CN111549158A - Method for detecting one-class integron gene intI1 in atmospheric environment - Google Patents
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Abstract
A method for detecting an integron gene intI1 in atmospheric environment. The method comprises the following steps: diluting an integrant gene intI1 standard sample with a known concentration to different concentrations, taking the standard sample as a template to perform PCR reaction to extract DNA, taking the DNA as the template, performing quantitative PCR amplification on the DNA by using a designed forward primer and a designed reverse primer, and recording the cycle number; determining the obtained cycle number according to a standard curve of a target gene intI1 plasmid standard sample by quantitative PCR; a standard curve of the target gene is made by using a standard sample with the initial copy number of a template with known concentration by using a 10-fold gradient dilution method; determining the cycle number of one type of integron gene intI1 in the atmospheric environment sample, and calculating the content of the gene intI1 according to a standard curve. The target gene primer sequence and the method provided by the invention can be used for quickly and accurately quantifying one type of integron gene intI1 in an atmospheric environment sample.
Description
Technical Field
The invention relates to the field of environmental microbiology, in particular to a method for detecting mobile genetic factor intI1 in atmospheric environment.
Background
Antibiotics are secondary metabolites produced by microorganisms (including bacteria, fungi, actinomycetes) that inhibit the growth and survival of other types of microorganisms, and chemically synthesized or semi-synthesized compounds, and almost all kinds of antibiotics are based on antibiotic structures naturally found in environmental microorganisms, and are mainly used for treating bacterial infectious diseases. The resulting increase in antibiotic-resistant bacterial infections and multi-environmental spread of antibiotic-resistant genes is posing a serious health threat to the world.
With the wide use of antibiotics in clinical anti-infective therapy, the problem of bacterial drug resistance is becoming more serious, and the horizontal transfer of genes among bacteria is an important reason for the formation and wide spread of bacterial multi-drug resistance. The integration subsystem has the capacity of capturing and expressing an external drug-resistant gene cassette, and bacteria can continuously capture and accumulate the drug-resistant gene cassette from the surrounding environment through integrase coded by an integrase gene intI1 in the system to form a huge drug-resistant gene multi-locus. The integrants can be present on heritable gene modules such as plasmids, transposons, etc., and perform horizontal gene transfer between bacteria of the same or different species, and play an important role in the formation of multiple drug-resistant bacteria and horizontal diffusion of drug-resistant genes. However, currently, little research is done on intI1 in atmospheric environment, and intI1 can carry antibiotic resistance genes into the human body through air transmission, which affects human health.
Disclosure of Invention
The invention aims to solve the problem that genes of non-culturable microorganisms cannot be detected in an atmospheric environment, and provides a method for quickly and accurately detecting an integron gene intI1 in the atmospheric environment.
The real-time quantitative PCR technology is a method for detecting the change of the quantity of each cycle of amplified products in the PCR amplification process in real time by using the change of a fluorescent signal and finally carrying out accurate quantitative analysis on an initial template. The method has a good effect on detecting the genes contained in bacteria with low content in the ambient atmosphere, so that the method has important theoretical and practical significance for researching the influence of the integrase gene intI1 in the atmospheric environment on human health and environment by adopting a real-time quantitative PCR technology.
Technical scheme of the invention
A primer sequence for detecting one type of integron gene intI1 in atmospheric environment comprises a forward primer and a reverse primer, and the base sequence of the primer is as follows:
forward primer (SEQ ID NO 1): GGCTTCGTGATGCCTGCTT, respectively;
reverse primer (SEQ ID NO 2): CATTCCTGGCCGTGGTTCT are provided.
A method for detecting mobile genetic factors in atmospheric environment, namely one type of integrant gene intI1, specifically comprises the following steps:
(1) diluting standard samples of the known concentration of the integrant gene intI1 to different concentrations, and performing PCR reaction by using the standard samples as templates; extracting DNA in a standard sample, taking the extracted DNA as a template, performing quantitative PCR amplification on the DNA by using the forward primer (SEQ ID NO 1) and the reverse primer (SEQ ID NO 2) designed above, and recording the cycle number when the fluorescence threshold is reached;
the reaction system of the quantitative PCR is 25 muL of qRT qPCR Supermix solution with the concentration of 1 × and 12.5 muL of 0.2 muM forward primer and 0.3 muL of 0.2 muM reverse primer, and 10.9 muL of ddH20，1.0μLTemplate DNA。
The extraction method of the DNA comprises the following steps: extracted with MO-BIO PowerSoil DNAIsolation kit. The method for extracting DNA can purify DNA in a short time, is suitable for DNA extraction in various difficult environments, and can extract enough DNA from an atmospheric sample with less microorganism. The patented technology can remove one hundred percent of humus and PCR inhibitors.
(2) Determining the number of cycles obtained by the PCR quantitative amplification in the step (1) based on the quantitative PCRThe standard curve of the gene intI1 plasmid standard sample; using 10-fold gradient dilution method, making a standard curve of the target gene by using a standard sample with known concentration template initial copy number, taking the logarithm value of the copy number of the standard sample as an abscissa, and measuring CTValues are plotted on the ordinate, giving standard plasmid concentrations of: 102、103、104、105、106、107，108Ensure R2>0.99, the effective curve is obtained when the amplification efficiency is between 90% and 110%;
the preparation method of the target gene intI1 plasmid standard sample comprises the following steps:
preparation of a target gene intI1 plasmid standard sample: through the forward primer (SEQ ID NO 1) and the reverse primer (SEQ ID NO 2) designed above, a target gene PCR product is obtained through PCR amplification, and then a target DNA fragment is obtained by utilizing a gel recovery kit.
Adding the purified DNA fragment into a competent Escherichia coli cell DH5 alpha by using a vector, adding the competent Escherichia coli cell DH5 alpha into an ampicillin-free LB culture medium for culture, recovering the competent Escherichia coli cell DH for 1h, culturing the competent Escherichia coli cell DH in an ampicillin-containing solid LB culture medium for one night, selecting a single colony white spot by a blue-white spot screening method, and adding the single colony white spot into an ampicillin-containing LB liquid culture medium for culture for 12 h.
Extracting plasmid with a plasmid extraction kit, cutting the gel, and then sending the gel to a sample for sequencing to detect the sequence of the inserted gene fragment. The plasmids meeting the requirements were used as standard samples.
(3) Determining the cycle number of one type of integron gene intI1 in the atmospheric environment sample according to the method in the step (1), and calculating the content of the gene intI1 according to the standard curve in the step (2).
And (3) carrying out real-time quantitative PCR detection on the unknown DNA sample under the same system to obtain the cycle number (Ct value) value of the unknown sample, and substituting the cycle number (Ct value) value into the standard curve to calculate the initial concentration of the target gene DNA of the sample to be detected.
Ensuring R2>0.99, the effective curve is obtained when the amplification efficiency is between 90% and 110%.
The invention has the advantages and beneficial effects that:
(1) the invention utilizes the MO-BIO Power soil DNA Isolation kit to completely remove PCR reaction inhibitors, thereby greatly improving the sensitivity and the success rate of PCR reaction. The pure DNA can be directly used for downstream experiments such as PCR and the like.
(2) Through common PCR reaction, the existence of one kind of integron gene intI1 in the atmospheric environment can be determined; the content of one type of integron gene intI1 in the atmospheric environment can be determined through real-time quantitative PCR reaction, and technical guidance is provided for the research of target DNA in the atmospheric environment;
(3) compared with the traditional DNA extraction method, the method has the advantages of short time for extracting the DNA, high DNA purity, high practicability and high reliability, and is beneficial to later-stage DNA detection.
Drawings
FIG. 1 is an electropherogram of DNA extracted from a sample of an atmospheric environment;
lane 1: DNA of microorganisms in atmospheric samples of schools,
lane 2: microbial DNA in a market atmospheric environment sample,
lane 3: DNA of microorganisms in a sample of the atmospheric environment of a hospital,
lane 4: the DNA of the microorganism in the atmospheric environment sample of the station,
lane 5: microbial DNA in atmospheric environment sample of residential area
Blank lane: and (5) negative control.
FIG. 2 is an electropherogram of intI1 of a DNA amplification target gene extracted from an atmospheric environment sample;
lane 1: target gene intI1 in the atmospheric environmental sample of school,
lane 2: the target gene intI1 in the market atmospheric environment sample,
lane 3: the target gene intI1 in the atmospheric environmental sample of the hospital,
lane 4: a target gene intI1 in a station atmospheric environment sample,
lane 5: target gene intI1 in atmospheric environment sample of residential area
Blank lane: and (5) negative control.
FIG. 3 is a quantitative standard curve for the target gene intI 1.
Detailed Description
The invention is further illustrated by the following detailed description of embodiments in connection with the accompanying drawings.
Example 1: and (3) extracting DNA from the atmospheric environment sample.
The steps of extracting the DNA in the atmospheric environment sample by utilizing the MO-BIO PowerSoil DNA Isolation Kit are as follows:
(1) the filters were placed in a beaker containing 50ml of 1 XPBS buffer and centrifuged at 200 Xg for 3h at 4 ℃.
(2) After gentle vortexing, the resuspension was poured into a funnel containing 0.2um Supor 200 PES membrane, and 500ml was run over within 2 min.
(3) Cutting PES membrane after air drying, placing into test tube of kit, adding 60 μ L of solutionC1, vortexing at maximum speed for 15min, centrifuging at 10000 × g for 30s, and transferring supernatant into new test tube.
(4) Add 250. mu.L of SolutionC2, vortex 5s, centrifuge 10000 Xg for 60s, and transfer the supernatant to a new tube.
(5) Adding 200 μ L of solutionC3, vortex mixing, standing at 4 deg.C for 5min, centrifuging at 10000 × g for 60s, and transferring the supernatant to a new test tube.
(6) Adding 1200 μ L of SolutionC4, vortex mixing, adding the supernatant into Spin Filter, and centrifuging at 10000 × g for 30 s.
(7) Then 500. mu.L of solutionC5 was added thereto, and the mixture was centrifuged at 10000 Xg for 30 seconds, and the supernatant was discarded, followed by centrifugation at 10000 Xg for 60 seconds.
(8) Spin Filter was transferred to a new tube, 100. mu.L of SolutionC6 was added, 10000 Xg was centrifuged for 30s, and Spin Filter was discarded, at which time the DNA was ready for the experiment without purification.
The electrophoresis chart of the extracted target DNA for PCR qualitative detection is shown in figure 1, and the result shows that the concentration of the DNA is higher, thereby meeting the requirements of subsequent experiments.
Example 2: design of primers
(1) Fromhttp://www.ncbi.nlm.nih.gov/Downloading sulfanilamide drug resistance gene sulI gene sequence (Accession No. AY063901.1, AF135182.5, CP002685.1, CP000499.1, CP017627.1, CH466550.2, CH474035.2, CM 0)04444.1, DS995704.1, FJ626843.1, FM992692.1, JN983043.1, JN983049.1, HK616890.1, X56845.1). The primer sequences are all introduced into PrimePremier 5.0, and alignment is carried out to find the conserved region of intI1 of the integrant genes from different sources. This was used as the DNA sequence of the target gene intI 1.
(2) Inputting the obtained conserved region sequence into DNAMAN software, designing a primer meeting the quantitative PCR requirement, wherein the length of the amplified product fragment is generally in the range of 100-200bp, and determining the primer with the length of 146bp of the amplified product fragment of the gene intI1 through index optimization selection and verification, wherein the annealing temperature is 60 ℃.
Forward primer intI 1-F: 5'-GGCTTCGTGATGCCTGCTT-3' (SEQ ID NO:1),
reverse primer intI 1-F: 5'-CATTCCTGGCCGTGGTTCT-3' (SEQ ID NO: 2).
Example 3: qualitative detection
In the experiment, 20 mu L of reaction system is selected as the PCR reaction system, and the qualitative PCR reaction system (table 1) and the qualitative PCR reaction program (table 2) are respectively as follows:
TABLE 1 qualitative PCR reaction System
TABLE 2 qualitative PCR reaction procedure
(1) The MO-BIO PowerSoil DNA Isolation kit is used for extracting DNA in an atmospheric sample, and qualitative PCR amplification is carried out by using a designed integrant gene intI1 primer sequence under the conditions of a qualitative PCR reaction system and a reaction program. And 5. mu.L of PCR product after reaction is subjected to agarose gel electrophoresis detection. The PCR amplification result was detected by electrophoresis in a 2% agarose gel in 1 XTAE electrophoresis buffer under 90mV for 30 min. The gel was observed under a gel imager for the presence of the target gene intI1 based on the known standard DNA molecular band size, and the results are shown in FIG. 2. The results showed the presence of the desired gene intI1 in the PCR product.
(2) Cutting the gel under a gel imager to obtain gel containing the target gene intI1 fragment, and recovering and purifying the gel by using a gel recovery kit. The recovered DNA is subjected to electrophoresis, and the concentration of the DNA is determined by observing the brightness of the target DNA fragment, so that the subsequent PCR cloning can be performed.
(3) The purified PCR product was ligated with a PMD-18-T vector and placed in a refrigerator at 4 ℃ overnight. Then 200. mu.L of competent E.coli cell DH 5. alpha. was added.
(4) Adding into LB culture medium without ampicillin, culturing for 1h, culturing overnight on solid LB culture medium containing ampicillin, selecting single colony white spot by blue white spot screening method, adding into LB liquid culture medium containing aminobenzyl, and culturing for 12 h. Extracting plasmid with the plasmid extracting kit, cutting gel, and sequencing to insert gene fragment. The sizes of 2 DNA sequence fragments obtained by sequencing are all 146bp (the base sequences are shown as SEQ ID NO: 3 and SEQ ID NO: 4).
(5) The sequencing sequence result is subjected to BLAST sequence comparison on an NCBI webpage, the homology of the nucleic acid sequence is directly compared, and the homology is found to be 100 percent, so that the designed primer has high specificity.
Example 4: preparation of Standard Curve
(1) The copy number is calculated by the formula of (copies/. mu.L) ═ plasmid concentration/molecular weight × 6.02.02 6.02 × 1023Wherein 6.02 × 1023The average molecular weight of each base in the experiment is 330, the average molecular weight of each base in the experiment is 660, the plasmid stock solution is C (ng/mu L), the length of the target gene is L (bp), and the copy number formula of the target gene (copies/mu L) is [ C/(3829+ L) × 660]×6.02×1023And calculating the initial copy number of the target gene.
(2) Taking the plasmid meeting the requirements as a standard sample, using a 10-fold gradient dilution method, making a standard curve of the target gene by using a standard sample with the initial copy number of a template with known concentration, and taking the logarithm value of the copy number of the standard sample as an abscissa to measureTo obtain CTThe values are on the ordinate. The standard plasmid concentrations were made as follows: 102、103、104、105、106、107，108. Ensuring R2>0.99, the effective curve is obtained when the amplification efficiency is between 90% and 110%.
(3) The standard curve reaction system is 1.0 muL of DNA standard, 12.5 muL of qRTqPCRSuperMix solution with the concentration of 1 ×, 0.3 muL of 0.2 muM forward primer, 0.3 muL of 0.2 muM reverse primer and 10.9 muL of ddH20。
The standard curve of the target gene (see fig. 3) obtained by the experiment is: ct ═ 3.962lgC + 39.536; amplification efficiency: 93.8 percent; coefficient of correlation R20.997, meets the requirement.
Example 5: quantitative detection
In the quantitative PCR reaction system of the present experiment, a 25. mu.L reaction system was selected, and the quantitative PCR reaction system (Table 3) and the quantitative PCR reaction program (Table 4) were respectively:
TABLE 3 real-time fluorescent quantitative PCR reaction System
TABLE 4 real-time fluorescent quantitative PCR reaction procedure
(1) Collecting an atmospheric environment sample of school, extracting DNA by using the method in the embodiment 1, determining the concentration to be 15 ng/mu L, meeting the concentration requirement, and storing to be detected;
(2) the forward primer and the reverse primer designed in the example 2 are used for detecting the concentration content of the integron gene intI1 of the atmospheric environmental sample of school by using the fluorescent quantitative PCR technology according to the reaction system and the reaction program in the table 3 and the reaction program in the table 4.
(3) And carrying out fluorescent quantitative PCR detection on the gene to be detected intI 1. Transferring the reaction system to a reaction hole of a 96-hole reaction plate, carrying out fluorescent quantitative PCR detection on the gene intI1 to be detected through a quantitative PCR system, recording the signal intensity scanned in each amplification cycle, obtaining the cycle threshold Ct of the gene intI1, substituting the cycle threshold Ct into the standard curve of the embodiment 4, and calculating to obtain the initial concentration of the target gene intI1 of the experimental sample.
(4) Repeatedly detecting the DNA of the sample to be detected for three times, respectively substituting the obtained Ct values into the standard curve to calculate the initial concentration of the target gene intI1 of the experimental sample to be 2.18 × 104copies/uL、4.54×104copies/uL、4.18×104copies/uL, which indicates that the target atmospheric environment sample contains one type of integron gene intI 1. Therefore, the primer sequence designed in the embodiment 2 is utilized, and the quantitative PCR technology is utilized to quickly detect the concentration content of the integron gene intI1 in the target atmospheric environment sample.
Sequence listing
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<120> method for detecting one type of integron gene intI1 in atmospheric environment
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ccctcactga tccgcatgcc cgttccatac agaagctggg cgaacaaacg atgctcgcct 60
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gacggccgag gtcttccgat ctcctg 146
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ccctcactga tccgcatgcc cgttccatac agaagctggg cgaacaaacg atgctcgcct 60
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gacggccgag gtcttccgat ctcctg 146
Claims (4)
1. A primer sequence for detecting one type of integron gene intI1 in atmospheric environment is characterized by comprising a forward primer and a reverse primer, wherein the base sequence is as follows:
a forward primer: GGCTTCGTGATGCCTGCTT, respectively;
reverse primer: CATTCCTGGCCGTGGTTCT are provided.
2. A method for detecting an integrant gene intI1 in atmospheric environment by using a target gene primer sequence as defined in claim 1, which comprises the following steps:
(1) diluting standard samples of the known concentration of the integrant gene intI1 to different concentrations, and performing PCR reaction by using the standard samples as templates; extracting DNA in a standard sample, and carrying out PCR quantitative amplification by using the extracted DNA as a template and using the forward primer and the reverse primer in claim 1 to obtain the cycle number of a PCR product;
(2) determining the cycle number obtained by PCR quantitative amplification in the step (1) according to a standard curve of a target gene intI1 plasmid standard sample by quantitative PCR; using 10-fold gradient dilution method, making a standard curve of the target gene by using a standard sample with known concentration template initial copy number, taking the logarithm value of the copy number of the standard sample as an abscissa, and measuring CTValues are plotted on the ordinate, giving standard plasmid concentrations of: 102、103、104、105、106、107，108Ensure R2>0.99, the effective curve is obtained when the amplification efficiency is between 90% and 110%;
(3) determining the cycle number of one type of integron gene intI1 in the atmospheric environment sample according to the method in the step (1), and calculating the content of the gene intI1 according to the standard curve in the step (2).
3. The method of claim 2, wherein the quantitative PCR reaction system is 25 μ L of qRT qPCR Supermix solution with concentration of 1 × at 12.5 μ L, 0.3 μ L of 0.2 μ M forward primer, 0.3 μ L of 0.2 μ M reverse primer, 10.9 μ L ddH20，1.0μL Template DNA。
4. The method according to claim 2 or 3, wherein said target gene intI1 plasmid standard sample is prepared as follows:
preparation of a target gene intI1 plasmid standard sample: through the forward primer and the reverse primer designed in the claim 1, PCR amplification is carried out to obtain a target gene PCR product, and then a glue recovery kit is utilized to obtain a target DNA fragment;
adding the purified DNA fragment into a competent Escherichia coli cell DH5 alpha by using a carrier, adding the competent Escherichia coli cell DH5 alpha into an ampicillin-free LB culture medium for culture, recovering the competent Escherichia coli cell DH for 1h, culturing the competent Escherichia coli cell DH in a solid LB culture medium containing ampicillin overnight, selecting a single colony white spot by a blue-white spot screening method, and adding the single colony white spot into an ampicillin-containing LB liquid culture medium for culture for 12 h;
and extracting plasmids in the bacterial liquid by using a plasmid extraction kit, qualitatively detecting whether the plasmids exist or not by using PCR amplification, and carrying out sample sequencing after cutting a gel, wherein the bands are clear and visible.
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