CN110760600A - Rapid detection method for enterococcus in offshore water area - Google Patents
Rapid detection method for enterococcus in offshore water area Download PDFInfo
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- CN110760600A CN110760600A CN201910644095.3A CN201910644095A CN110760600A CN 110760600 A CN110760600 A CN 110760600A CN 201910644095 A CN201910644095 A CN 201910644095A CN 110760600 A CN110760600 A CN 110760600A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a method for quickly detecting enterococcus in an offshore water area, which mainly comprises the following steps of firstly, monitoring the acquisition of total DNA of bacteria in the marine water environment; secondly, obtaining a target fragment; constructing a standard plasmid and establishing a Q-PCR quantitative standard curve; and finally, measuring the abundance of the enterococcus by adopting a Q-PCR technology. The invention shortens the detection time, overcomes the problem that the original method can not detect the unculturable enterococcus and improves the identification accuracy.
Description
Technical Field
The invention relates to the technical field of detection of common pathogenic bacteria, and relates to a q-PCR detection method of enterococcus gene specific fragments.
Background
Fecal contamination of recreational waters can destroy recreational water quality, in the severe cases can result in bathing grounds being closed and posing a threat to public health. Therefore, the real-time detection of pathogen species and abundance in recreational water bodies plays an important role in water quality assessment. The water environment has a large amount of unknown pathogens and the concentration of the unknown pathogens is low, which causes the problem that the direct monitoring of the water-borne pathogens is high in cost and has certain technical difficulty. Therefore, indicator bacteria having close relationship with pathogenic microorganisms can be selected to reflect the number and distribution of pathogenic microorganisms. Enterococcus (Enterococcus) is a facultative anaerobic gram-positive bacterium, can be used as a fecal indicator, and has been used as an important index for measuring seawater quality and evaluating health risk by various national and international organizations. Enterococcus can effectively indicate fecal pollution and fecal-derived pathogenic bacteria, viruses, protozoa and the like in water. And the enterococcus content has strong correlation with the incidence of gastrointestinal diseases of recreational seawater-exposed people. However, the detection method for enterococcus in China is usually based on a culture method, and the method achieves the purpose of detection by separating, identifying and counting a selective culture medium. However, the detection method is tedious and time-consuming, results can be obtained only within 48-72 hours generally, the method is low in sensitivity and easy to generate false positive phenomena, and has great limitation in the practical application process, bacteria enrichment culture is often needed, and the number of enterococcus in an initial sample cannot be accurately quantified. Therefore, the invention provides a rapid, sensitive and specific enterococcus detection method.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for quickly detecting enterococcus in offshore water areas.
In order to solve the technical problems in the background technology, the invention adopts the technical scheme that: a method for rapidly detecting enterococcus in offshore water areas comprises the following steps:
1) acquisition of total DNA of bacteria for monitoring sea water environment
Total bacteria in a certain volume (500ml) of seawater sample are all trapped on a 0.22 μm microporous filter membrane by a suction filtration method, and the total DNA of the bacteria on the filter membrane is extracted by a Water DNA Kit (Omega, USA) at-80 ℃.
2) Acquisition of target fragment
Looking up relevant documents to search or Design a characteristic Primer through GenScript Real-time PCR (TaqMan) Primer Design, the method designs a specific Primer in a conservative permissive sequence of the enterococcus 16s rDNA gene sequence according to GenBank, the size of a target fragment is about 144bp, and the Primer information is as follows:
an upstream primer: 5'-CCCTTATTGTTAGTTGCCATC-3'
A downstream primer: 5'-ACTCGTTGTATTTCCCATTGT-3'
The PCR experiment was performed using enterococcus strain (positive control) and environmental DNA samples, the system was: 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 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 59 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; final extension at 72 ℃ for 2 min. Recovering and purifying PCR product by using common DNA purification kit and obtaining target fragment
3) Construction of Standard plasmids
Connecting the purified target product with a Ptopo-T carrier at room temperature or constant temperature of 20 ℃ for 5-10 minutes;
(ii) taking 5ul of the ligation product, adding the ligation product into a centrifuge tube (the centrifuge tube containing the competent cells is unfrozen on ice) containing 50ul of E.coli DH5 α competent cells by 1.5ml, stirring the mixture gently and mixing the mixture evenly, and carrying out ice bath for 30 min;
(iii) placing the centrifuge tube on a metal bath, performing heat shock at 42 ℃ for 45-90 s, and then placing the centrifuge tube on ice for 2 min;
(iv) adding 800ul LB culture medium, culturing for 1-2h on a shaking table at 37 ℃ and 170rpm, taking 30ul and 100ul of LB solid culture medium containing ampicillin, and culturing for 12-16h at 37 ℃;
(v) picking single colony, and shake culturing overnight at 37 ℃ and 200rpm in LB liquid culture medium containing ampicillin; then, the plasmid in the bacterial liquid is extracted by using the small plasmid extraction kit, and the plasmid is sent to a sequencing 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.). Diluting the mixture in a gradient of 3.72 × 100 to 3.72 × 104copiA quantitative standard curve is established in the range of es/mu L. The procedure for Q-PCR was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s, for 40 cycles. The system is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH2O 3.5.5 μ L, upstream primer (10 μ M)0.25 μ L, downstream primer (10 μ M)0.25 μ L, DNA 1 μ L. After completion of the reaction, a standard curve was established with Ct values and log copy number.
5) Determination of enterococcus number in environmental sample
The enterococcus DNA concentration was determined by Q-PCR technique. Reaction system: 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; Q-PCR procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s, for 40 cycles. The Ct value is then converted to copy number according to a quantitative standard curve to quantify the concentration of enterococci in the environmental sample.
Advantageous effects
Compared with the current detection method based on selective culture, the method has the following advantages: firstly, the detection time is shortened from 2-3 days of the original method to 1 day, and only 3-4 hours are needed when the standard curve is finished. Secondly, the method overcomes the problem that the original method can not determine the unculturable enterococcus and improves the identification accuracy.
1. The fluorescent quantitative PCR detection method is a rapid, sensitive and specific detection method, can make up for the defect of long time consumption (separation, identification and counting) of the traditional culture counting method, and can enlarge the detection range of environmental samples.
2. Traditional culture counting methods can only detect culturable pathogenic bacteria, however, researches show that bacteria can enter a 'viable but non-culturable' state under environmental pressure, and fluorescence quantitative PCR is based on a gene level, so that the defects in the aspect can be compensated, and the abundance of the pathogenic bacteria in the environment can be more accurately quantified.
Drawings
FIG. 1 is an agarose gel electrophoresis image, from left to right, of marker blank enterococcus (positive control) Shenzhen great Yazhen bay estuary D1, D2.
FIG. 2 is a graph at 5.20X 102～5.20×106Q-PCR quantitative amplification curves were established in the copies/μ L range.
FIG. 3 is a graph at 5.20X 102～5.20×106A Q-PCR dissolution curve was established in the copies/μ L range, where the dissolution curve exhibited a single peak, indicating that the amplification product in the template was a single product.
FIG. 4 is a linear relationship between Ct values obtained by Q-PCR and log copy number (i.e., standard curve).
FIG. 5 is a survey of 2018.11 and 2019.3 \28390andenterococcus abundance in the estuary sea area.
FIG. 6 shows the determination of the abundance of enterococcus in the Weak Australian estuary of Shenzhen Daizn Bay in 2018.11 and 2019.1.
Detailed Description
The invention is further illustrated by the following specific examples and the accompanying drawings. The examples are intended to better enable those skilled in the art to better understand the present invention and are not intended to limit the present invention in any way.
The method established in the research is suitable for quantifying the enterococcus in the seawater sample, and the method specification is as follows:
1. collecting a water sample at a position 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 the sample, storing the sample in an ice box, immediately conveying the sample back to a laboratory for treatment at low temperature after the collection of all the samples is finished, conveying the sample back to the laboratory, filtering part of the water sample by using microporous 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, storing the sample in a refrigerator at the temperature of-80 ℃ and extracting subsequent DNA
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, downstreamPrimer (10. mu.M) 0.25. mu. L, DNA 1. mu.L. The procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s, for 40 cycles. The Ct value is then converted to copy number according to a quantitative standard curve to quantify the concentration of enterococci in the environmental sample.
4. The detection method is used for evaluating the abundance of enterococcus in 11 months in 2018, 1 month in 2019 and 3 months in the Huang island Luan estuary sea area and the Weak Australian estuary sea area in Shenzhen great asia bay, and a good effect is obtained.
FIG. 1 shows the acquisition of target genes of enterococci, and the blank control and the environment have other band differences.
FIG. 2 shows the amplification curve of the standard sample, and the CT values of the same dilution sample are different by 0.5 cycle, the cycle numbers of the gradient sample are well spaced, and the amplification curve is normal because the sample is negative and has no amplification.
FIG. 3 shows a single peak in the melting curve, indicating that the amplified product in the template is a single product, the amplified product is the gene to be detected, and no primer dimer or other products are present.
FIG. 4 is a graph showing a regression equation of CT value and log copy number, which is applied to the detection of bacteria to be detected in environmental samples. Wherein R2 is 0.9997>0.9, which shows good regressiveness, and can be used for detecting the abundance of pathogenic bacteria.
FIG. 5 shows the abundance of enterococcus in the estuary sea area investigated 2018.11 and 2019.3 \28390.
FIG. 6 shows the determination of the abundance of enterococcus in the Weak Australian estuary of Shenzhen Daizn Bay in 2018.11 and 2019.1.
It should be understood that the embodiments and examples discussed herein are illustrative only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
Sequence listing
<110> Tianjin university
<120> method for rapidly detecting enterococcus in offshore water area
<160>2
<170>SIPOSequenceListing 1.0
<210>2
<211>21
<212>DNA
<213> Enterococcus faecalis (Enterococcus faecalis)
<400>2
cccttattgt tagttgccat c 21
<210>2
<211>21
<212>DNA
<213> Enterococcus faecalis (Enterococcus faecalis)
<400>2
actcgttgta tttcccattg t 21
Claims (6)
1. A method for rapidly detecting enterococcus in an offshore water area, comprising the steps of:
1) monitoring the acquisition of total DNA of the bacteria in the sea water environment;
2) obtaining a target fragment: according to GenBank, the gene sequence of enterococcus 16srDNA is published, specific primers are designed in the conservative allowable sequence, the size of a target fragment is about 144bp, and the primer information is as follows:
an upstream primer: 5'-CCCTTATTGTTAGTTGCCATC-3'
A downstream primer: 5'-ACTCGTTGTATTTCCCATTGT-3'
3) Construction of a standard plasmid:
4) establishing a Q-PCR quantitative standard curve:
after extraction is finished, use
The concentration of 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.) and was diluted in a gradient at 5.20X 102～5.20×106Establishing a quantitative standard curve in a copes/mu L range;
5) determination of enterococcus number of environmental sample: the enterococcus abundance was determined by Q-PCR technique.
2. The method for rapid detection of enterococcus in offshore waters according to claim 1, wherein said step 1) comprises: the total bacteria in the seawater sample were all retained on a 0.22 μm microporous filter by suction filtration, stored at-80 ℃ and the total DNA of the bacteria on the filter was extracted using the Water DNA Kit (Omega, USA).
3. The method for rapid detection of enterococcus in offshore waters according to claim 1, wherein said step 2) employs a PCR procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 1min, annealing at 59 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; final extension at 72 deg.C for 2 min;
and recovering and purifying the PCR product by using a common DNA purification kit and obtaining a target fragment.
4. The method for rapid detection of enterococcus in offshore waters according to claim 1, wherein said step 3) comprises constructing a standard plasmid:
connecting the purified target product with a Ptopo-T carrier at room temperature or constant temperature of 20 ℃ for 5-10 minutes;
(ii) taking 5ul of the ligation product, adding the ligation product into a centrifuge tube (the centrifuge tube containing the competent cells is unfrozen on ice) containing 50ul of E.coli DH5 α competent cells by 1.5ml, stirring the mixture gently and mixing the mixture evenly, and carrying out ice bath for 30 min;
(iii) placing the centrifuge tube on a metal bath, performing heat shock at 42 ℃ for 45-90 s, and then placing the centrifuge tube on ice for 2 min;
(iv) adding 800ul LB culture medium, culturing for 1-2h on a shaking table at 37 ℃ and 170rpm, taking 30ul and 100ul of LB solid culture medium containing ampicillin, and culturing for 12-16h at 37 ℃;
(v) picking single colony, and shake culturing overnight at 37 ℃ and 200rpm in LB liquid culture medium containing ampicillin; the plasmid in the cell culture solution will then be extracted with small volumes and sent to sequencing.
5. The method for rapid detection of enterococcus in offshore waters according to claim 1, wherein the Q-PCR in step 4) is performed by: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles;
the system is as follows: 2 × SYBR Green QPCR Mix 5 μ L, ddH2O 3.5.5 μ L, upstream primer (10 μ M)0.25 μ L, downstream primer (10 μ M)0.25 μ L, DNA 1 μ L;
after completion of the reaction, a standard curve was established with Ct values and log copy number.
6. The method for rapid detection of enterococcus in offshore waters according to claim 1, wherein the reaction system in step 5): 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; Q-PCR procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles;
the Ct value is then converted to copy number according to a quantitative standard curve to quantify the concentration of enterococci in the environmental sample.
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| CN111349682A (en) * | 2020-03-13 | 2020-06-30 | 国家海洋环境监测中心 | Method for identifying enterococcus in seawater and marine sediments |
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