CN104862394A - Primer for detecting Escherichia coli, and method and application of primer - Google Patents

Primer for detecting Escherichia coli, and method and application of primer Download PDF

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CN104862394A
CN104862394A CN201510236760.7A CN201510236760A CN104862394A CN 104862394 A CN104862394 A CN 104862394A CN 201510236760 A CN201510236760 A CN 201510236760A CN 104862394 A CN104862394 A CN 104862394A
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primer
adsorption column
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colibacillary
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CN104862394B (en
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毛小琴
刘有福
宋玉竹
牛华
夏雪山
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Kunming University of Science and Technology
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention discloses a primer for detecting Escherichia coli, and a method and an application of the primer. The nucleotide sequence of the primer is indicated in SEQ ID NO: 1 and SEQ ID NO: 2, an Escherichia coli detection method is established for the involved primer, and the detection method has the advantages of fine specificity, good sensitivity, rapidness and reliability in detection and simplicity in operation. A kit comprising the primer is designed, and the Escherichia coli is conveniently detected. The specific primer is designed for specific genes LafF of the Escherichia coli and can be used for rapidly, simply, conveniently and accurately detecting the Escherichia coli.

Description

A kind of for detecting colibacillary primer and methods and applications thereof
Technical field
The invention belongs to biological technical field, relating to a kind of for detecting colibacillary primer and application thereof.
Background technology
Colon bacillus (Escherichia coli) custom is called intestinal bacteria, classifies in enterobacteriaceae, belongs to Escherichia.Major part intestinal bacteria are the bacterium that normally live away from home in people and various animal intestinal, and in food or water, colibacillary detecting means direct or indirect recent fecal pollution.Intestinal bacteria are as the excrement source contact scar hygieneic bacteriology index of drinking-water, food etc.; And it is close with some primary bowel pathogenic bacterias in the extraneous survival time.Its appearance also may indicate the existence of some enteric pathogenic bacteria (as Salmonellas, Shigellae).Intestinal bacteria are the monitoring of hygiene indicators of generally acknowledging in the world, and therefore colibacillary detection technique seems very important.
Traditional colibacillary detection method has routine smear Microscopical Method For Detection, culture method etc., but these methods exist certain defect, and if conventional smear for microscopic examination is the most basic bacteriology checking method, but susceptibility is low, poor specificity; Culture method is the main path and the means that find contagium at present clinically, but generally needs the time of 4 to 7 days, and complex operation, time-consuming effort, can not realize object fast.
The present invention utilizes information biology to find out intestinal bacteria specific gene, and for its design primer, establish colibacillary detection method, achieve easy, detect colibacillary object fast and accurately.
Summary of the invention
The present invention is directed to the defect of prior art, proposing a kind of for detecting colibacillary gene, obtaining this specific gene that can be used for identification of escherichia coli by information biology means, and relating to primer for this gene.
Another object of the present invention is to, for involved primer, establish colibacillary detection method, its have specificity good, sensitive better, detect feature fast and reliable, simple to operate.
3rd object of the present invention is that providing a kind of detects colibacillary test kit, is convenient to colibacillary detection.
The present invention realizes especially by following technical scheme:
A kind of for detecting colibacillary primer, comprise upstream primer and downstream primer, described upstream primer nucleotide sequence is as shown in SEQ ID NO:1, and described downstream primer nucleotide sequence is as shown in SEQ ID NO:2.
Prove by experiment, detection primer pair intestinal bacteria provided by the invention have good specificity, this detection primer for target fragment LafF gene.
A kind of colibacillary method of detection, comprises the following steps:
1) template DNA of detected sample is extracted;
2) primer described above is designed;
3) pcr amplification:
Reaction system: Taq 0.125 μ L, 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH 2o supplies 25 μ L;
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 1min;
4) agarose gel electrophoresis method for detecting is utilized to judge detected result.
A kind of for detecting colibacillary test kit, comprise primer SEQ ID NO:1 and SEQ ID NO:2, be specially: 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 1 μ L, upstream and downstream primer each 1 μ L, ddH 2o 25 μ L.
Beneficial effect of the present invention is: design special primer for colibacillary specific gene LafF, this primer can quick, easy, detect intestinal bacteria accurately.
Accompanying drawing explanation
Fig. 1 is the impact that different annealing temperature and MgCl2 concentration are reacted colibacillus PCR; M:2000bp DNA Marker; 1:MgCl 2concentration 0.8mM; 2:MgCl 2concentration 1.6mM; 3:MgCl 2concentration 2.4mM; 4:MgCl 2concentration 3.2mM; 5:MgCl 2concentration 4.0mM;
Fig. 2 is that large intestine bar LafF bacterium gene specific detects electrophorogram; M:2000bp DNAMarker; The coli strain that 1 ~ 3: three strains are different; 4: Klebsiella Pneumoniae; 5: not labor Di Shi citrobacter; 6: Corynebacterium diphtheriae swimming lane; 7: Acinetobacter junii; 8: staphylococcus epidermidis; 9: enterococcus faecalis; 10: staphylococcus haemolyticus; 11: streptococcus aureus; 12: morganella morganii;
Fig. 3 is intestinal bacteria LafF gene sensitivity technique electrophorogram; M:2000bp DNAMarker; 1 ~ 10: the number of plasmid in every microlitre template, 1 ~ 10 difference corresponding 10 9individual/μ L ~ 10 0individual/μ L; 11: negative control.
Embodiment
Below in conjunction with embodiment, the present invention is described further, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, any simple modification done following examples according to technical spirit of the present invention or equivalent variations, all drop in protection scope of the present invention.
The screening of embodiment 1 specific target gene and design of primers:
1) Local BLAST analysis
The intestinal bacteria whole genome sequence downloaded from NCBI (http://www.ncbi.nlm.nih.gov/genome/), Local BLAST retrieval is carried out to the nucleic acid database of this locality, acquires each sequence fragment of intestinal bacteria and database comparison result.
2) BLAST heavily confirms
Use online BLAST function, use 2 kinds of strategies to confirm its bacterial classification specificity and intestinal bacteria qualification operability.A kind of strategy is for getting rid of intestinal bacteria during BLAST, and result display is intestinal bacteria specific gene without any similar sequences person; Select when the second strategy is BLAST to retrieve in intestinal bacteria, it is different coli strain consensus sequences that result returns a large amount of similar sequences person.In conjunction with two kinds of strategies, finally show that gene LafF is as shown in SEQ ID NO:3, meets two kinds of policy criteria.
3) design of primers
For intestinal bacteria LafF gene, design special primer: gaagaaaatcgtggtggcgg and gtcggttttccttttccggc.
The foundation of embodiment 2 detection method
1) extraction of DNA
Carry out extracting and reclaiming with the bacterial genomes DNA Mini Kit of border biological gene Science and Technology Ltd. of the village, Beijing ally, step is as follows:
(1) get inoculum 5mL, centrifugal 1 minute of 12,000rpm, exhaust supernatant as far as possible.
(2) in the centrifuge tube leaving bacterial sediment, add 500 μ L cell suspending liquids, use pipettor or the thorough suspended bacterial cell precipitation of turbula shaker, 37 DEG C of temperature baths 30 minutes.Mixing is put upside down for several times every 10 minutes.Centrifugal 2 minutes of 12,000rpm, exhaust supernatant as far as possible.
(3) in bacterial sediment, add 225 μ L buffer A, vibrate to thalline and thoroughly suspend.
(4) Xiang Guanzhong adds 6 μ L RNaseA solution, and vibrate 15 seconds, room temperature places 5 minutes.
(5) Xiang Guanzhong adds 10 μ L Proteinase K Solution, puts upside down mixing.
(6) add 25 μ L lysis buffer S, put upside down mixing.
(7) add 250 μ L buffer B, vibrate 10 seconds, place 10 minutes for 70 DEG C.Centrifugal 1 minute of 12,000rpm, gets supernatant and puts into a new pipe, discard precipitation.
(8) add 250 μ L dehydrated alcohols, fully vibration mixing 15 seconds, now may occur flocks, brief centrifugation is to remove the globule of cap wall.
(9) all add in an adsorption column (adsorption column puts into collection tube) by previous step gained solution and flocks, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column is put into collection tube.
(10) in adsorption column, add 500 μ L damping fluid C, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column is put into collection tube.
(11) in adsorption column, add 700 μ L rinsing liquid W2, centrifugal 30 seconds of 12,000rpm, outwell waste liquid, adsorption column puts into collection tube.
(12) in adsorption column, add 500 μ L rinsing liquid W2,12,000rpm, from 30 seconds, outwells waste liquid, is put back in collection tube by adsorption column.Then 12,000rpm centrifugal 2 minutes.Adsorption column is placed in a new 1.5mL centrifuge tube, room temperature places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(13) proceeded to by adsorption column in a clean centrifuge tube, the unsettled dropping 100 in the middle part to adsorption film μ L elution buffer TE, room temperature places 2 minutes, and centrifugal 2 minutes of 12,000rpm, by solution collection in centrifuge tube.
2) PCR condition optimizing:
One, to annealing temperature and Mg 2+be optimized simultaneously.Annealing temperature is 6 gradients, 49,51,53,55,57,59 DEG C.Mg 2+concentration is 5 gradients, 0.8,1.6,2.4,3.2 and 4.0mM.Described is 25 μ L by polymerase chain reaction detection intestinal bacteria condition optimizing amplification system.Concrete reaction system is: Taq DNA polymerase 0.125 μ L, 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+be respectively 0.8,1.6,2.4,3.2 and 4.0mM, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH 2o supplies 25 μ L.
Two, increase by following program in PCR instrument:
(1) denaturation
95 DEG C 5 minutes
(2) 30 circulations:
95 DEG C of 30 second
49,51,53,55,57,59 DEG C of 30 second
72 DEG C of 20 second
(3) increase afterwards
72 DEG C 1 minute
Final establishment Mg 2+for 4.0M, annealing temperature is 57 DEG C of conditions is the optimal condition detected.Result as shown in Figure 1.
Embodiment 3 specific detection
One, the strain of intestinal bacteria clinical separation strain 3 is collected, the strain of non-intestinal bacteria 9.These bacterial strains bacterial genomes DNA Mini Kit of border biological gene Science and Technology Ltd. of the village, Beijing ally is extracted genome, and step is as follows:
(1) get inoculum 5mL, centrifugal 1 minute of 12,000rpm, exhaust supernatant as far as possible.
(2) in the centrifuge tube leaving bacterial sediment, add 500 μ L cell suspending liquids, use pipettor or the thorough suspended bacterial cell precipitation of turbula shaker, 37 DEG C of temperature baths 30 minutes.Mixing is put upside down for several times every 10 minutes.Centrifugal 2 minutes of 12,000rpm, exhaust supernatant as far as possible.
(3) in bacterial sediment, add 225 μ L buffer A, vibrate to thalline and thoroughly suspend.
(4) Xiang Guanzhong adds 6 μ L RNaseA solution, and vibrate 15 seconds, room temperature places 5 minutes.
(5) Xiang Guanzhong adds 10 μ L Proteinase K Solution, puts upside down mixing.
(6) add 25 μ L lysis buffer S, put upside down mixing.
(7) add 250 μ L buffer B, vibrate 10 seconds, place 10 minutes for 70 DEG C.Centrifugal 1 minute of 12,000rpm, gets supernatant and puts into a new pipe, discard precipitation.
(8) add 250 μ L dehydrated alcohols, fully vibration mixing 15 seconds, now may occur flocks, brief centrifugation is to remove the globule of cap wall.
(9) all add in an adsorption column (adsorption column puts into collection tube) by previous step gained solution and flocks, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column is put into collection tube.
(10) in adsorption column, add 500 μ L damping fluid C, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column is put into collection tube.
(11) in adsorption column, add 700 μ L rinsing liquid W2, centrifugal 30 seconds of 12,000rpm, outwell waste liquid, adsorption column puts into collection tube.
(12) in adsorption column, add 500 μ L rinsing liquid W2,12,000rpm, from 30 seconds, outwells waste liquid, is put back in collection tube by adsorption column.Then 12,000rpm centrifugal 2 minutes.Adsorption column is placed in a new 1.5mL centrifuge tube, room temperature places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(13) proceeded to by adsorption column in a clean centrifuge tube, the unsettled dropping 100 in the middle part to adsorption film μ L elution buffer TE, room temperature places 2 minutes, and centrifugal 2 minutes of 12,000rpm, by solution collection in centrifuge tube.
Two, PCR reaction system:
The genome of each bacterial strain is used separately as template and carries out polymerase chain reaction.Reaction system is 25 μ L, Taq 0.125 μ L, 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH 2o supplies 25 μ L.
Three, increase by following program in PCR instrument:
(1) denaturation
95 DEG C 5 minutes
(2) 30 circulations:
95 DEG C of 30 second
57 DEG C of 30 second
72 DEG C of 20 second
(3) increase afterwards
72 DEG C 1 minute
At Mg 2+for 4.0mM, annealing temperature is under the condition of 57 DEG C, and this detection method specificly can detect intestinal bacteria, and does not affect by other bacterial classifications, as shown in Figure 2.
Embodiment 4 sensitivity technique
One, positive plasmid vector construction
1) take genome of E.coli as template, carry out pcr amplification with above-mentioned special primer, reaction system is 100 μ L, Taq 0.5 μ L, 10 × polymerase buffer 10 μ L, dNTP 8 μ L, Mg 2+6.4 μ L, template 4 μ L, each 4 μ L of upstream and downstream primer, add ddH 2o supplies 100 μ L.
2) increase by following program in PCR instrument:
(1) denaturation
95 DEG C 5 minutes
(2) 30 circulations:
95 DEG C of 30 second
57 DEG C of 30 second
72 DEG C of 20 second
(3) increase afterwards
72 DEG C 1 minute
3) after loop ends, PCR primer is used 1.5% agarose gel electrophoresis, and carry out extracting and reclaiming with a small amount of sepharose DNA recovery test kit of border biological gene Science and Technology Ltd. of the village, Beijing ally, step is as follows:
(1) single target DNA band is cut (excising redundance) from sepharose as far as possible
Put into clean centrifuge tube, take weight.
(2) in blob of viscose, add 2 times of volume sol solutionses, 55 DEG C of water-baths 10 minutes, constantly leniently spin upside down centrifuge tube therebetween, to guarantee that blob of viscose fully dissolves.
(3) add in an adsorption column by previous step gained solution, centrifugal 1 minute of 12,000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.
(4) in adsorption column, add 700 μ L rinsing liquid W2, centrifugal 1 minute of 12,000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.
(5) in adsorption column, add 50 μ L rinsing liquid W2, centrifugal 1 minute of 12,000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.
(6) adsorption column is put into collection tube, centrifugal 2 minutes of 12,000rpm, removes rinsing liquid as far as possible.Adsorption column is placed in room temperature and places several minutes, thoroughly dry.
(7) adsorption column is put into a clean centrifuge tube, to the elution buffer TE of the unsettled dropping 30 in adsorption film mid-way μ L, room temperature places 2 minutes.Centrifugal 2 minutes of 12,000rpm, collects DNA solution.
4) preparation of bacillus coli DH 5 alpha competent cell:
(1) picking single DH5 α bacterium colony, be inoculated in 3mL containing penbritin LB substratum in, 37 DEG C of overnight incubation, get next day above-mentioned bacterium liquid in proportion 1:100 be inoculated in 50mLLB substratum, 37 DEG C vibration 2 hours.When OD600 value reaches 0.35, results bacterial cultures.
(2) bacterium is transferred in the sterile polypropylene tube of a 50mL precooling, place 10 minutes on ice, culture is cooled.
(3) centrifugal 10 minutes of 4100rpm at 4 DEG C, sucking-off nutrient solution, and pipe is inverted within l minute, flows to end to make the substratum remained.
(4) every 50mL initial incubation liquid and the 0.1mol/L CaCl2-MgCl of 30mL precooling 2molten (80mmol/L MgCl 2, 20mmol/L CaCl 2) re-suspended cell precipitation.
(5) centrifugal 10 minutes of 4100rpm at 4 DEG C, sucking-off supernatant liquor, and pipe is inverted within l minute, flows to end to make the liquid remained.
(6) every 50mL initial incubation thing 0.1mol/L CaCl of 2mL precooling 2the resuspended cell precipitation of solution, for subsequent use after packing.
5) conversion of connection and connection product:
(1) in Eppendorf tube, add the ligase enzyme buffer mixture of 1 μ L Takara pMD19-T simple carrier, 4 μ LLafF gene PCR product and 5 μ L.
(2) 16 DEG C are reacted 3 hours.
(3) full dose (10 μ L) is added in 100 μ L DH5 α competent cells, places 30 minutes in ice.
(4) 42 DEG C heating 90 seconds after, then in ice place 1 minute.
(5) 37 DEG C of LB substratum 890 μ L of bathing of temperature are added, 37 DEG C of slow shaking culture 60 minutes.
(6) get on LB substratum that 200 μ L coat containing penbritin, cultivate 16h to form single bacterium colony for 37 DEG C.
6) screening of LafF gene pcr product and qualification
The above-mentioned single bacterium colony of picking is in the LB substratum containing penbritin, and 37 DEG C of slow shaking culture 4h, carry out pcr amplification.Amplimer and amplification condition are with aforementioned optimum reaction condition.After the positive colony of confirming through PCR is carried out plasmid extraction, carry out the mensuration of nucleotide sequence with the full-automatic nucleotide sequencing instrument of U.S. Applied Biosystems3730A.Sequencing primer is BcaBESTTMSequencingPrimer ML3-47 (5 ' CGCCAGGGTTTTCCCAGTCACGAC 3 '), and object sequencing fragment result from 5 ' end to 3 ' terminal sequence is: atgaagaaaatcgtggtggcgggcgtggtcagcgccgttctggcgttggtggtggg ggccggagctggatggggtgtctggcatcattatgccggaaaaggaaaaccg.
7) positive plasmid extracts:
(1) get the bacterium liquid of 5mL incubated overnight, add in centrifuge tube, centrifugal 1 minute of 12,000rpm, absorbs supernatant as far as possible.
(2) in the centrifuge tube leaving bacterial sediment, add 250 μ L solution 1, use pipettor or the thorough suspended bacterial precipitation of turbula shaker.
(3) in centrifuge tube, add 250 μ L solution 2, leniently spin upside down and make the abundant cracking of thalline for 6-8 time.
(4) in centrifuge tube, add 350 μ L solution 3, leniently spin upside down 6 – 8 times immediately, fully mix, namely occur white flock precipitate.Centrifugal 10 minutes of 12,000rpm, now forms precipitation bottom centrifuge tube.Transfer in adsorption column by the supernatant liquor pipettor that previous step is collected, centrifugal 60 seconds of 12,000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.
(5) in adsorption column, add 700 μ L rinsing liquid W2,12,000rpm centrifugal 30-60 second, outwell the waste liquid in collection tube, adsorption column is put into collection tube.
(6) in adsorption column, add 500 μ L rinsing liquid W2, centrifugal 60 seconds of 12,000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.
(7) adsorption column is put into collection tube, centrifugal 2 minutes of 12,000rpm, is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(8) adsorption column is placed in a clean centrifuge tube, the middle part to adsorption film drips 60 μ L elution buffer TE, and room temperature places 2 minutes, and plasmid solution is collected in centrifuge tube by 12,000rpm for centrifugal 1 minute.
Two, intestinal bacteria sensitivity
1) plasmid number converts
Recording plasmid concentration with ultraviolet spectrophotometer is: 158ng/ μ L.
Plasmid number reduction formula: (note: X is plasmid concentration; Series logarithm for the purpose of Y; NA is Avogadro constant; 2692 is carrier base logarithm; 660 for base molecular-weight average) number that obtains plasmid is 5.06 × 10 10individual/μ L.
2) get 1 μ L LafF positive plasmid and be diluted to 1.0 × 10 9individual/μ L, gets the positive plasmid that 2 μ L have diluted and carries out 10 times of gradient dilutions, be diluted to 1.0 × 10 8to 1.0 × 10 09 gradients.Getting each gradient dilution liquid 2 μ L is respectively template.Reaction system is 25 μ L, Taq0.125 μ L, 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 2 μ L, each 1 μ L of upstream and downstream primer, adds ddH 2o supplies 25 μ L.
3. increase by following program in PCR instrument:
(1) denaturation
95 DEG C 5 minutes
(2) 30 circulations:
95 DEG C of 30 second
57 DEG C of 30 second
72 DEG C of 20 second
(3) increase afterwards
72 DEG C 1 minute
At Mg 2+for 4.0mM, annealing temperature is under the condition of 57 DEG C, and this detection method can detect that colibacillary minimum concentration is 1/μ L, as shown in Figure 3.

Claims (4)

1. for detecting a colibacillary primer, comprising upstream primer and downstream primer, it is characterized in that: described upstream primer nucleotide sequence is as shown in SEQ ID NO:1, and described downstream primer nucleotide sequence is as shown in SEQ ID NO:2.
2. detect a colibacillary method, it is characterized in that: comprise the following steps:
1) template DNA of detected sample is extracted;
2) primer according to claim 1 is designed;
3) pcr amplification:
Reaction system: Taq 0.125 μ L, 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 1 μ L, each 1 μ L of upstream and downstream primer, adds ddH 2o supplies 25 μ L;
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 1min;
4) agarose gel electrophoresis method for detecting is utilized to judge detected result.
3. for detecting a colibacillary test kit, it is characterized in that: comprise primer SEQID NO:1 and SEQ ID NO:2.
4. one according to claim 3 is for detecting colibacillary test kit, it is characterized in that: 10 × polymerase buffer 2.5 μ L, dNTP 2 μ L, Mg 2+1.6 μ L, template 1 μ L, upstream and downstream primer each 1 μ L, ddH 2o 25 μ L.
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CN108796099A (en) * 2018-06-13 2018-11-13 华南理工大学 A kind of Escherichia coli O 157:PSR detection primers, kit and its detection method of H7
CN110229916A (en) * 2019-06-06 2019-09-13 昆明理工大学 A kind of primer of Escherichia coli specific gene and the method for detecting Escherichia coli

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