CN110438246A - The rapid detection method of staphylococcus aureus in offshore waters - Google Patents

The rapid detection method of staphylococcus aureus in offshore waters Download PDF

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Publication number
CN110438246A
CN110438246A CN201910644094.9A CN201910644094A CN110438246A CN 110438246 A CN110438246 A CN 110438246A CN 201910644094 A CN201910644094 A CN 201910644094A CN 110438246 A CN110438246 A CN 110438246A
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staphylococcus aureus
detection method
pcr
rapid detection
dna
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汪光义
李佳倩
郭嘉懿
王国柱
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Tianjin University
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Tianjin University
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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
    • 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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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
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  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention discloses a kind of rapid detection method of staphylococcus aureus in offshore waters, key step are as follows: 1) monitors the acquisition of seawater environment bacteria total DNA；2) acquisition of target fragment；3) standard plasmid is constructed；4) Q-PCR quantitation curves are established；5) staphylococcus aureus abundance is measured using Q-PCR technology.Present invention reduces detection time, detection time was shorten to 1 day by 2-3 days of original method, only needed 3-4 hour in the case where completing standard curve, and was overcome original method and can not be measured and can not cultivate enterococcal problem, and the accuracy of identification is improved.

Description

The rapid detection method of staphylococcus aureus in offshore waters
Technical field
The present invention relates to encountered pathogenic bacteria detection technique fields, are related to staphylococcus aureus conservative genetic fragment A kind of q-PCR detection method.
Background technique
Staphylococcus aureus (Staphylococcus aureus) is ball-type diameter about 0.8um or so, under the microscope In single, in pairs and be arranged in thyrsiform, belong to staphylococcus, no brood cell and flagellum, and most of no pod membranes.It is golden yellow Color staphylococcus is widely present in nature (excreta of such as empty gas and water, humans and animals).Staphylococcus aureus intestines poison Element is a world health problem.The infection as caused by staphylococcus aureus accounts for second, is only second to Escherichia coli.The U.S. by Food poisoning, accounts for the 33% of entire food posioning caused by Staphylococcus aureus enterotoxin, Canadian then more, accounts for To 45%, food poisoning caused by Chinese staphylococcus aureus also happens occasionally, and as mankind's activity is to coastal waters water The influence of matter and the development of coastal tourism, the hygienic issues that staphylococcus aureus causes in the seawater also increasingly by The attention of people.Traditional culture counting method is mainly but passed through for the assessment of staphylococcus aureus content in seawater at present (such as fermentation method, filter membrane method, maximum possible counting method), although these methods also can achieve accurate counting, compares consumption When effort.And strong influence is brought for the development of the monitoring of offshore waters pathogen from now on.So inventing a kind of quick, letter Just, sensitive detection method.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide staphylococcus aureuses in a kind of offshore waters Rapid detection method.
Technical scheme is as follows: the rapid detection method of staphylococcus aureus in offshore waters, including as follows Step:
1) acquisition of seawater environment bacteria total DNA is monitored
The micropore that bacterium total in certain volume (500ml) seawater sample is all trapped in 0.22 μm is filtered by filtering method It on film, is saved at -80 DEG C, extracts bacteria total DNA on filter membrane with Water DNA Kit kit (Omega, USA), it is specific to walk Suddenly referring to kit specification.
2) acquisition of target fragment
Pertinent literature is consulted to search or set by GenScript Real-time PCR (TaqMan) Primer Design Characteristic primer is counted, this method designs specific primer, purpose piece for staphylococcus aureus specific heat stable nuclease gene Duan great little about 130bp, primer information are as follows:
Upstream primer: 5 '-CGATTGATGGTGATACGGTT-3 '
Downstream primer: 5 '-GCACTTGCTTCAGGACCAT-3 '
PCR experiment, system are as follows: 2 × PCR are carried out using staphylococcus aureus (positive control) and environment DNA sample Master Mix 12.5μL、ddH29.5 μ L of O, upstream primer (10 μM) 1 μ L, downstream primer (10 μM) 1 μ L, 1 DNA μ L.It adopts PCR program: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 1min, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 recycle；72℃ Extend 2min eventually.By common DNA purification kit recovery purifying PCR product and obtain target fragment
3) standard plasmid is constructed
The purpose product of (I) purifying is attached with pTOPO-T carrier, at room temperature connection or 20 DEG C of 5-10 points of constant temperature connections Clock；
(II) takes 5ul connection product, and addition is placed in the centrifuge tube containing 50ulE.coli DH5 α competent cell 1.5ml In (be put on ice for thawing containing competent cell centrifuge tube), after being gently mixed mixing, ice bath 30min；
Centrifuge tube is placed on metal bath by (III), 42 DEG C of thermal shock 45s~90s, then puts back to 2min. (IV) addition on ice 800ul LB culture medium, 37 DEG C, cultivate 1-2h on 170rpm shaking table after, take 30ul, 100ul to be coated on the LB containing parasiticin Solid medium, 37 DEG C of culture 12-16h；
(V) picking single colonie, in the LB liquid medium containing ampicillin, 37 DEG C, 200rpm shaking table culture mistake Night culture.Then the plasmid that will be extracted with miniplasmids extracts kit in bacterium solution, and plasmid is sent into sequencing company sequencing.
4) Q-PCR quantitation curves are established
It is used after the completion of extractingND-1000 UV-VisSpectrophotometer(Thermo Fisher Scientific, USA) nucleic acid content analyzer measures the concentration of plasmid, and be used in line computation software (http: // Cels.uri.edu/gsc/cndna.html the accurate copy number of plasmid template) is calculated.To it into gradient dilution, In The built-in vertical quantitation curves of copies/ μ L range.The program of Q-PCR are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 10s, 62 DEG C Anneal 30s, 72 DEG C of extension 30s, 40 circulations；System are as follows: 2 × SYBR Green QPCR Mix, 5 μ L, 3.5 ddH2O μ L, on Swim primer (10 μM) 0.25 μ L, downstream primer (10 μM) 0.25 μ L, 1 DNA μ L.It is built after the reaction was completed with Ct value and log copy number Day-mark directrix curve.
5) environmental sample staphylococcus aureus number measures
Enterococcus DNA concentration is measured using Q-PCR technology.Reaction system: 2 × SYBR Green QPCR Mix, 5 μ l, ddH23.5 μ L of O, upstream primer (10 μM) 0.25 μ L, downstream primer (10 μM) 0.25 μ L, 1 DNA μ L；Q-PCR program: 95 DEG C Initial denaturation 3min；95 DEG C of denaturation 10s, 62 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 recycle.Then according to quantitation curves Ct value is converted into copy number, thus the concentration of quantitative environment Gold Samples staphylococcus aureus.
The utility model has the advantages that fluorescent quantitative PCR detection method is a kind of quick, sensitive, method for detecting specificity, biography can be made up Defect that time-consuming for system culture counting method (separation is identified, is counted), can detect environmental sample on a large scale.Tradition culture counting method Educable pathogen can only be detected, however, some researches show that, bacterium can enter under ambient pressure is " active but not Can cultivate the state of (viable but nonculturable, VBNC), quantitative fluorescent PCR be based on gene level, can be with The defect for making up this respect more accurately quantifies the abundance of the pathogen in environment.Detection method is cultivated with selectivity is based on instantly It compares, this method has the advantage that detection time was shorten to 1 day by 2-3 days of original method firstly, shortening detection time, 3-4 hour is only needed in the case where completing standard curve.Secondly, overcome original method can not measure can not cultivate it is enterococcal Problem improves the accuracy of identification.
Detailed description of the invention
Fig. 1 is agarose gel electrophoresis figure, is successively from left to right marker, Daya Gulf D1, D2, blank and golden yellow Staphylococcus is positive.
Fig. 2 is in 1.8x103~1.8x107Q-PCR quantitative amplification curve is established within the scope of copies/ μ L.
Fig. 3 is in 1.8x103~1.8x107Q-PCR solubility curve is established within the scope of copies/ μ L, wherein solubility curve is in It is existing unimodal, show that amplified production is single product in template.
Fig. 4 is Ct value obtained by Q-PCR and the linear relationship (i.e. standard curve) between log copy number.
Fig. 5 is the measurement light Australia's Tidal resuspension Staphylococcus aureus abundance of 2018.11 and 2019.1 Shenzhen Daya Gulfs.
Fig. 6 is the measurement light Australia's Tidal resuspension staphylococcus aureus abundance of 2018.11 and 2019.1 Shenzhen Daya Gulfs.
Specific embodiment
Below by specific embodiments and the drawings, the present invention is further illustrated.The embodiment of the present invention is in order to more So that those skilled in the art is more fully understood the present invention well, any limitation is not made to the present invention.
The method that this research is established is suitable for quantifying the staphylococcus aureus seawater sample, method specification It is as follows:
1, in the sampled point of setting, water sample sample is acquired at the 0.5m of underwater with the special sample collector of water sample, is filled Enter in the sterile glass vials that volume is 1L, be stored in ice chest after number, after the completion of all samples acquisition, is immediately transported its low temperature It sends laboratory treatment back to, after transporting laboratory back, part water sample sample is filtered with the miillpore filter that aperture is 0.22 μm, Every membrane filtration water sample 500mL, each sampled point filter three filter membranes, save in -80 DEG C of refrigerators and for subsequent DNA's It extracts.
2, sample DNA is extracted, the DNA of water sample sample, which is extracted, selects E.N.Z.A.TM Water DNA Kit kit (Omega, USA), for the pollution interference for ensuring to exclude extraneous miscellaneous bacteria, operation need to carry out in superclean bench below, specifically mention Step is taken to see kit specification.
3, after extracting DNA, according to the progress Q-PCR experiment of following system: 2 × SYBR Green QPCR Mix, 5 μ L, ddH23.5 μ L of O, upstream primer (10 μM) 0.25 μ L, downstream primer (10 μM) 0.25 μ L, 1 DNA μ L.Program: 95 DEG C of initial denaturations 3min；95 DEG C of denaturation 10s, 62 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 recycle.Then according to quantitation curves by Ct value It is converted into copy number, thus the concentration of quantitative environment Gold Samples staphylococcus aureus.
With the detection method to January 2019 November in 2018, the big Asia in Qinhuangdao Luan River Mouth sea area in March and Shenzhen Wan Dan Australia Tidal resuspension staphylococcus aureus abundance is assessed, and obtains good result.
Fig. 1 is the acquisition of staphylococcus aureus target gene, and blank control and environment are other band differences occur.
The amplification curve of Fig. 2 standard sample thus sees that same dilution sample CT value differs 0.5 circulation, gradient sample Recurring number interval is good, and negative without amplification, indicates that amplification curve is good.
Fig. 3 is that solubility curve presentation is unimodal, shows that amplified production is single product in template, the product of amplification is to be detected Gene does not occur primer dimer or other products.
Fig. 4 establishes the regression equation of CT value Yu log copy number, the detection applied to bacterium to be measured in environmental sample.Wherein R2 =0.9934 > 0.9 indicates that regression is good, can be used for actually detected pathogen abundance.
Fig. 5 is the measurement light Australia's Tidal resuspension Staphylococcus aureus abundance of 2018.11 and 2019.1 Shenzhen Daya Gulfs.
Fig. 6 is the measurement light Australia's Tidal resuspension staphylococcus aureus abundance of 2018.11 and 2019.1 Shenzhen Daya Gulfs.
It should be understood that embodiment and example discussed herein simply to illustrate that, to those skilled in the art For, it can be improved or converted, and all these modifications and variations all should belong to the protection of appended claims of the present invention Range.
Sequence table
<110>University Of Tianjin
<120>in offshore waters staphylococcus aureus rapid detection method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>staphylococcus aureus (Staphylococcus aureus)
<400> 1
cgattgatgg tgatacggtt 20
<210> 3
<211> 19
<212> DNA
<213>staphylococcus aureus (Staphylococcus aureus)
<400> 3
gcacttgctt caggaccat 19

Claims (6)

1. the rapid detection method of staphylococcus aureus in offshore waters, it is characterised in that, include the following steps:
1) acquisition of seawater environment bacteria total DNA is monitored；
2) acquisition of target fragment:
For staphylococcus aureus specific heat stable nuclease gene design specific primer, target fragment size about 130bp, Primer information is as follows:
Upstream primer: 5 '-CGATTGATGGTGATACGGTT-3 '
Downstream primer: 5 '-GCACTTGCTTCAGGACCAT-3 '
3) standard plasmid is constructed；
4) Q-PCR quantitation curves are established: being used after the completion of extractingND-1000 UV- VisSpectrophotometer (Thermo Fisher Scientific, USA) nucleic acid content analyzer measures the dense of plasmid Degree, and it is used in the accurate copy number that line computation software calculates plasmid template；To it into gradient dilution, in 1.8x103~ 1.8x107The built-in vertical quantitation curves of copies/ μ L copies/ μ L range；
5) environmental sample staphylococcus aureus number measures: measuring staphylococcus aureus abundance using Q-PCR technology.
2. the rapid detection method of staphylococcus aureus in offshore waters according to claim 1, it is characterised in that, institute State step 1) specifically: bacterium total in seawater sample is all trapped on 0.22 μm of miillpore filter by suction filtration method ,- It is saved at 80 DEG C, extracts bacteria total DNA on filter membrane with Water DNA Kit kit (Omega, USA).
3. the rapid detection method of staphylococcus aureus in offshore waters according to claim 1, it is characterised in that, institute State step 2) specifically: the PCR program of use: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 1min, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations；72 DEG C extend 2min eventually；
By common DNA purification kit recovery purifying PCR product and obtain target fragment.
4. the rapid detection method of staphylococcus aureus in offshore waters according to claim 1, it is characterised in that, institute Stating step 3) is specially to construct standard plasmid:
The purpose product of (I) purifying is attached with pTOPO-T carrier, and connection or 20 DEG C of constant temperature connect 5-10 minutes at room temperature；
(II) takes 5ul connection product, and addition, which is placed in the centrifuge tube containing 50ulE.coli DH5 α competent cell 1.5ml, (to be contained There is competent cell centrifuge tube to be put on ice for thawing), after being gently mixed mixing, ice bath 30min；
Centrifuge tube is placed on metal bath by (III), 42 DEG C of thermal shock 45s~90s, then puts back to 2min. (IV) on ice and 800ul is added LB culture medium, 37 DEG C, cultivate 1-2h on 170rpm shaking table after, take 30ul, 100ul to be coated on the LB solid training containing parasiticin Support base, 37 DEG C of culture 12-16h；
(V) picking single colonie, in the LB liquid medium containing ampicillin, 37 DEG C, 200rpm shaking table culture trains overnight It supports；The plasmid in bacterium solution is extracted, and plasmid is sent into sequencing.
5. the rapid detection method of staphylococcus aureus in offshore waters according to claim 1, it is characterised in that, institute State step 4) specifically: the program of Q-PCR are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 10s, 62 DEG C of annealing 30s, 72 DEG C extend 30s, 40 circulations；
System are as follows: 2 × SYBR Green QPCR Mix, 5 μ L, 3.5 ddH2O μ L, upstream primer (10 μM) 0.25 μ L, downstream are drawn Object (10 μM) 0.25 μ L, 1 DNA μ L；
Standard curve is established with Ct value and log copy number after the reaction was completed.
6. the rapid detection method of staphylococcus aureus in offshore waters according to claim 1, it is characterised in that, institute State step 5) specifically: reaction system: 2 × SYBR Green QPCR Mix, 5 μ l, ddH23.5 μ L of O, upstream primer (10 μM) 0.25 μ L, downstream primer (10 μM) 0.25 μ L, 1 DNA μ L；Q-PCR program: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 10s, 62 DEG C Anneal 30s, 72 DEG C of extension 30s, 40 circulations；
Then Ct value is converted into copy number according to quantitation curves, thus quantitative environment Gold Samples staphylococcus aureus Concentration.
CN201910644094.9A 2019-07-17 2019-07-17 The rapid detection method of staphylococcus aureus in offshore waters Pending CN110438246A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104726550A (en) * 2014-11-11 2015-06-24 南昌大学 Kit for detecting staphylococcus aureus viable cells in food
CN106434917A (en) * 2016-09-24 2017-02-22 中华人民共和国广州机场出入境检验检疫局 LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104726550A (en) * 2014-11-11 2015-06-24 南昌大学 Kit for detecting staphylococcus aureus viable cells in food
CN106434917A (en) * 2016-09-24 2017-02-22 中华人民共和国广州机场出入境检验检疫局 LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHIHONG ZHANG 等: "Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products.", 《J. DAIRY SCI.》 *

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Application publication date: 20191112