CN102140514B - Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia - Google Patents
Specific primers and kit for detecting various serotype pathogenic bacteria of legionella pneumophilia Download PDFInfo
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Abstract
The invention relates to specific polymerase chain reaction (PCR) primers for detecting various serotype pathogenic bacteria of legionella pneumophilia, a method for quickly detecting by adopting a multi-PCR kit, and application. The kit comprises 10*PCR reaction liquid, MgCl2, dNTP, primers, and DNA polymerase; wherein the sequences of the primers is one or both of (1) and (2), wherein (1) wzt or wzm specific nucleotide sequences of legionella pneumophila serogroups 1, 4, 6, 10 and 13; and (2) complementary DNA sequences of DNA sequences selected from (1). The primers have high practicability for wzt specific nucleotide of common legionella pneumophila serogroups, and a PCR kit, a gene chip or a microarray comprising the nucleotide; and the PCR kit is easy and convenient to prepare, short in detection period, high in speed and accuracy, and strong in operability, detection cost is reduced, and the PCR kit is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method and application thereof of multiplex PCR rapid detection, relate in particular to a kind of can be simultaneously for detection of legionella pneumophilia serotype 1 type (Legionella pneumophila serogroup 1), 4 types (Legionellapneumophila serogroup 4), 6 types (Legionella pneumophila serogroup 6), 10 types (Legionella pneumophila serogroup 10) and the round pcr of five kinds of pathogenic bacterium of 13 types (Legionella pneumophila serogroup13) and the sequence of used PCR primer thereof.
Background technology
Legionella (Legionella) was found in " Philadelphia legionnaires disease event " first in 1976, and therefore gained the name.Legionella is a kind of facultative intracellular parasitic bacteria, extensively be present in nature and artificial water, the edatope, suck pollution has the aerosol of legionella, drink pollution or surface of a wound contact stain water source etc., all might cause the infection of legionella, by legionella cause the febris acuta pulmonary disorder---legionnaires disease is zoonosis, generation that this disease can be in the animals such as horse, ox, sheep, pig and dog and popular is reported in existing a plurality of countries and regions in succession.Legionella contaminated environment, soil and water source that infected humans and animals is discharged become this sick source of infection.The U.S., Britain, Canada, Holland, Sweden and Spain etc. have reported more than 30 countries and regions the generation of legionnaires disease in the animals such as horse, ox, sheep, pig and dog and popular in succession.It is reported, the serosurvey of China Shenyang, In Chengdu part livestock and poultry, ox, sheep, pig, chicken, duck, goose and dog all detect in various degree legionnaires disease positive antibody, and infection rate is 10.3%-55.5%.
Confirmed that at present legionella has 50 kinds, more than 70 serotype, wherein legionella pneumophilia and human diseases relation are the closest.According to the difference of O-antigen, legionella pneumophilia can be divided into 15 serotypes.Wherein nearly 70% infection of legionella is caused by legionella pneumophilia O1, and 20-30% causes by other serotype, and non-legionella pneumophilia causes the approximately infection of legionella of 5-10%.
At present, a lot of molecule parting methods are applied to the research of legionella somatotype, but most of method does not have stdn, the weak point that traditional serological typing is identified is the impact that it easily is subjected to environmental factors, and may and his bacterial classification between have common antigen and cross reaction occurs, and the poor defective of the same existence and stability of monoclonal antibody somatotype that develops thus.
Along with the particularly development of round pcr of molecular engineering, many molecular biology methods are used to Molecular Subtyping of Legionella and epidemiological study.4 kinds of molecule parting technology commonly used that are used at present the legionella somatotype are: randomly amplified polymorphic DNA (Random Amplified Polymorphic DNA, RAPD), pulsed field gel electrophoresis (Pulsed-Field GelElectrophoresis, PFGE), amplified fragment length polymorphism (Amplified fragment lengthpolymorphism, AFLP), Gene identification (sequence-based typing, SBT).Because it once can isolate a large amount of dna fragmentations, and have easy and simple to handlely, the advantage such as repetition rate is good is successively for Molecular Subtyping of Legionella and epidemiology survey.
1988, since Chamberlain et al at first reports employing multiplex PCR diagnosis duchenne muscular dystrophy (DMD), a plurality of DNA detection fields such as genome structure and functional gene, sudden change and polymorphism, and quantitative analysis and reverse transcription PCR have been successfully used in.[Deletion screening of the Duchenne muscular dystrophylocus via multiplex DNA amplification。Nucleic Acids Res, 1988,16:11141～11156], can in same reaction system, add more than one pair of special primer pair, if there is the template with the special complementation of each primer pair, then can in the consubstantiality reaction system, amplify the target DNA fragment of one or more simultaneously, thereby detect corresponding pathogenic bacterium.The method has advantages of fast detecting pathogenic bacterium, responsive, high specificity.
Summary of the invention
The object of the present invention is to provide a kind of multiplex PCR Fast Detection Technique to common serotype in the legionella pneumophilia, and set up corresponding multiple PCR detection kit, for rapid detection and evaluation common pathogen provide effective means.The sequence of the primer comprises one or more that choose in the following sequence:
(1) legionella pneumophilia serotype 1 type and 6 type wzt genes, 4 types, the specific nucleotide sequence of 10 types and 13 type wzm genes;
The complementary dna sequence of the nucleotide sequence of (2) choosing in above-mentioned (1).
Above-mentioned primer sequence can be used for preparation and detects legionella pneumophilia various serotype PCR test kit, gene chip or microarray.Described legionella pneumophilia serotype is taken a sample in the crude extract of the culture of tap water, mineral water, air conditioning cooling water, or legionella pneumophilia 1 type, 4 types, 6 types, the crude extract of the pure growth of 10 types and 13 types.
The present invention also provides a kind of multiple PCR reagent kit, comprises 10 * PCR enzyme spcificity reaction buffer, MgCl
2, dNTP, PCR primer, archaeal dna polymerase, described PCR primer comprises above-mentioned nucleotide sequence; Wherein said PCR primer is preferably the nucleotide sequence shown in SEQ ID NO:1-SEQ ID NO:10.
The upstream primer of SEQ NO:1 ACACTTTTAGGCTTTGGT specific amplified legionella pneumophilia 10 types
The downstream primer of SEQ NO:2 CCCAAGCATAAAAACAATA specific amplified legionella pneumophilia 10 types
The upstream primer of SEQ NO:3 TAAAGATATTGTAGAGAGCCAGC specific amplified legionella pneumophilia 6 types
The downstream primer of SEQ NO:4 CATAGAGAGATAACCCTCACATT specific amplified legionella pneumophilia 6 types
The upstream primer of SEQ NO:5 TGCAGCAAGCAAAAGTTCAG specific amplified legionella pneumophilia 1 type
The downstream primer of SEQ NO:6 AATAAGGGTGAATACAAAGTACATC specific amplified legionella pneumophilia 1 type
The upstream primer of SEQ NO:7 TTGTCATTTGTGCCACAG specific amplified legionella pneumophilia 13 types
The downstream primer of SEQ NO:8 GCCAATTACCCTTTAAAC specific amplified legionella pneumophilia 13 types
The upstream primer of SEQ NO:9 CAACTCCGGATTGGTAAA specific amplified legionella pneumophilia 4 types
The downstream primer of SEQ NO:10 TTCAAATCGCGGTACCTG specific amplified legionella pneumophilia 4 types
Above-mentioned multiple PCR detection kit can comprise following reagent: 10mM dNTP 20 μ l; 10 * enzyme spcificity reaction buffer, 50 μ l; 5U/ μ l hot resistant DNA polymerase 8 μ l; Each 10 μ l of primer; Positive reference substance 10 μ l, negative control product 10 μ l; DdH
2O 5ml.
Above-mentioned for legionella pneumophilia various serotype PCR test kit, whole detecting step comprises sample pretreatment-amplification-electrophoresis detection result.Primer and the needed reagent of PCR reaction system add in the amplification pipe in advance, and the user only needs that pretreated sample is added the amplification pipe and starts amplified reaction and get final product, Simple fast finish testing.
Used nucleotide sequence also can be used for the development of chip among the present invention, comprises solid phase carrier and the oligonucleotide probe that is fixed on the solid phase carrier, and wherein said oligonucleotide probe comprises above-mentioned Nucleotide.
As seen from the above technical solutions, a kind of multi-PRC reaction system that the present invention sets up can detect 5 kinds of serotype legionella pneumophilias simultaneously, and this test kit has the following advantages:
(1) method is easy, and the cycle is short, speed is fast, workable: the PCR test kit compound method that the present invention prepares is easy, and sense cycle is short, speed is fast, workable, be easy to industrialization production, and testing cost is relatively low, market application foreground is wide.If this multiple PCR reagent kit is with legionella pneumophilia 1 type, 4 types, 6 types, the bacteria suspension of 10 types and 13 types carries out pcr amplification, consistent as the template amplification acquired results with the DNA that obtains through extraction, and susceptibility and specificity indifference, like this, can save the extraction step of template DNA, working method is simplified.Simultaneously, compare the routine biochemistry detection method, the testing sample that present method adopts 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) detection sensitivity is high: multiple PCR detection kit provided by the invention and detection method susceptibility thereof are high, and accuracy of detection is high, can detect the dna profiling of 1pg/ μ l.Can carry out comprehensively, system, detect accurately and identify common pathogen.
(2) testing cost is relatively low: can be applied to the fields such as environmental monitoring, Food Hygiene Surveillance, commodity inspection quarantine, and provide technology mode for other different pathogenic microbes detects make up.
(3) accuracy is high: the present invention is according to the special district of the wzt gene of legionella pneumophilia 1 type and 6 types, and 4 types, and the special district of the wzm gene of 10 types and 13 types designs primer.Then utilize primer sets to synthesize the composite PCR detection system, can directly these five kinds of legionella pneumophilias and its nearly source bacterial classification be separated.
Description of drawings
Fig. 1 is test kit detected result figure of the present invention, and wherein: M is DL2000DNA marker; The negative reference substance of N; The positive reference substance of P; 1 is legionella pneumophilia 10 type G2818; 2 is legionella pneumophilia 6 types 62771; 3 is legionella pneumophilia 1 type G2756; 4 is legionella pneumophilia 13 type G2819; 5 is legionella pneumophilia 4 type G2781;
Fig. 2 is that test kit of the present invention detects other serotype reference culture electrophoresis result of legionella pneumophilia figure, wherein: 1, legionella pneumophilia 10 type G2818; 2, legionella pneumophilia 6 type G2771; 3 is legionella pneumophilia 1 type G2756; 4 is legionella pneumophilia 13 type G2819; 5 is legionella pneumophilia 4 type G2781; 6, legionella pneumophilia 7 type G3410; 7, legionella pneumophilia 12 type G2822; 8, legionella pneumophilia 11 type G2824; 9, legionella pneumophilia 14 type G2817; 10), legionella pneumophilia 2 type G2773; M, DL2000DNA marker;
Fig. 3 is the continuation of Fig. 2, wherein: 11, legionella pneumophilia 3 type G2772; 12, legionella pneumophilia 8 type G2820; 13, legionella pneumophilia 5 type G3408; 14, legionella pneumophilia 15 type G2829; 15, legionella pneumophilia 9 type G2774; M, DL2000 DNA marker;
Fig. 4 is that test kit of the present invention detects the non-legionella pneumophilia electrophoresis result of legionella figure, wherein: 1, legionella pneumophilia 10 type G2818; 2, legionella pneumophilia 6 type G2771; 3 is legionella pneumophilia 1 type G2756; 4 is legionella pneumophilia 13 type G2819; 5 is legionella pneumophilia 4 type G2781; 6, Legionella waltersii; 7, Legionellamicdadei; 8, Legionella longbeachae; 9, Legionella anisa; M, DL2000 DNA marker;
Fig. 5 is the continuation of Fig. 4, wherein: 10, Legionella gormanii; 11, Legionella bozemanii; 12, Legionella steigerwaltii; M, DL2000 DNA marker;
Fig. 6 is that the present invention detects legionella clinical strains electrophoresis result figure, wherein: 1, legionella pneumophilia 10 type G2818; 2, legionella pneumophilia 6 type G2771; 3 is legionella pneumophilia 1 type G2756; 4, be legionella pneumophilia 13 type G2819; 5, be legionella pneumophilia 4 type G2781; 6, legionella pneumophilia 1 type G2759; 7, legionella pneumophilia 3 type G2762; 8, legionella pneumophilia 7 type G2763; 9, legionella pneumophilia 7 type G2764; 10, legionella pneumophilia 5 type G2765; M, DL2000 DNA marker;
Fig. 7 is that test kit of the present invention detects the nearly source of other kind bacterial standard bacterial strain electrophoresis result figure, wherein: 1, legionella pneumophilia 10 type G2818; 2, legionella pneumophilia 6 type G2771; 3 is legionella pneumophilia 1 type G2756; 4 is legionella pneumophilia 13 type G2819; 5 is legionella pneumophilia 4 type G2781; 6, shigella; 7, Salmonellas; 8, streptococcus aureus; 9, Yersinia enterocolitica; M, DL2000DNA marker;
Fig. 8 is the continuation of Fig. 7, wherein: 10, Pseudomonas aeruginosa; 11, Aeromonas hydrophila; M, DL2000DNA marker;
Fig. 9 is the electrophoresis result figure that the present invention screens the special primer of legionella pneumophilia 10 types, wherein: 1, legionella pneumophilia 10 type G2818; 2, legionella pneumophilia 6 type G2771; 3 is legionella pneumophilia 1 type G2756; 4 is legionella pneumophilia 13 type G2819; 5 is legionella pneumophilia 4 type G2781; M, DL2000DNA marker;
Figure 10 is the electrophoresis result figure that the present invention screens the special primer of legionella pneumophilia 10 types, wherein: 6, legionella pneumophilia 7 type G3410; 7, legionella pneumophilia 12 type G2822; 8, legionella pneumophilia 11 type G2824; 9, legionella pneumophilia 14 type G2817; 10, legionella pneumophilia 2 type G2773; 11, legionella pneumophilia 3 type G2772; 12, legionella pneumophilia 8 type G2820; M, DL2000 DNA marker.
Embodiment:
For guaranteeing that the above and other objects of the present invention feature and advantage become apparent, the below is especially exemplified by preferred embodiment, and the cooperation Figure of description, in conjunction with specific examples the present invention is described in further detail.
Embodiment 1: genomic extraction
1) get a little bacterium liquid from strain preservative tube, streak inoculation is dull and stereotyped in legionella BCYE growth; 2.5%CO
2, cultivated 5-7 days for 37 ℃.
2) add 1mL 50mM Tris-HCl (pH8.0) in dull and stereotyped, scrape bacterium with the rod that is coated with of the bacterium of going out, get an amount of bacterium liquid to the 1.5mL centrifuge tube, centrifugal 5 minutes long-pending bacterium of 10000rpm are removed supernatant.
3) add 250 μ L 50mM Tris-HCl (pH8.0) resuspended, add 10 μ L 0.5M EDTA (pH8.0), fully mixing.
4) add 15 μ L 20mg/mL N,O-Diacetylmuramidases, abundant mixing, 37 ℃ are incubated 20 minutes.
5) add 3 μ L 20mg/mL Proteinase Ks, gentle mixing.
6) add 20 μ L 10%SDS, 50 ℃ of water-baths 1 hour to solution is clarified.
7) add 2 μ L 25mg/mL RNAase, 65 ℃ of water-baths 20 minutes.
8) add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1), centrifugal 10 minutes of 10000rpm, supernatant liquor is put new pipe, repeats extracting once.
9) add the equal-volume chloroform: primary isoamyl alcohol (24: 1), centrifugal 10 minutes of 10000rpm, supernatant liquor is put new pipe.
10) add the dehydrated alcohol of 2.5 times of precoolings, jog, precipitation DNA.
11) with kapillary around playing a DNA, and ice washing with alcohol with 70%.
12) 65 ℃ of oven dry, 30 μ L TE return molten, and the agarose gel electrophoresis by 0.4% detects.
Embodiment 2: the design of primer
The sequence of downloading from NCBI, in conjunction with the laboratory sequence of testing oneself, for the wzt gene of legionella pneumophilia 1 and 6 types, special district's design primer of the wzm gene of legionella pneumophilia 4,10 and 13 types, primer sequence is as shown in table 1 below:
The special primer sequence of table 1 legionella pneumophilia serotype 1 type, 4 types, 6 types, 10 types and 13 types
Embodiment 3: the screening of special primer
For the wzt gene of legionella pneumophilia 1 and 6 types, special district's design primer of the wzm gene of legionella pneumophilia 4,10 and 13 types, primer sequence (SEQ ID NO:1-SEQ ID NO:10) as shown in table 1.Collected 1 strain legionella pneumophilia, 1 type reference culture, the specificity of the nearly edge bacterium checking of the reference culture of 14 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 4 strain legionellas and 6 strains primer, strain number and source see the following form 2.
Table 2 is for the reference culture of examination
The employed clinical strains of this patent is as shown in table 3 below:
Table 3 is for the biochemical investigation result of examination clinical strains
The PCR system be the testing sample template of 10uM primer 0.3 μ l, 10 * buffer, 2.5 μ l, 10mM dNTP 0.25 μ l, 5U/ μ l Taq polysaccharase 0.2 μ l and 3 μ l in the thin-walled PCR pipe of 0.2ml, use at last ddH
2O complements to 25 μ l.All primers are all obtaining positive findings in the legionella pneumophilia template separately, do not obtain any PCR product band in other group, so these oligonucleotide are high specials.Screening legionella pneumophilia 10 types the results are shown in Figure 9 and Figure 10, only have the bacterial strain of 10 types that the band of 645bp is arranged, other serotypes illustrate that without amplified production this primer is special.
Embodiment 4:PCR detection kit
One, the preparation of test experience material requested and equipment
1. test kit forms:
Each test kit can be used for detecting 10 samples.
Wherein 10 * buffer, dNTP, Taq polysaccharase are provided by precious biotechnology (Dalian) company limited; It is synthetic that primer mixture is that the sequence of designed, designed offers Shanghai Ying Jun biotech company; Positive reference substance (sample that contains legionella pneumophilia 13 types), negative control product (change template into ddH
2O) and ddH
2O is prepared voluntarily by us.
The method of using above-mentioned multiple PCR reagent kit to detect legionella pneumophilia 1 type, 4 types, 6 types, 10 types and 13 types may further comprise the steps:
(1) extraction of environmental sample template to be measured:
The environmental sample template is generally 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 mutually body fluid of the pure growth of legionella pneumophilia 1 type, 4 types, 6 types, 10 types and 13 types, or pure dna, or positive reference substance and negative control product.
With 1.5ml 50mM Tris-HCl (pH8.0) scraping culture, under the 12000rpm condition centrifugal 1 minute, remove supernatant liquor; DdH with 500 μ l
2The resuspended precipitation of O, under the 8000rpm condition centrifugal 5 minutes, remove supernatant liquor, control is done; With 100 μ l ddH
2The resuspended precipitation of O, water-bath is 10 minutes in 100 ℃ of boiling water; Place again on ice after 10 minutes under the 12000rpm condition centrifugal 2 minutes; With the template of 3 μ l middle layer supernatant as the PCR reaction.
(2) in the PCR thin-walled tube, add dNTP, 10 * enzyme spcificity reaction buffer, Taq polysaccharase, primer, testing sample template and ddH
2O, mixing increases at the PCR instrument; The reaction cycle parameter comprises sex change, renaturation, the temperature and time of extension, the cycle index of DNA, is specially: be 94 ℃ 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 58 ℃, 40 seconds;
Elongating temperature and time are 72 ℃, 30 seconds;
The cycle index of sex change, renaturation, extension is 33 circulations;
The temperature and time that carries out a circulation for stablizing amplified production is 72 ℃, 5 minutes.
(3) electrophoresis amplified production in electrophoresis equipment, the record result
1. getting 2 μ l amplified productions mixes with 9: 1 volume ratio with 10 * tetrabromophenol sulfonphthalein sample-loading buffer;
2. mixed solution is splined on 2% the sepharose;
3. with agarose gel electrophoresis 120v voltage stabilizing electrophoresis approximately 10 minutes, contrast with DL2000Marker;
4. observe and record the result.
(4) analyze and carry out the result and judge.
But the present invention is by preparing a kind of PCR test kit that five kinds of serotype industrialization of legionella pneumophilia are produced that detects, the combination of components that the PCR detection method need to be used together, during use, extract testing sample, simultaneously through comparatively simple operation program just can carry out fast, consumption and the concentration of each component is the test gained in sensitive, the easy detection, test kit, detect legionella pneumophilia 1 type, 4 types, 6 types, 10 types and the employed testing installation of 13 types with this test kit simple, testing cost is low.
Using the purpose of positive and negative control product is for the whole operating process of Quality Control, judges in order to draw accurately.
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 4, and the dna profiling amount is 0.5 μ l.
Table 4 one-time detection is tested the amount of reagent in the employed test kit
| Composition | Concentration | Application of sample amount (μ l) | |
| ddH2O | - | 15.2 | |
| 10 * enzyme |
10× | 2.5 | |
| | 25mM | 3 | |
| dNTP | 10mM | 0.4 | |
| The upstream primer mixture | 10μM | 1.5 | |
| The downstream primer mixture | 10μM | 1.5 | |
| The Taq enzyme | 5U/μl | 0.4 |
Annotate: on have the mixture of primer to comprise primer P-1, P-3, P-5, P-7, P-9; The mixture of downstream primer comprises primer P-2, P-4, P-6, P-8, P-10.
Hot resistant DNA polymerase among the present invention is the Taq polysaccharase.
Above-mentioned positive reference substance is for having determined to contain one or more sample of legionella pneumophilia 1 type, 4 types, 6 types, 10 types and 13 types, and the negative control product then are ddH
2O.
2. plant and instrument
PCR instrument (being 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
The used experimental strain of the present invention as shown in Table 1 and Table 2.
Two, detect the concrete operations of implementing: under similarity condition, adopt multiple PCR detection kit provided by the present invention to carry out the detection of PCR method above-mentioned 28 strain reference cultures and 5 strain legionnella clinical bacterial strains.
1. the extraction of testing sample template
A. for reference culture
1) get 3 μ l and connect the bacterium amount, under aseptic technique, streak inoculation is in the BCYE substratum, and 37 ℃, 5%CO2 are cultivated 24-48h;
2) add 1mL 50mM Tris-HCl (pH8.0) in dull and stereotyped, scrape bacterium with the rod that is coated with of the bacterium of going out, get an amount of bacterium liquid to the 1.5mL centrifuge tube, centrifugal 5 minutes long-pending bacterium of 10000rpm are removed supernatant;
3) the resuspended precipitation of 500ul ddH2O, 8000rpm removes supernatant, and control is done as far as possible;
4) the resuspended precipitation of 100ul ddH2O, mixing, 100 ℃ of boiling water baths 10 minutes;
5) put on ice after 10 minutes centrifugal 2 minutes of 12000rpm;
6) get 3 μ l middle layer supernatant as the template of PCR reaction.
B. for environment culture (blood, phlegm, segmental bronchus extract, hydrothorax lung tissue, tap water, mineral water, air conditioner condensate water etc.)
1) add 1mL 50mM Tris-HCl (pH8.0), scrape bacterium with the rod that is coated with of the bacterium of going out, centrifugal 5 minutes of 8000rpm removes supernatant;
2) the resuspended precipitation of 500ul ddH2O, centrifugal 5 minutes of 8000rpm removes supernatant, and control is done as far as possible;
3) the resuspended precipitation of 100ul ddH2O, mixing, 100 ℃ of boiling water baths 10 minutes;
4) put on ice after 10 minutes centrifugal 2 minutes of 12000rpm;
5) get 3 μ l middle layer supernatant as the template of PCR reaction.
2. draw respectively with micropipet that the 10uM primer respectively adds 0.3 μ l in the PCR test kit, the MgCl23 μ l of 10 * buffer2.5 μ l, the 25mM of 25mM, the dNTP 0.4 μ l of 10mM, the testing sample template of 5U/ μ l Taq polysaccharase 0.4 μ l and 3 μ l is in the thin-walled PCR pipe of 0.2ml, complement to 25 μ l with ddH2O at last, fully mixing;
3. will increase according to following temperature and time at the PCR instrument after the mixture high speed centrifugation several seconds: 94 ℃ of 1 circulations in 5 minutes; 94 ℃ 30 seconds, 58 ℃ 40 seconds, 72 ℃ 30 seconds, 33 circulations; 72 ℃ of 1 circulations in 5 minutes.
4. electrophoresis amplified production in electrophoresis equipment records the result
1. getting 2 μ l amplified productions mixes with 9: 1 volume ratio with 10 * tetrabromophenol sulfonphthalein sample-loading buffer;
2. mixed solution is splined on 2% the sepharose;
3. with agarose gel electrophoresis 120v voltage stabilizing electrophoresis approximately 10 minutes, contrast with DL2000Marker;
4. observe and record the result, when the forward position bromophenol blue indicator migrates to apart from well 3cm stop electrophoresis at least, observe and log at the gel imaging instrument.
Carry out the result according to following condition and judge the multiplex PCR detected result
Legionella pneumophilia 10 types should have at the 645bp place band, legionella pneumophilia 6 types have a band at the 517bp place, legionella pneumophilia 1 type has a band at the 369bp place, and legionella pneumophilia 13 types have a band at the 297bp place, and legionella pneumophilia 4 types should have at the 243bp place band.
The test of positive control and negative control is as long as change the testing sample template into a kind of in legionella pneumophilia 10 types, 6 types, 1 type, 13 types or 4 types, negative template (ddH
2O) get final product, one or more the sample template that contains legionella pneumophilia 10 types, 6 types, 1 type, 13 types or 4 types has the fragment of corresponding size, does not contain the sample template of above-mentioned five kinds of legionella pneumophilias without this fragment.Fig. 2 and shown in Figure 3 seen in reference culture electrophoresis result record: all reference cultures have the fragment of corresponding size except legionella pneumophilia 10 types, 6 types, 1 type, 13 types and 4 types, and other bacterial strains are all without amplified production.The clinical strains electrophoresis result records as shown in Figure 6: add up to 10 strains, except legionella pneumophilia 1 type clinical strain has the 369bp fragment, other clinical strains are all without amplified production.This explanation adopts the PCR detection method can get rid of false positive reaction, identifies according to the detection box that has or not above fragment to carry out pathogenic bacteria, and accuracy in detection has improved, and has avoided the unnecessary loss that causes because detecting error.
The hybridization kit that utilizes above-mentioned experimental procedure to obtain can be used for detecting simultaneously one or more of legionella pneumophilia 10 types, 6 types, 1 type, 13 types and 4 types.
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 does.
Claims (4)
1. for detection of the specific PCR primer of legionella pneumophilia (Legionella pneumophila) various serotype pathogenic bacteria, it is characterized in that the nucleotide sequence of described Auele Specific Primer shown in SEQ ID NO:1-SEQ ID NO:10.
2. specific PCR primer claimed in claim 1, Auele Specific Primer wherein is:
The upstream primer of SEQ NO:1 ACACTTTTAGGCTTTGGT specific amplified legionella pneumophilia 10 types;
The downstream primer of SEQ NO:2 CCCAAGCATAAAAACAATA specific amplified legionella pneumophilia 10 types;
The upstream primer of SEQ NO:3 TAAAGATATTGTAGAGAGCCAGC specific amplified legionella pneumophilia 6 types;
The downstream primer of SEQ NO:4CATAGAGAGATAACCCTCACATT specific amplified legionella pneumophilia 6 types;
The upstream primer of SEQ NO:5 TGCAGCAAGCAAAAGTTCAG specific amplified legionella pneumophilia 1 type;
The downstream primer of SEQ NO:6 AATAAGGGTGAATACAAAGTACATC specific amplified legionella pneumophilia 1 type;
The upstream primer of SEQ NO:7 TTGTCATTTGTGCCACAG specific amplified legionella pneumophilia 13 types;
The downstream primer of SEQ NO:8 GCCAATTACCCTTTAAAC specific amplified legionella pneumophilia 13 types;
The upstream primer of SEQ NO:9 CAACTCCGGATTGGTAAA specific amplified legionella pneumophilia 4 types;
The downstream primer of SEQ NO:10 TTCAAATCGCGGTACCTG specific amplified legionella pneumophilia 4 types.
3. a test kit that contains the described specific PCR primer of claim 1 is characterized in that it comprises 10 * PCR enzyme spcificity reaction buffer, MgCl
2, dNTP, PCR primer, archaeal dna polymerase; The nucleotide sequence of described PCR primer shown in SEQ ID NO:1-SEQ ID NO:10.
4. test kit claimed in claim 3, wherein it is comprised of following reagent:
1)dNTP 10mM 50μl；
2) 10 * enzyme spcificity reaction buffer, 300 μ l;
3)MgCl
2 25mM 300μl；
4) Taq polysaccharase 5U/ul 5 μ l;
5) primer mixture 5uM 70 μ l;
6) positive reference substance 10 μ l;
7) negative control product 10 μ l;
8)ddH
2O 5ml；
Described positive reference substance is one or more of legionella pneumophilia 10 types, 6 types, 1 type, 13 types and 4 types; The negative control product are water.
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