CN102311950A - Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof - Google Patents
Nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof Download PDFInfo
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Abstract
The invention relates to a nucleotide specific to wzt of Legionella pneumophila serogroup 1 and application thereof. The nucleotide is a nucleotide shown as SEQ ID NO: 1 and/or a nucleotide shown as SEQ ID NO: 2 and is complementary with the nucleotides. The nucleotides can be used for preparing a PCR (Polymerase Chain Reaction) kit for detecting Legionella pneumophila serogroup 1, a gene chip or a micro-array. The nucleotide specific to the wzt of the Legionella pneumophila serogroup 1 provided by the invention as well as the PCR kit, the gene chip or the micro-array including the nucleotide have the advantages of strong practical applicability, simple preparation method of the PCR kit, short detection period, high speed, strong maneuverability, easiness for industrial production, low detection cost, high accuracy and high sensitivity.
Description
Technical field
The present invention relates to a kind of Nucleotide and application thereof; Relate in particular to a kind of wzt (antigenic abc transporter of LPS O-to legionella pneumophilia 1 type (Legionella pneumophila serogroup 1); ABC transporter of LPS O-antigen is hereinafter to be referred as wzt) special Nucleotide and application thereof.
Background technology
Legionella (legionella) is a kind of gram negative bacillus; Extensively be present in the water body, soil of nature and artificial environment; Can suck through contain that the aerocolloidal mode of bacterium is sent out in air and by human body and cause the human infection, cause febris acuta property pulmonary disorder---legionnaires disease.First since the U.S. finds legionnaires disease, this disease demonstrated worldwide distribution and two high big characteristics of case fatality rate from 1976.The growth and breeding of legionella and environment are because of closely related, and cooling tower water coolant, water of condensation and the thermal water of central air conditioning is the legionella living environment suitable in the external world.Legionella in Environmental Water is bred finite concentration, then can form aerosol and then infected person.Host infection mainly receives the influence of its susceptibility and bacterial virulence; Any age all can take place; But person in middle and old age, fundamental immunity chump, long distance travelers, smoker are the high risk population, and are prone to dye legionella with public places such as hospital, hotel, heavy construction building sites.
At present, legionella (Legionellace) has found that more than 50 is planted, more than 70 serotype, and constantly have new bacterial classification to come to light.Wherein with mankind's relation the closest legionella pneumophilia, from environment, separate the earliest, be the representative species of legionella.According to the antigenic difference of O-, legionella pneumophilia can be divided into 15 serotypes.Wherein nearly 70% infection of legionella is caused by legionella pneumophilia 1 type; 20-30% is caused by other serotype; Non-legionella pneumophilia causes the infection of legionella of about 5-10%, from then on can learn, legionella pneumophilia 1 type occupies sizable ratio in the microbial case of legion; Be the main monoid that causes a disease of legionella, in the research of legionella, occupy an important position.
In addition, the legionella kind is various, and conventional microbial culture and serum are identified somatotype, length consuming time, and workload is big, and flux is low, can not satisfy epidemiology survey and reach the incident real-time analysis needs that cause a disease that happen suddenly; The monoclonal antibody classifying method is because the MAbs somatotype needs special technique, and expensive plant and instrument also can only use in specific laboratory.Therefore; In recent years; Increasing molecular engineering is used for evaluation, detection and the disease screening of pathogenic bacteria, comprises specific gene sequence analysis, random amplification length polymorphum (RAPD) analysis, rDNA restriction fragment length polymorphism (RFLP) analysis etc.With the traditional detection compared with techniques, these are based on the molecular detection technology of polymerase chain reaction (PCR), need not pass through the processes such as separation, pure culture of pathogenic bacteria, and have fast, advantages such as sensitivity, high specificity.
1993; Luk; J.M.C et.al chooses the specific nucleotide sequence of Salmonellas (S.enterica) O-antigen gene bunch, has identified the O-antigen [Luk, J.M.C.et.al. (1993) " Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of S.enterica major serogroups (A; B; C2, andD) ", J.Clin.Microbiol.31:2118-2123] of Salmonellas through PCR method; Opened the beginning of screening special molecular sign from O antigen gene bunch, for the various O-Detection of antigen of bacterium provides that high-throughput, detection speed are fast, high specificity, highly sensitive molecular detecting method.Bacterium O antigen type is various, and general different bacterium has different O-antigen, and the bacterium in the same kind also has different O-antigen mostly.Research shows; The antigenic variety of bacterium O-is to be caused by the genetics variety of being responsible for its synthetic gene cluster; Some specific genes in the O-antigen gene bunch have high specificity for the surface antigen of its coding; Thus for inspiring, can be used for the legionella molecule parting from screening special molecular sign the O antigen gene bunch.
Summary of the invention
The object of the present invention is to provide a kind of special Nucleotide of wzt to legionella pneumophilia 1 type, said Nucleotide is:
1) Nucleotide shown in Nucleotide shown in the SEQ ID NO:1 and/or the SEQ ID NO:2;
2) with 1) shown in Nucleotide complementary Nucleotide.
Above-mentioned Nucleotide can be used for preparation and detect legionella pneumophilia 1 type with PCR test kit, gene chip or microarray; Said legionella pneumophilia 1 type can be taken a sample in the crude extract of the culture of tap water, mineral water, air conditioning cooling water, or the crude extract of the pure growth of legionella pneumophilia 1 type etc.
The present invention also provides a kind of PCR test kit, comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, and said PCR primer comprises above-mentioned Nucleotide; Wherein said PCR primer is preferably the Nucleotide shown in SEQ ID NO:1 and/or SEQ ID NO:2.
Above-mentioned PCR detection kit can comprise following reagent: 10mM dNTP 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 article 10 μ l; DdH
2O 1ml.
Above-mentioned PCR detection kit to legionella pneumophilia 1 type, electrophoresis detection result that whole detection step comprises sample pretreatment---amplification---.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 accomplishes testing simply fast.
The present invention also provides a kind of gene chip, comprises solid phase carrier and is fixed on the oligonucleotide probe on the solid phase carrier, and wherein said oligonucleotide probe comprises above-mentioned Nucleotide; Wherein said oligonucleotide probe is preferably the Nucleotide shown in SEQ ID NO:1 and/or SEQ ID NO:2.
The present invention also provides a kind of little array, and it comprises above-mentioned Nucleotide; Wherein said Nucleotide is preferably the Nucleotide shown in SEQ ID NO:1 and/or SEQ ID NO:2.
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.This PCR test kit carries out pcr amplification as if the bacteria suspension with legionella pneumophilia 1 type, with consistent as template amplification gained result through extracting the DNA that obtains.Susceptibility and specificity indifference like this, can be saved the extraction step of template DNA, make working method be able to simplify.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.
(2) accuracy is high
The present invention compares through the wzt gene to the legionella pneumophilia among all GeneBank, finds the special district of the wzt gene of legionella pneumophilia 1 type, designs primer.Then utilize primer sets to synthesize the composite PCR detection architecture, can directly legionella pneumophilia 1 type and its nearly source bacterial classification be separated.
(3) highly sensitive
For let above and other objects of the present invention, feature and advantage can be more obviously understandable, hereinafter is special lifts preferred embodiment, and conjunction with figs., elaborates as follows.
Description of drawings
Fig. 1 is test kit detected result figure of the present invention, wherein:
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 detects legionella pneumophilia reference culture electrophoresis result figure for test kit of the present invention, wherein:
1, legionella pneumophilia 1 type G2756; 2, legionella pneumophilia 2 type G2773; 3, legionella pneumophilia 3 type G2772; 4, legionella pneumophilia 4 type G2781; 5, legionella pneumophilia 6 type G2771; 6, legionella pneumophilia 8 type G2820; 7, legionella pneumophilia 9 type G2774; 8, legionella pneumophilia 10 type G2818; 9, legionella pneumophilia 11 type G2824; 10, legionella pneumophilia 12 type G2822; 11, legionella pneumophilia 13 type G2819; 12, legionella pneumophilia 14 type G2817; 13, legionella pneumophilia 15 type G2829; M, DL2000 DNA marker;
Fig. 3 detects the non-legionella pneumophilia electrophoresis result of legionella figure for test kit of the present invention, wherein:
1, legionella pneumophilia 1 type G2756; 2, Legionella waltersii; 3, Legionella micdadei; 4, Legionella longbeachae; 5, Legionella anisa; 6, Legionella gormanii; 7, Legionella bozemanii; 8, Legionella steigerwaltii; M, DL2000DNA marker;
Fig. 4 detects legionella clinical strains electrophoresis result figure for the present invention, wherein:
1, legionella pneumophilia 1 type G2756; 2, legionella pneumophilia 1 type G2759; 3, legionella pneumophilia 2 type G2761; 4, legionella pneumophilia 3 type G2762; 5, legionella pneumophilia 7 type G2763; 6, legionella pneumophilia 7 type G2764; 7, legionella pneumophilia 5 type G2765; 8, non-legionella pneumophilia 19 type G2766; 9, non-legionella pneumophilia 352 type G2767; M, DL2000 DNA marker;
Fig. 5 detects other kind nearly source bacterial standard bacterial strain electrophoresis result figure for test kit of the present invention, wherein:
1, legionella pneumophilia 1 type G2756; 2, Salmonellas; 3, shigella; 4, streptococcus aureus; 5, YE; 6, Pseudomonas aeruginosa; 7, Aeromonas hydrophila; M, DL2000 DNA marker.
Embodiment
Below in conjunction with specific examples, further set forth the present invention.Should understand these embodiment only be used to the present invention is described and be not used in the restriction scope of the present 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 ℃, 5%CO in the BCYE plate culture medium
2Incubated overnight legionella pneumophilia 1 type bacterial strain, 50mM Tris-HCl (pH8.0) scrapes and gets bacterium colony, centrifugal collection thalline.With 250ul 50mMTris-HCl (pH8.0) and 10ul 0.4M EDTA re-suspended cell, the N,O-Diacetylmuramidase that adds 10ul 10mg/ml continues insulation 20 minutes.The Proteinase K, the 15ul 10%SDS that add 3ul 20mg/ml afterwards, 50 ℃ of incubations 2 hours add the RNase of 3ul10mg/ml again, 65 ℃ of incubations 20 minutes.Use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) solution extracting twice, get supernatant, use isopyknic chloroform again: primary isoamyl alcohol (49: 1) extracting.Supernatant 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 through 0.4% agarose gel electrophoresis.
Embodiment 2: the screening of special primer
Special district's design primer to the wzt gene of legionella pneumophilia 1 type; (upstream primer is 5 '-GTACTACTACAACCGCAGCAGA-3 ', sees sequence SEQ IDNO:1 to primer with this; Downstream primer is 5 '-CGAGTGATATCCATGACTGAAC-3 ', sees sequence SEQ ID NO:2).Reference culture with 1 strain legionella pneumophilia, 1 type; Other serotype reference culture of 12 strain legionella pneumophilias, 7 other bacterial strains of strain legionella, 1 strain legionella pneumophilia, 1 type clinical separation strain, other serotype clinical separation strain of 7 strain legionellas and the nearly edge bacterium of 6 strains are that template is carried out PCR; System be the testing sample template of 5uM primer 0.4ul, 10 * buffer2.5ul, 10mM dNTP 0.25ul, 5U/ulTaq polysaccharase 0.2ul and 3ul in the thin-walled PCR of 0.2ml pipe, use ddH at last
2O complements to 25ul.All primers all obtain positive findings in legionella pneumophilia 1 type, in other groups, do not obtain any PCR product band, so this oligonucleotide all is a high special to legionella pneumophilia 1 type and wzt thereof.
Embodiment 3: the PCR detection kit
One, the preparation of test experience material requested and equipment
1. test kit is formed:
1)dNTP(10mM) 30ul；
2) 10 * buffer (10 * enzyme spcificity reaction buffer) 50ul;
3) Taq polysaccharase (5U/ul) 5ul;
4) primer mixture (5uM) 10ul;
5) positive reference substance (KP) 10ul;
6) negative control article (KN) 10ul;
7)ddH
2O 5ml。
Each test kit can be used for detecting 10 samples.
Wherein 10 * buffer, dNTP, Taq polysaccharase are provided by precious biotechnology (Dalian) ltd; Primer mixture is synthetic for the sequence that designs voluntarily offers Shanghai Ying Jun biotech company; Positive reference substance, negative control article and ddH
2O is prepared by us voluntarily.
The upstream primer that detects employed primer is 5 '-GTACTACTACAACCGCAGCAGA-3 ' (SEQ ID NO:1), and downstream primer is 5 '-CGAGTGATATCCATGACTGAAC-3 ' (SEQ ID NO:2), and ratio is 1: 1.
The PCR detection method of using above-mentioned PCR test kit to detect legionella pneumophilia 1 type may further comprise the steps:
(1) extracts environmental sample template to be measured;
(2) in the PCR thin-walled tube, add dNTP, 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 appearance;
(4) electrophoresis amplified production in electrophoresis equipment, the record result;
(5) analyze and carry out the result and judge.
Environmental sample template in the above-mentioned steps (1) is the crude extract of the culture of blood, phlegm, segmental bronchus extract, hydrothorax lung tissue, tap water sampling, mineral water sampling, air conditioning cooling water sampling etc.; Or the coarse body fluid of the pure growth of legionella pneumophilia 1 type; Or pure dna, or positive reference substance and negative control article.
The process for extracting of the environmental sample template in the above-mentioned steps (1) is:
(1) 1.5ml 50mM Tris-HCl (pH8.0) scrapes and gets culture, under the 12000rpm condition centrifugal 1 minute, removes supernatant;
(2) get the ddH of 500 μ l
2The resuspended deposition of O, under the 8000rpm condition centrifugal 5 minutes, remove supernatant, control is done;
(3) get 100 μ l ddH
2The resuspended deposition 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 appearance 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 95 ℃, 40 seconds;
The renaturation temperature and time is 56 ℃, 40 seconds;
Elongating temperature and time are 72 ℃, 40 seconds;
The cycle index of sex change, renaturation, extension is 33 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 2～5 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 DL2000Marker;
(4) observe and write down the result.
But the present invention is through preparing the PCR test kit that a kind of industrialization that detects legionella pneumophilia 1 type 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 the sensitivity, easy detection, test kit; It is simple to detect the employed testing installation of legionella pneumophilia 1 type with this test kit, and it is low to detect cost.
Using the purpose of positive and negative control article is to be used for Quality Control entire operation process, judges so that draw accurately.If contain legionella pneumophilia 1 type, then from electrophoresis result, can observe band with the positive reference substance same position; If do not contain legionella pneumophilia 1 type, then the same with the negative control article do not have this band.
The amount of reagent that one-time detection of the present invention is tested in the employed test kit sees the following form shown in 1, and the dna profiling amount is 3 μ l.
Table 1 one-time detection is tested the amount of reagent in the employed test kit
Hot resistant DNA polymerase among the present invention is the Taq polysaccharase.
Above-mentioned positive reference substance is for to confirm the being sample of legionella pneumophilia 1 type, and the negative control article are not the samples of legionella pneumophilia 1 type for confirming through the laboratory then, like intestinal bacteria.
This PCR test kit carries out pcr amplification as if the bacteria suspension with legionella pneumophilia 1 type, with consistent as template amplification gained result through extracting the DNA that obtains.Susceptibility and specificity indifference like this, can be saved the extraction step of template DNA, make working method be able to simplify.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.
2. plant and instrument
PCR appearance (having another name called DNA thermal cycling amplification appearance), electrophoresis equipment (comprising electrophoresis apparatus and electrophoresis chamber), gel imaging appearance ,-20 ℃ of refrigerators, supercentrifuge, micropipet and 0.2ml PCR thin-walled tubes.
3. testing sample provides
Collected 1 strain legionella pneumophilia, 1 type reference culture; The specificity of the nearly edge bacterium checking of the reference culture of 12 other serotypes of strain legionella pneumophilia, 7 other bacterial strains of strain legionella, 1 strain legionella pneumophilia, 1 type clinical separation strain, other serotype clinical separation strain of 7 strain legionellas and 6 strains primer, strain number and source see the following form 2.
Table 2 supplies the reference culture of examination
The applicant has collected the pathogenic bacteria that the separation of 8 strain Center of Diseases Prevention & Control, Shenzhen City obtains simultaneously, and the result sees table 3:
Table 3 supplies the biochemical investigation result of examination clinical strains
| Strain name | The biochemical identification type | The laboratory numbering | The biochemical identification type |
| G2759 | L.pneumophila(01) | G2764 | L.pneumophilas (07) |
| G2761 | L.pneumophila(02) | G2765 | L.pneumophila(05) |
| G2762 | L.pneumophila(03) | G2766 | L.spp(019) |
| G2763 | L.pneumophila(07) | G2767 | L.spp(0352) |
The present invention compares through the wzt gene to the legionella pneumophilia among all GeneBank, finds the special district of the wzt gene of legionella pneumophilia 1 type, designs primer.Then utilize primer sets to synthesize the composite PCR detection architecture, can directly legionella pneumophilia 1 type and its nearly source bacterial classification be separated.1 strain legionella pneumophilia, 1 type reference culture, other serotype reference culture of 12 strain legionella pneumophilias, 7 other bacterial strains of strain legionella, 1 strain legionella pneumophilia, 1 type clinical separation strain, other serotype clinical separation strain of 7 strain legionellas and the nearly edge type strain of 6 strains are increased; The result finds that the accuracy rate of detection method of the present invention is up to 100%.
Two, detect the concrete operations of implementing: under similarity condition, adopt PCR detection kit provided by the present invention to carry out the detection of PCR method above-mentioned 26 strain reference cultures and 8 strain legionnella clinical bacterial strains.
1. the extraction of testing sample template
A. to reference culture
1) 3uL connects bacterium amount, aseptic technique, streak inoculation in the BCYE substratum, 37 ℃, 5%CO
2Cultivate 24-48h;
2) 50mM Tris-HCl (pH8.0) scrapes and gets bacterium colony, and centrifugal 5 minutes of 8000rpm removes supernatant.
3) 500ul ddH
2The resuspended deposition of O, centrifugal 5 minutes of 8000rpm removes supernatant, and control is done as far as possible.
4) 100ul ddH
2The resuspended deposition 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. to environment culture (blood, phlegm, segmental bronchus extract, hydrothorax lung tissue, tap water, mineral water, air conditioner condensate water etc.)
1) 50mM Tris-HCl (pH8.0) scrapes and gets bacterium colony, and centrifugal 5 minutes of 8000rpm removes supernatant.
2) 500ul ddH
2The resuspended deposition of O, centrifugal 5 minutes of 8000rpm removes supernatant, and control is done as far as possible.
3) 100ul ddH
2The resuspended deposition 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 5uM primer 0.4ul in the PCR test kit respectively with micropipet, 10 * buffer 2.5ul of 25mM, the dNTP 0.25ul of 10mM, the testing sample template of 5U/ul Taq polysaccharase 0.2ul and 3ul is used ddH at last in the thin-walled PCR pipe of 0.2ml
2O complements to 25ul, fully mixing;
3. with on the PCR appearance, increasing after the mixture high speed centrifugation several seconds: 95 ℃ of 1 circulations in 5 minutes according to following temperature and time; 95 ℃ 40 seconds, 56 ℃ 40 seconds, 72 ℃ 40 seconds, 33 circulations; 72 ℃ of 1 circulations in 5 minutes.
4. electrophoresis pcr amplification product in electrophoresis equipment writes down the result.
1) getting the 2ul 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 DL2000Marker;
4) observe forward position tetrabromophenol sulfonphthalein indicator migrate to apart from well at least 3cm stop electrophoresis, on the gel imaging appearance, observe and log.
5. carry out the result according to following condition and judge that legionella pneumophilia 1 type should have a band at the 796bp place.
Positive control and negative control test are as long as change the testing sample template into legionella pneumophilia 1 type positive template and the negative template (containing intestinal bacteria) of legionella pneumophilia 1 type; The sample template that contains legionella pneumophilia 1 type has the 796bp fragment, and the sample template that does not contain legionella pneumophilia 1 type does not have this fragment.Reference culture electrophoresis result record is seen shown in Figure 2: all reference cultures have the 796bp fragment except that legionella pneumophilia 1 type, and other bacterial strains all do not have amplified production.Clinical strains electrophoresis result record is seen shown in Figure 3: add up to 8 strains, except that legionella pneumophilia 1 type has the 796bp fragment, other bacterial strains all do not 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 legionella pneumophilia 1 type.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 scheme of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Claims (9)
1. special Nucleotide of wzt to legionella pneumophilia 1 type, said Nucleotide is:
1) Nucleotide shown in Nucleotide shown in the SEQ ID NO:1 and/or the SEQ ID NO:2;
2) with 1) shown in Nucleotide complementary Nucleotide.
2. the described Nucleotide of claim 1 detects legionella pneumophilia 1 type with the application in PCR test kit, gene chip or the microarray in preparation.
3. application according to claim 2 is characterized in that, said legionella pneumophilia 1 type is taken a sample in the crude extract of the culture of tap water, mineral water, air conditioning cooling water, or the crude extract of the pure growth of legionella pneumophilia 1 type.
4. a PCR test kit comprises PCR primer, dNTP, damping fluid and archaeal dna polymerase, it is characterized in that, said PCR primer comprises the described Nucleotide of claim 1.
5. PCR test kit according to claim 4 is characterized in that, described PCR primer is shown in SEQ ID NO:1 and/or SEQ ID NO:2.
6. a gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe on the solid phase carrier, it is characterized in that said oligonucleotide probe comprises the described Nucleotide of claim 1.
7. gene chip according to claim 6 is characterized in that, said oligonucleotide probe is shown in SEQ ID NO:1 and/or SEQ ID NO:2.
8. a little array is characterized in that, comprises the described Nucleotide of claim 1.
9. little array according to claim 8 is characterized in that, said Nucleotide is shown in SEQ IDNO:1 and/or SEQ ID NO:2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604945A (en) * | 2012-04-12 | 2012-07-25 | 天津生物芯片技术有限责任公司 | Nucleotides specific for legionella pneumophila O5 type wzt gene, and application thereof |
| CN102628042A (en) * | 2012-04-12 | 2012-08-08 | 天津生物芯片技术有限责任公司 | Specific ribonucleotide of legionella pneumophilia O9 wzm gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597647A (en) * | 2009-07-09 | 2009-12-09 | 广州金域医学检验中心有限公司 | Species of legionella quick detection kit and detection method thereof |
| CN101748192A (en) * | 2008-12-05 | 2010-06-23 | 南开大学 | Gene chip of main pathogenic microorganism in drinking water and testing kit |
-
2010
- 2010-07-08 CN CN2010102266251A patent/CN102311950A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748192A (en) * | 2008-12-05 | 2010-06-23 | 南开大学 | Gene chip of main pathogenic microorganism in drinking water and testing kit |
| CN101597647A (en) * | 2009-07-09 | 2009-12-09 | 广州金域医学检验中心有限公司 | Species of legionella quick detection kit and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 《GenBank ACCESSION: AJ007311》 20050415 Lueneberg,E. Legionella pneumophila serogroup 1 lipopolysaccharide biosynthesis gene cluster 全部 1-9 , * |
| 《Genome Res.》 20081231 Christel Cazalet et al. Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species 431-441 1-9 第18卷, * |
| 《食品与发酵工业》 20071231 陈广全等 用寡核普酸微阵列芯片方法检测常见的食源性致病微生物 122 8-9 , 第12期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604945A (en) * | 2012-04-12 | 2012-07-25 | 天津生物芯片技术有限责任公司 | Nucleotides specific for legionella pneumophila O5 type wzt gene, and application thereof |
| CN102628042A (en) * | 2012-04-12 | 2012-08-08 | 天津生物芯片技术有限责任公司 | Specific ribonucleotide of legionella pneumophilia O9 wzm gene and application thereof |
| CN102604945B (en) * | 2012-04-12 | 2013-06-26 | 天津生物芯片技术有限责任公司 | Nucleotides specific for legionella pneumophila O5 type wzt gene, and application thereof |
| CN102628042B (en) * | 2012-04-12 | 2013-06-26 | 天津生物芯片技术有限责任公司 | Specific ribonucleotide of legionella pneumophilia O9 wzm gene and application thereof |
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