CN101469326B - Nucleotide specific to ITS of Balcillus proteus mirabilis and use thereof - Google Patents
Nucleotide specific to ITS of Balcillus proteus mirabilis and use thereof Download PDFInfo
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Abstract
The invention relates to nucleotide special to an Internal transcribed spacer (ITS for short) of a 16S rRNA-23S rRNA gene in Proteus mirabilis, in particular to oligonucleotide special to the ITS in the Proteus mirabilis and application thereof. The invention also provides a PCR detection reagent taking an oligonucleotide pair as a primer and a detection method thereof. The detection method utilizes the PCR reagent to detect the Proteus mirabilis in human body and the environment, is simple, convenient and quick, has good specificity and high sensitivity, can be used in the fields of supervision and detection of food and clinical samples, detection of pathogen in the food, microbial classification and epidemiological investigation and so on, and has deep social benefit and large economic benefit.
Description
Technical field
The present invention relates to a kind of to Proteus mirabilis (Proteus mirabilis)) in 16S rRNA-23SrRNA gene between the district (Internal transcribed spacer, hereinafter to be referred as ITS) special Nucleotide, particularly relate to oligonucleotide and the application thereof special to the ITS in the Proteus mirabilis.
Background technology
Proteus mirabilis is a kind of conditioned pathogen, be present in human intestinal (carrying rate is up to 25%) and the hospital environment, can cause people's primary and secondary infection, go into septicemia as wound infection, respiratory tract infection, diarrhoea, urinary tract infections, peritonitis, otitis media, mastoiditis, endocarditis, meningitis, also can cause food poisoning.Proteus mirabilis is urinary tract infection (Urinary TractInfection, UTI) main pathogenic bacterium, in complicated urinary tract infection (complicated UTI), be only second to intestinal bacteria and Klebsiella Pneumoniae (causing 12% infection), in long term indwelling catheter patient's bacteruria, be only second to Si Shi Providian bacterium (causing 15% infection) (R ó zalski A, Sidorczyk Z, 1997), also be one of the pathogenic bacteria of baby's enteritis.
At present, it is main that Proteus mirabilis is mainly adopted conventional biological chemistry detection method, too much false positive, false negative phenomenon appear in this detection method easily, usually bacterial strain to be measured need be detected through cultivating one's ability of long period, detecting operation process complexity, sense cycle is long, needs two to three days ability to detect testing sample one time usually, and need higher technology and equipment condition, detect the cost height.Thereby need highly sensitive, quick, the easy detection method of development.
In recent years, increasing molecular engineering is used for evaluation, detection and the disease screening of pathogenic bacteria, comprises transcribed spacer (ITS) sequential analysis, random amplification length polymorphism (RAPD) analysis, rDNA restriction fragment length polymorphism (RFLP) analysis etc.Compare with the traditional detection technology, these are based on the molecular detection technology of polymerase chain reaction (PCR), do not need the processes such as separation, pure culture through pathogenic bacteria, and have fast, advantages such as sensitivity, high specificity.
Rrna internal transcribed spacer district (ITS) is the zone between 16S rRNA and 23S rRNA, and all the form with single copy or multiple copied exists in the genome of nearly all bacterium, relatively lacks (200bp-1000bp).This zone evolutionary rate is very fast, has hv sites, and its variation speed is equivalent to 16S rRNA or 23S rRNA about ten times, high conservative between different strains in planting, and between the kind of bacterium, exist abundant variation.Therefore have very high resolving power, the easier very near bacterial classification of evolutionary relationship that distinguishes has strengthened the specificity that detects greatly.Can provide prolific hereditary information for phylogeny, classification evaluation and the Molecular Detection of research bacterium.
Polymerase chain reaction technology (Polymerase chain reaction, the abbreviation round pcr) technology as microorganism detection is obtaining approval and popularization at present, this technology has advantages such as high-throughput, detection speed are fast, high specificity, sensitivity height with respect to traditional method, only need sample is increased bacterium simply in advance or increases the bacterium process, prepare the DNA of bacteria template by centrifugal and cracking again, the target sequence that just can increase in the PCR process under the high specific primer mediation reaches the purpose that whether contains invasive organism to be measured in the test sample.The amplification procedure of PCR only needs 2 hours.This has greatly improved working speed undoubtedly and has reduced job costs inspection and quarantine department and Clinical Laboratory.
Summary of the invention
An object of the present invention is to provide a kind of Nucleotide special to the ITS of Proteus mirabilis;
Another object of the present invention provides the application of this specific nucleotide, with its design primer, by optimizing and design, provides a kind of PCR test kit that is used to detect Proteus mirabilis, and utilizes this PCR test kit to detect the existence of Proteus mirabilis.
Purpose of the present invention is achieved through the following technical solutions:
A kind of Nucleotide special to the ITS of Proteus mirabilis, described Nucleotide comprises:
A) nucleotide sequence shown in SEQ ID NO:1 or the SEQ ID NO:2 or its complementary DNA or RNA sequence;
B) be different from a) but amino acid sequence coded and a) the coded identical nucleotide sequence of RNA sequence;
C) above-mentioned a) or b) in disappearance, replace or insert one or more bases, still have a nucleotide sequence of described functional nucleotide.
Isolating Nucleotide shown in described SEQ ID NO:1 and the SEQ ID NO:2, total length is respectively 398 and 524 bases;
The Nucleotide of above-mentioned a kind of ITS high special to Proteus mirabilis is the oligonucleotide that comes from ITS, sequence with SEQ ID NO:5 is 186 to 205 the base that is arranged in 186 to 205 the base of SEQ ID NO:1 and is arranged in SEQ ID NO:2.
The separation method of the Nucleotide that the above-mentioned ITS to Proteus mirabilis is special comprises the steps: (1) genomic extraction; (2) by the ITS gene cluster in the pcr amplification Proteus mirabilis; (3) make up the ITS clone; (4) to the ITS cloning and sequencing; (5) splicing of nucleotide sequence and analysis; (6) screening of special primer.
The application of above-mentioned Nucleotide can be used for described Nucleotide the PCR test kit, be used for hybridization and fluoroscopic examination as probe as primer, perhaps is used to make gene chip or microarray with bacterial detection.
Above-mentioned PCR test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase,
Wherein the PCR primer comprises
D) nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4;
E) be different from d) but amino acid sequence coded and d) the coded identical nucleotide sequence of aminoacid sequence;
F) disappearance above-mentioned e) or e), replace or insert one or more bases, still have a nucleotide sequence of described functional nucleotide.
SEQ ID NO:3 wherein (5 '-TGTACACACCGCCCGTC-3 ') be upstream primer, SEQ ID NO:4 (5 '-GACTCTCGTCAGATAAGTGG-3 ') be downstream primer.
Above-mentioned upstream primer SEQ ID NO:3 is the 16S rRNA gene conserved regions design synthetic according to bacterium, downstream primer SEQ ID NO:4 is the ITS type design synthetic Oligonucleolide primers that contains tRAN-glu according to Proteus mirabilis, and it is positioned at the 186-205 base position on the ITS sequence.This primer can be in test tube optionally duplicates synthetic DNA section between two primers by archaeal dna polymerase.
Above-mentioned PCR detection kit comprises following reagent: 25 mM MgCl
250 μ l; 10mMdNTP 30 μ l; 10 * enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 5 μ l; Each 10 μ l of primer; Positive reference substance 10 μ l, negative control product 10 μ l; DdH
2O 1ml.
The invention still further relates to the application of above-mentioned PCR test kit in detecting Proteus mirabilis.
---amplification---electrophoresis detection result that above-mentioned PCR detection kit at Proteus mirabilis, whole detection step comprise sample pretreatment.Primer and the needed reagent of PCR reaction system add in the amplification pipe in advance, and the user only needs that pretreated sample adding amplification pipe is started amplified reaction and gets final product, and finishes testing simply fast.
The PCR detection method of using above-mentioned PCR test kit to detect Proteus mirabilis may further comprise the steps:
(1) extracts clinical sample template to be measured;
(2) in the PCR thin-walled tube, add dNTP, MgCl
2, 10 * enzyme spcificity reaction buffer, Taq polysaccharase, primer, testing sample template and ddH
2O, mixing;
(3) mixture with mixing among the thin-walled PCR increases on the PCR instrument;
(4) electrophoresis amplified production in electrophoresis equipment, the record result;
(5) analyze and carry out the result and judge.
Clinical sample template in the above-mentioned steps (1) is to be the crude extract of hemoculture thing, phlegm culture, urine culture or cerebrospinal fluid culture in the clinical sample, or the coarse body fluid of the pure growth of Proteus mirabilis, or pure dna, or positive reference substance and negative control product.
The extracting method of the clinical sample template in the above-mentioned steps (1) is:
(1) gets culture 1.5ml, under the 8000rpm condition centrifugal 5 minutes, remove supernatant liquor;
(2) get the ddH of 500 μ l
2The resuspended precipitation of O, under the 8000rpm condition centrifugal 5 minutes, remove supernatant liquor, control is done;
(3) get 100 μ l ddH
2The resuspended precipitation of O, water-bath is 10 minutes in 100 ℃ of boiling water;
(4) place on ice after 10 minutes under the 12000rpm condition centrifugal 2 minutes again;
(5) get the template of 3 μ l middle layer supernatant as the PCR reaction.
Reaction cycle parameter on the PCR instrument in the above-mentioned steps (3) comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially:
Be 95 ℃ for a circulation that makes sex change can reach treating processes in temperature required and essential early stage early stage, 5 minutes;
Denaturation temperature and time are 94 ℃, 30 seconds;
The renaturation temperature and time is 50 ℃, 30 seconds;
Elongating temperature and time are 72 ℃, 30 seconds;
The cycle index of sex change, renaturation, extension is 30 circulations;
To carry out a round-robin temperature and time be 72 ℃ in order to stablize amplified production, 5 minutes.
Above-mentioned steps (4) electrophoresis amplified production in electrophoresis equipment, record result's concrete steps are:
(1) getting 5～10 μ l amplified productions mixes with 5: 1 volume ratio with 6 * tetrabromophenol sulfonphthalein sample-loading buffer;
(2) mixed solution is splined on 1% the sepharose;
(3) with about 10 minutes of agarose gel electrophoresis 120v voltage stabilizing electrophoresis, contrast with DL2000 Marker;
(4) observe and write down the result.
But the present invention is by preparing the PCR test kit that a kind of industrialization that detects Proteus mirabilis is produced, the combination of components that the PCR detection method need be used together, during use, extract testing sample, simultaneously through comparatively simple operation program just can carry out fast, the consumption and the concentration of each component is the test gained in sensitive, the easy detection, test kit, it is simple to detect the employed testing installation of Proteus mirabilis with this test kit, and it is low to detect cost.
Using the purpose of positive and negative control product is to be used for Quality Control entire operation process, judges so that draw accurately.If contain Proteus mirabilis, then from electrophoresis result, can observe band with the positive reference substance same position; If do not contain Proteus mirabilis, then the same with the negative control product do not have this band.
The amount of reagent that one-time detection of the present invention is tested in the employed actual box is each 1 μ l of primer, 10mM dNTP 0.25 μ l, 10 * enzyme spcificity reaction buffer, 2.5 μ l, 5U/ μ l hot resistant DNA polymerase 0.2 μ l, 25mM MgCl
22.5 μ l, remaining volume is used ddH after the amount of removing 3 μ l testing samples
2O complements to 25 μ l.
Hot resistant DNA polymerase among the present invention is the Taq polysaccharase.
Above-mentioned positive reference substance is for to determine the being sample of Proteus mirabilis, and the negative control product are not the samples of Proteus mirabilis for determining through the laboratory then, as intestinal bacteria.
This PCR test kit carries out pcr amplification as if the bacteria suspension with Proteus mirabilis, and is consistent as template amplification gained result with the DNA that obtains through extraction.Susceptibility and specificity indifference like this, can be saved the extraction step of template DNA, and working method is simplified.Simultaneously, compare the routine biochemistry detection method, the testing sample that present method adopted can directly be the clinical sample nutrient solution, perhaps test sample is carried out the simple separation cultivation and just can detect, thereby saved manpower and materials.
The Proteus mirabilis of adopting PCR detection method provided by the present invention to increase should have a band at the 357bp place.
Result according to design of primers, the Proteus mirabilis fragment length that amplifies should be 357bp, therefore if amplification has the band with the positive control same position about 357bp, can judge that then this testing sample carries the germs of a disease, see Fig. 1 for details, wherein, A is a testing sample, detects germ purpose band; + positive control;-negative control; M is DNA marker.
Compared with prior art, the present invention has following advantage:
(1) practical
The PCR test kit compound method that the present invention prepared is easy, and sense cycle is short, speed is fast, and is workable, is easy to industrialization production, and it is relatively low to detect cost, and market application foreground is wide.
(2) accuracy height
The present invention compares the ITS that obtains among sequence and the GeneBank by ITS clone, order-checking to other 3 kinds in Proteus mirabilis and the proteus, finds the special district of Proteus mirabilis ITS, designs primer.Then utilize primer sets to synthesize the composite PCR detection architecture, can directly Proteus mirabilis and its nearly source bacterial classification be separated.The present inventor increases to reference culture, 53 other bacterial strains of strain proteus and the nearly edge type strain of 13 strains of 31 strain Proteus mirabilises, found that the accuracy rate of detection method of the present invention is up to 100%.
(3) highly sensitive
Proteus mirabilis detection kit provided by the invention and detection method thereof have quite high susceptibility, and the accuracy of detection height can detect the dna profiling of 1pg/ μ l.
State with other purpose, feature and advantage and can become apparent on the present invention for allowing, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.
Description of drawings
Fig. 1 is test kit detected result figure of the present invention,
A is a testing sample, detects pathogenic bacteria; The positive reference substance of P; The negative reference substance of N; M is DL2000 DNA marker;
Fig. 2 is clinical strains part electrophoresis result figure of the present invention,
1, Proteus mirabilis C1875; 2, Proteus mirabilis C2297; 3, Proteus mirabilis C2299; 4, Proteus mirabilis C2381; 5, Proteus mirabilis C3725; M, DL2000DNA marker;
Fig. 3 is clinical strains part electrophoresis result figure of the present invention,
1, proteus vulgaris C4305; 2, Pan Shi Bacillus proteus C2179; 3, Proteus mirabilis C3725; 4, Proteus mirabilis C3759; 5, Proteus mirabilis C5735; M, DL2000DNA marker;
Fig. 4 is test kit examination criteria bacterial strain part electrophoresis result figure of the present invention,
1, Proteus mirabilis G2292; 2, Proteus mirabilis G2293; 3, Proteus mirabilis G2294; 4, Proteus mirabilis G2295; 5, Proteus mirabilis G2296; 6, Proteus mirabilis G2298; 7, proteus vulgaris G1811; M, DL2000 DNA marker;
Fig. 5 is test kit examination criteria bacterial strain part electrophoresis result figure of the present invention;
1, Proteus mirabilis G2299; 2, Proteus mirabilis G2372; 3, Proteus mirabilis G2609; 4, Proteus mirabilis G2645; M, DL2000 DNA marker;
Fig. 6 is test kit examination criteria bacterial strain part electrophoresis result figure of the present invention,
1, Pan Shi Bacillus proteus G2620; 2, produce sticking Bacillus proteus G2676; 3, Proteus mirabilis G2621; 4, Proteus mirabilis G2627; 5, Proteus mirabilis G2631; M, DL2000DNA marker;
Fig. 7 is test kit examination criteria bacterial strain part electrophoresis result figure of the present invention,
1, Proteus mirabilis; 2, shigella dysenteriae; 3, citrobacter freundii; 4, Salmonellas; 5, intestinal bacteria; 6, streptococcus aureus; 7, vibrio cholerae; M, DL2000 DNAmarker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment only is used to the present invention is described and is not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).
Embodiment 1: genomic extraction
37 ℃ of incubated overnight Proteus mirabilises in the LB of 5mL substratum, centrifugal collecting cell.With 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, 37 ℃ of incubations 20 minutes, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml then continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul 10mg/ml again, 65 ℃ of incubations 30 minutes.Add equal-volume phenol extracting mixture, get supernatant liquor, use isopyknic phenol again: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant liquor, again with isopyknic ether extracting to remove remaining phenol.Supernatant liquor rolls out DNA and washes DNA with 70% ethanol with glass yarn with 2 times of volume ethanol deposit D NA, at last DNA is resuspended among the 30ul TE.Genomic dna detects by 0.4% agarose gel electrophoresis.
Embodiment 2: by the ITS in the pcr amplification Proteus mirabilis
Genome with Proteus mirabilis is that template is passed through its ITS of pcr amplification.At first, design downstream primer (5 '-GGTACT TAG ATG TTT CAG TTC-3 ') according to 23S rRNA gene conservative district again according to the 16S rRNA gene conservative district design upstream primer (5 '-TGTACA CAC CGCCCG TC-3 ') of ITS one end.The PCR response procedures was as follows: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change are 30 seconds then, 50 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute, and carried out 30 circulations like this; At last, continue to extend 5 minutes at 72 ℃, obtain the PCR product, the agarose gel electrophoresis with 0.8% detects the size and the specificity thereof of PCR product.Merge 3 pipe long PCR products, and reclaim the shortest band in the test kit recovery purified pcr product with the UNIQ-10 pillar DNA glue of the living worker's biotechnology in Shanghai company;
Embodiment 3: make up the ITS clone
At first be the acquisition that connects product:
With 3 * 10 of PCR purified product and Promega company
-3The pGEM-T-Easy carrier connect 24 hours in 16 ℃, cumulative volume is 10 μ l, and the 10 * buffer of 1 μ l and the T4DNA ligase enzyme of 0.5 unit are wherein arranged, and obtains connecting product.
Next is the preparation of competent cell:
The method that provides with reference to Bio-Rad company prepares the competent cell bacillus coli DH 5 alpha.Get a ring bacillus coli DH 5 alpha list bacterium colony in the LB of 5ml substratum, 180rpm cultivated after 10 hours, got in the LB substratum that the 2ml culture is transferred to 200ml, and 37 ℃ of 250rpm thermal agitations are cultivated OD600 about 0.5, ice bath cooling was 20 minutes then, in centrifugal 15 minutes of 4 ℃ of 4000rpm.Confide all supernatant liquor, dispel thalline, in centrifugal 15 minutes of 4 ℃ of 4000rpm with the deionization aqua sterilisa 200ml of cold ice precooling.Deionization aqua sterilisa 100ml with cold ice precooling dispelled thalline again, in centrifugal 15 minutes of 4 ℃ of 4000rpm.With 10% glycerine suspension cell of cold ice precooling, centrifugal 10 minutes of 4 ℃ of 6000rpm abandon supernatant liquor, precipitate 10% glycerine suspension cell with the precooling of 1ml ice at last, are competent cell.The competent cell that makes is packed as 60ul one pipe ,-70 ℃ of preservations.
Be electric transformed competence colibacillus cell at last:
Get after 2-3ul connects product and 60ul competence bacillus coli DH 5 alpha mixes, forward in the electric shock cup of 0.2cm of Bio-Rad company and shock by electricity, voltage is 2.5 kilovolts, and the time is 5.0 milliseconds-6.0 milliseconds.The SOC substratum that adds 1ml after the electric shock immediately in cup makes the bacterium recovery.Immediately bacterium is coated in 37 ℃ of inversion incubated overnight on the LB solid medium that contains penbritin, X-Gal and IPTG then, obtains blue white bacterium colony next day.With the white colony that obtains promptly the white clone forward on the LB solid medium that contains penbritin and cultivate, from each clone, extract plasmid and cut the segmental size of evaluation insertion wherein simultaneously, obtain the ITS clone that white clone group is Proteus mirabilis with EcoR I enzyme.
Embodiment 4: to the ITS cloning and sequencing
Select insert each minimum and time little 8 clone of fragment by with ABI377 type automatic dna sequencer to two-way order-checking of insertion fragment among the clone, thereby acquisition contains the sequence of the ITS of tRNA-Glu gene.
Embodiment 5: the screening of special primer
Special district's design primer at the ITS of Proteus mirabilis; Combine with upstream primer in the 16S rRNA gene conserved regions at downstream primer of special district design, (upstream primer is 5 '-TGTACACACCGCCCGTC-3 ', sees sequence SEQ ID NO:3 to primer with this; Downstream primer is 5 '-GACTCTCGTCAGATAAGTGG, see sequence SEQ ID NO:4) genome that produces sticking Bacillus proteus and 40 strain Proteus mirabilises with the proteus vulgaris of 25 strains, 26 strain Pan Shi Bacillus proteuss, 2 strains is that template is carried out PCR, system be the testing sample template of 10uM primer 1ul, 25mM MgCl2 2.5ul, 10 * buffer2.5ul, 10mM dNTP 0.25ul, 5U/ul Taq polysaccharase 0.25ul and 3ul in the thin-walled PCR pipe of 0.2ml, use ddH at last
2O complements to 25ul.All primers all obtain positive findings in Proteus mirabilis, in other groups, all do not amplify the correct band of size, that is to say, in other groups, do not obtain any PCR product band, so this oligonucleotide all is a high special to Proteus mirabilis and ITS thereof.
Embodiment 6: the PCR detection kit
The preparation of test experience material requested and equipment
1. test kit is formed:
1)MgCl
2(25mM) 50ul；
2)dNTP(10mM) 30ul；
3) 10 * buffer (10 * enzyme spcificity reaction buffer) 50ul;
4) Taq polysaccharase (5U/ul) 5ul;
5) primer mixture (10uM) 10ul;
6) positive reference substance (KP) 10ul;
7) negative control product (KN) 10ul;
8)ddH
2O 5ml。
Each test kit can be used for detecting 10 samples.
Wherein MgCl2,10 * buffer, dNTP, Taq polysaccharase are given birth to the worker by Shanghai and are provided; Primer mixture is synthetic for the sequence that designs voluntarily offers Shanghai Ying Jun biotech company; Positive reference substance, negative control product and ddH
2O is prepared voluntarily by us.
The upstream primer that detects employed primer is 5 '-TGTACACACCGCCCGTC-3 ' (SEQ ID NO:3), and downstream primer is 5 '-GACTCTCGTCAGATAAGTGG (SEQID NO:4), and ratio is 1: 1.
2. plant and instrument
PCR instrument (having another name called DNA thermal cycling amplification instrument), electrophoresis equipment (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging instrument ,-20 ℃ of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes.
3. testing sample provides
We have collected the specificity of the nearly edge type strain checking of reference culture, 53 other bacterial strains of strain proteus and the 12 strains primer of 31 strain Proteus mirabilises, and strain number and source see the following form 1.
Table 1: for the reference culture of examination
Simultaneously the applicant has collected the Tianjin 6 tame hospitals of 10 strains since 2005 and has separated the pathogenic bacteria that obtains on one's body from patient, all clinical strains all through hospital the Bacteria Identification system--the VITEK system has carried out biochemical identification, its biochemical identification the results are shown in Table 2:
Table 2: for the biochemical investigation result of examination clinical strains
| Strain name | The biochemical identification type | The laboratory numbering | The biochemical identification type |
| C1875 | ?P.mirabilis | ?C3731 | ?P.mirabilis |
| ?C2297 | ?P.mirabilis | ?C3759 | ?P.mirabilis |
| ?C2299 | ?P.mirabi1is | ?C5735 | ?P.mirabilis |
| ?C2381 | ?P.mirabilis | ?C2179 | ?P.penneri |
| ?C3725 | ?P.mirabilis | ?C4305 | ?P.vulgaris |
Two, detect the concrete operations of implementing: carried out adopting under the similarity condition PCR detection kit provided by the present invention to carry out the detection of PCR method with above-mentioned 94 strain reference cultures with through 10 strain proteus clinical strains of common biochemical identification.
1. the extraction of testing sample template
A. at reference culture
1) 1% connects the aseptic technique of bacterium amount, be inoculated in the 3ml LB substratum 200rpm, 37 ℃ of incubated overnight;
2) collect the 1.0ml overnight culture, centrifugal 5 minutes of 8000rpm removes supernatant.
3) 500ul ddH
2The resuspended precipitation of O, centrifugal 5 minutes of 8000rpm removes supernatant, and control is done as far as possible.
4) 100ul ddH
2The resuspended precipitation of O, mixing, 100 ℃ of boiling water baths 10 minutes,
5) put on ice after 10 minutes centrifugal 2 minutes of 12000rpm.
6) get the template of 3ul middle layer supernatant as the PCR reaction.
B. at clinical culture (hemoculture thing, phlegm culture, urine culture etc.)
1) collect positive culture 1.0ml, centrifugal 5 minutes of 8000rpm removes supernatant.
2) 500ul ddH
2The resuspended precipitation of O, centrifugal 5 minutes of 8000rpm removes supernatant, and control is done as far as possible.
3) 100ul ddH
2The resuspended precipitation of O, mixing, 100 ℃ of boiling water baths 10 minutes,
4) put on ice after 10 minutes centrifugal 2 minutes of 12000rpm.
5) get the template of 3ul middle layer supernatant as the PCR reaction.
2. draw 10uM primer 1ul in the PCR test kit, the MgCl of 25mM respectively with micropipet
22.5ul, the dNTP0.25ul of 10 * buffer 2.5ul, 10mM, the testing sample template of 5U/ul Taq polysaccharase 0.25ul and 3ul is used ddH at last in the thin-walled PCR pipe of 0.2ml
2O complements to 25ul, fully mixing;
3. will on the PCR instrument, increase after the mixture high speed centrifugation several seconds: 95 ℃ of 1 circulations in 5 minutes according to following temperature and time; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ of 1 circulations in 5 minutes.
4. electrophoresis pcr amplification product in electrophoresis equipment writes down the result.
1) getting the 3ul amplified production mixes with 6 * tetrabromophenol sulfonphthalein sample-loading buffer;
2) mixed solution is splined on 1.5% the sepharose;
3) with sepharose through about 10 minutes of 120v voltage voltage stabilizing electrophoresis, contrast with DL2000 Marker;
4) observe forward position tetrabromophenol sulfonphthalein indicator migrate to apart from well at least 3cm stop electrophoresis, on the gel imaging instrument, observe and log.
5. carry out the result according to following condition and judge that Proteus mirabilis should have a band at the 357bp place.
Positive control and negative control test are as long as change the testing sample template into Proteus mirabilis positive template and the negative template (containing intestinal bacteria) of Proteus mirabilis, the sample template that contains Proteus mirabilis has the 357bp fragment, and the sample template that does not contain Proteus mirabilis does not have this fragment.Reference culture part electrophoresis result record is seen shown in Figure 2: all reference cultures are except that Proteus mirabilis has the 357bp fragment, and other bacterial strains all do not have amplified production.Clinical strains electrophoresis result record is seen shown in Figure 3: add up to 10 strains, except that Proteus mirabilis had the 357bp fragment, other bacterial strains did not all have amplified production.This explanation adopts the PCR detection method can get rid of false positive reaction, identifies that according to the detection box that has or not above fragment to carry out pathogenic bacteria accuracy in detection has improved, and has avoided because of detecting the unnecessary loss that error causes.
The hybridization kit that utilizes above-mentioned experimental procedure to obtain can be used for detecting Proteus mirabilis.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Sequence table
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉a kind of Nucleotide and the application thereof special to the ITS of Proteus mirabilis
<130>7P13015-CN
<160>5
<170>PatentIn?version?3.2
<210>1
<211>392
<212>DNA
＜213〉nucleotide sequence special to the ITS of Proteus mirabilis
<400>1
cctaagagat?acgtgttatg?tgcagtgctc?acacagattg?tctgatgaag?aacgagcaaa 60
agcgcgtctg?cgaagctgac?aaaagtcccc?ttcgtctaga?ggcctaggac?accgcccttt 120
cacggcggta?acaggggttc?gaatccccta?ggggacgcca?atgcgcggta?tgagtgaaag 180
gcgtaccact?tatctgacga?gagtcagaga?ataactaagc?taattcaaat?gagttatctt 240
acttattatg?ctctttaaca?atctggaaca?agctgaaaaa?ttgaaaacaa?atcaatatat 300
caccgaggta?tattggtgag?tctctcaaaa?tctcaaacct?taaagtttgt?cacgcaaagt 360
ttatctttga?aaaagacact?ttcgggttgt?ga 392
<210>2
<211>524
<212>DNA
＜213〉nucleotide sequence special to the ITS of Proteus mirabilis
<400>2
cctaagagat?acgtgttatg?tgcagtgctc?acacagattg?tctgatgaag?aacgagcaaa 60
agcgcgtctg?cgaagctgac?agaagtcccc?ttcgtctaga?ggcctaggac?accgcccttt 120
cacggcggta?acaggggttc?gaatccccta?ggggacgcca?atgcgcggta?tgagtgaaag 180
gcgtaccact?tatctgacga?gagtcagaga?ataactaagc?taattcaaac?gagttatctt 240
atttattatg?ctctttaaca?atctggaaca?agctgaaaaa?ttgaaaacaa?atcaatatat 300
caacaacgag?gtatattgat?gagtctctca?aaatctcaaa?ctttgaatgt?gttttgacat 360
caaagtggga?tgagcgagca?atttacagtt?cgaggcggac?agcgcacagc?aagcgcagca 420
tacttaagta?tgtgagcatt?gcgagcactg?cccaacgaag?aaatgtaatc?tgcgcagcca 480
tcaccaccca?gatagtcttt?gaaagagaca?ctttcgggtt?gtga 524
<210>3
<211>17
<212>DNA
＜213〉the upstream primer sequence of the ITS of detection Proteus mirabilis
<400>3
tgtacacacc?gcccgtc 17
<210>4
<211>20
<212>DNA
＜213〉the downstream primer sequence of the ITS of detection Proteus mirabilis
<400>4
gactctcgtc?agataagtgg 20
<210>5
<211>20
<212>DNA
＜213〉to the nucleotide sequence of the ITS high special of Proteus mirabilis
<400>5
ccacttatct?gacgagagtc 20
Claims (2)
1. a PCR test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and the nucleotide sequence that it is characterized in that described PCR primer is shown in SEQ ID NO:3-4.
2. test kit according to claim 1, it is characterized in that, described PCR primer SEQID NO:3 is the 16S rRNA gene conserved regions design synthetic according to bacterium, and primer SEQ IDNO:4 is the ITS design synthetic that contains tRNA-glu according to Proteus mirabilis.
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| CN102559863A (en) * | 2011-09-23 | 2012-07-11 | 中国科学院烟台海岸带研究所 | Specific nucleic acid identification sequence used for detecting proteus mirabilis, and application thereof |
| CN103045514B (en) * | 2012-12-27 | 2014-07-09 | 江南大学 | Proteus mirabilis for synthetizing acidic ethyl urethane hydrolytic enzyme and application thereof |
| CN106566889B (en) * | 2016-11-07 | 2020-02-04 | 深圳市疾病预防控制中心 | Primer, probe, kit and method for detecting proteus mirabilis |
| CN110951895B (en) * | 2019-12-24 | 2021-03-23 | 重庆市畜牧科学院 | System and method for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pani |
| CN114317788A (en) * | 2021-12-30 | 2022-04-12 | 漳州傲农现代农业开发有限公司 | Probe primer combination for detecting swine proteus mirabilis, kit and method thereof |
| CN114752694A (en) * | 2022-05-31 | 2022-07-15 | 湖南大学 | 16SrRNA gene specific sequence fragment for identifying proteus and screening method thereof |
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| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probes and amplification primers for rapid detection and identification of common bacterial pathogens and antibiotic resistance genes in clinical samples in daily diagnosis in microbiology laboratories |
| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probes and amplification primers for rapid detection and identification of common bacterial pathogens and antibiotic resistance genes in clinical samples in daily diagnosis in microbiology laboratories |
| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
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