the plasmid template was calculated with an on-line calculation software (http:// cells. uri. edu/gsc/cndna. html.). The diluted solution is subjected to 10-fold gradient dilution, and a corresponding quantitative standard curve is established. The procedure for Q-PCR was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles. The system is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH₂O3.5. mu.L, forward primer (10. mu.M) 0.25. mu.L, reverse primer (10. mu.M) 0.25. mu. L, DNA template 1. mu.L. After completion of the reaction, a standard curve was established with Ct values and log copy number.

5) Determination of the resistance Gene intl1 in environmental samples

Determination of the intl1 gene by Q-PCRThe DNA concentration of the gene. The reaction system is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH₂O3.5. mu.L, forward primer (10. mu.M) 0.25. mu.L, reverse primer (10. mu.M) 0.25. mu. L, DNA 1. mu.L; the Q-PCR program was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles. The Ct value was then converted to copy number according to a quantitative standard curve to quantify the concentration of intl1 gene in the environmental sample.

Advantageous effects

Compared with the current detection method based on selective culture, the detection time can be effectively shortened by using the q-PCR method for detection, which is shortened from 3 to 4 days of the original method to 1 day, and only 2 to 3 hours are needed when a standard curve is completed. Meanwhile, the problem that the resistance gene intl1 carried by the non-culturable microorganism cannot be determined by the original method is solved, and the identification accuracy is improved.

Drawings

FIG. 1 is an agarose gel electrophoresis image with a blank on the far right and environmental samples on the rest.

FIG. 2 at 2.69X 10¹～2.69×10⁵Establishment of Q-PCR quantitative curves in the copies/μ L range

FIG. 3 at 2.69X 10¹～2.69×10⁵A Q-PCR dissolution curve is established in the range of copies/mu.L, wherein the dissolution curve presents a single peak, which indicates that the amplification product in the template is a single product and has specificity.

FIG. 4 Linear relationship between Ct value and log copy number obtained by Q-PCR

FIG. 52019 and 2020, 28390and four-season abundance of int I1 gene in the estuary sea area.

Detailed Description

The method established in the research is suitable for quantifying the resistance gene intl1 in the seawater sample, and the method specification is as follows:

1. collecting a water sample at a position about 0.5m below the water surface by using a special sample collector for the water sample at a set sampling point, filling the water sample into a sterile glass bottle with the volume of 1L, numbering and storing the sample in an ice box, immediately conveying the sample back to a laboratory at a low temperature of 4 ℃ for treatment after the collection of all the samples is finished, conveying the sample back to the laboratory, filtering part of the water sample by using millipore filter membranes with the pore diameter of 0.22 mu m, filtering 500mL of the water sample by using each filter membrane, filtering three filter membranes by using each sampling point, and storing the sample at the temperature of-80 ℃ until the subsequent DNA extraction.

2. And (2) extracting the DNA of the sample, wherein an E.N.Z.A.TM Water DNA Kit (Omega, USA) is selected for DNA extraction of the Water sample, the following operations are carried out in a super clean bench in order to ensure that pollution interference of external mixed bacteria is eliminated, and the specific extraction steps are shown in the Kit specification.

3. After DNA extraction, Q-PCR experiments were performed as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH₂O3.5. mu.L, forward primer (10. mu.M) 0.25. mu.L, and reverse primer (10. mu.M) 0.25. mu. L, DNA 1. mu.L. The procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles. The Ct value was then converted to copy number according to a quantitative standard curve to quantify the concentration of intl1 gene in the environmental sample.

4. The detection method is used for detecting the intl1 gene of the fixed station position of the culture area and the estuary area of the scallop in 2019, 10 months and 12 months, 2020, 4 months and 7 months, Qinhuang island \28390, and the estuary area, and has good effect.

Claims

1. A method for detecting an anti-resistance gene intl1 in offshore areas, which is characterized by comprising the following steps:

1) extracting DNA in an environmental sample;

2) amplification of the fragment of interest:

an upstream primer: 5'-GGCTTCGTGATGCCTGCTT-3', respectively;

a downstream primer: 5'-CATTCCTGGCCGTGGTTCT-3', respectively;

3) construction of Standard plasmid templates

(2) thermally shocking at 42 deg.C for 60s, ice-cooling for 2min, adding 800ul LB liquid culture medium, and shake-culturing at 37 deg.C and 170rpm for 1 h;

(5) extracting plasmids, and sequencing after extraction;

4) establishing a Q-PCR quantitative standard curve;

5) determination of the resistance Gene of environmental sample intl 1: the DNA concentration of the intl1 gene was determined by Q-PCR technique.

2. The method for detecting the resistance gene intl1 in offshore areas according to claim 1, wherein the step 1) comprises the steps of trapping a seawater sample on a microfiltration membrane with a pore size of 0.22 μm by using a filtration system, and extracting DNA on the membrane by using an E.N.Z.A.TM.Water DNA Kit (Omega, USA).

3. The method for detecting the resistance gene intl1 in the offshore area according to claim 1, wherein the PCR experiment in step 2) is performed by using blank and environmental samples according to the following system: 2 XPCR Master Mix 12.5. mu. L, ddH₂O9.5. mu.L, upstream primer (10. mu.M) 1. mu.L, downstream primer (10. mu.M) 1. mu. L, DNA 1. mu.L;

the PCR procedure used: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles; final extension at 72 deg.C for 5 min; and (3) carrying out 1% agarose gel electrophoresis detection on the PCR product, cutting and recovering the presented target band, and sequencing.

4. The method for detecting the resistance gene intl1 in the offshore area according to claim 1, wherein the method is used after the extraction in step 4)

ND-1000UV-VisSpectrophotometer(Thermo Fisher ScIENTIFIC, USA) nucleic acid content analyzer to determine the concentration of plasmid, and using on-line calculation software (http:// cells. uri. edu/gsc/cndna. html) to calculate the exact copy number of plasmid template. Carrying out gradient dilution by 10 times to establish a corresponding quantitative standard curve;

the procedure for Q-PCR was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles;

the system is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH₂O3.5. mu.L, forward primer (10. mu.M) 0.25. mu.L, reverse primer (10. mu.M) 0.25. mu. L, DNA template 1. mu.L;

after completion of the reaction, a standard curve was established with Ct values and log copy number.

5. The method for detecting the resistance gene intl1 in the offshore area according to claim 1, wherein the reaction system in step 5) is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH₂O3.5. mu.L, forward primer (10. mu.M) 0.25. mu.L, reverse primer (10. mu.M) 0.25. mu. L, DNA 1. mu.L;

the Q-PCR program was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and 39 cycles;

the Ct value was then converted to copy number according to a quantitative standard curve to quantify the concentration of intl1 gene in the environmental sample.