CN101020929A - Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene - Google Patents
Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene Download PDFInfo
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Abstract
The (Streptococcus suis Serotype 2 (SS2) virulence gene PCR amplification kit includes a SS2DNA contrast template, a PCR amplification detecting reagent, an agarose gel electrophoresis reagent and a DNA gradient standard. The PCR amplification detecting process includes the measurement of 7 important SS2 genes, diagnosing Streptococcus infection and SS2 infection based on whether to amplify specific Streptococcus gene, specific SS2 gene and 5 relevant important SS2 virulence genes, and predicting the virulence, invasiveness trend and disease prognosis fast and early. The present invention is favorable to the clinical diagnosis of borderline case and early warning, and is hopeful to be applied in emergency detection of human SS2 infection, etc.
Description
(1) technical field
The present invention relates to a kind of 2 type pig streptococcus virulence gene pcr amplification detection kit and detection methods.
(2) background technology
Streptococcus suis is a kind of important zoonosis, infected person mainly be streptococcus suis 2-type (Streptococcus suis Serotype 2, SS2).Can cause pneumonia, meningitis, microbemia, septicemia etc. after the people infects, weight person occur toxic shock syndrome (Toxic Shock Syndrome, TSS), and the most serious with the latter, onset acute disease gesture is dangerous, untimely treatment can cause very fast death.Studies show that the strong and weak relevant virulence factor with it of virulence is closely related.Since nineteen sixty-eight Denmark scholar reported first the human infection swine streptococcus cause meningitic case, the report of people's infection with streptococcus suis example has all been arranged in global many countries and regions successively.In China, the people had been reported in Jiangsu Province infection with streptococcus suis in 1998,25 examples of falling ill, dead 14 examples; People's infection with streptococcus suis outburst epidemic situation, 204 examples of falling ill took place in Sichuan Province to August in 2005 6, dead 38 examples, most toxic shocks that take place for being seen in the maximum-norm people infected pigs suis epidemic situation of report both at home and abroad up to now, have caused the extensive concern of various circles of society in the death.Etiological diagnosis promptly and accurately and virulence identify it is the emergent key that detects, instructs clinical application, prediction prognosis and influence the sensitivity of disease early warning system of people infected pigs suis.
Difference pathogenic between the SS2 different strains is big, and the important virulence factor of known comparatively sure SS2 has at present: hemolysin sly, muramidase-released protein mrp, capsular polysaccharide cps2J and extracellular protein factor ef.Sly is bringing into play important effect in the process of swine streptococcus intrusion and lysing cell; Mrp claims class M albumen again, is made up of 1208 radon base acid. molecular weight is 136kd, is the protein in cell wall of bacterium.The bacterial strain of M protein positive can be resisted the phagolysis of scavenger cell and can adhere to chrotoplast.It is reported that the strain that has only mrp+ef+ could survive in mouse macrophage.But these two kinds of albumen are not essential to the pathogenic of 2 type bacterial strains.Test shows that the mrp transgenation strain of 2 type bacterial strains is in full accord to the pathogenic of piglet and its original strain.Except that mrp and ef, also have other virulence factors to exist.Capsular polysaccharide cps is the important virulence factor of SS2 of having proved conclusively, and the allelic mutant strain of shortage cps enters in laboratory animal pig and the rat body and can be recycled removing very soon, to the laboratory animal nontoxicity.Its possible action pathway is to influence thalline to adhere to activity on epithelial cell and the scavenger cell.The glyceraldehyde-3-phosphate dehydrogenase gapdh of 39kDa is the SS2 surface protein, plays an important role in SS2 at first attaches to the process of host cell.
It is the object of the invention that its encoding gene is carried out simple and easy, quick, responsive, special diagnostic nucleic acid.The research report of existing at present utilization PCR (polymerase chain reaction) technology for detection SS2 virulence associated gene, but used PCR reacts and cycling condition is not quite similar, and need repeatedly increase; Though the report that also has multiplex PCR to detect, but still fail in 1 amplification, to detect 7 important gene of all SS2 simultaneously, be difficult to satisfy the emergent needs that detect.
(3) summary of the invention
The present invention can once detect 7 important gene of all SS2, simple and easy, 2 type pig streptococcus virulence gene pcr amplification detection kit and detection methods fast and accurately in the amplification simultaneously in order to provide a kind of.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of 2 type swine streptococcus (Streptococcus suis Serotype 2, SS2) virulence gene pcr amplification detection kit, comprise SS2 DNA contrast template (DNA 〉=100ng/ μ l, obtain by 2 type pig streptococcus bacterial strain extractings, contain SS2 and belong to (tuf), plant (16S rRNA), hemolysin (sly), muramidase-released protein (mrp), capsular polysaccharide (cps2J), extracellular protein factor (ef), Glycerose 3-phosphate dehydrogenase specific genes such as (gapdh)), the pcr amplification detection reagent, agarose gel electrophoresis reagent and dna ladder degree standard substance, described pcr amplification detection reagent mainly comprises the PCR damping fluid, specificity amplification primer, deoxidation nucleoside triphosphate mixture, archaeal dna polymerase and sterilization pure water; Described specificity amplification primer is totally 8 to (except that 7 pairs of SS2 Auele Specific Primers, also containing 1 couple of bacterium beta globin gene primer CI, Intemal Control):
16S-195(s)：5’-CAG TAT TTA CCG CAT GGT AGA TAT-3’
16S-489(as2)：5’-GTA AGA TAC CGT CAA GTG AGA A-3’
cps2J-s：5’-GTT GAG TCC TTA TAC ACC TGT T-3’
cps2J-as：5’-CAG AAA ATT CAT ATT GTC CAC C-3’
mrp-Fw：5’-GGT ATA CCT TGC TGG TAC CGT TC-3’
mrp-Re：5’-AGT CTC TAC AGC TGT AGC TGG-3’
sly-Fw：5’-AGT TCG CAC TTG ATT TTA AG-3’
sly-Re：5’-AAT ACA TTG CCA GAT TAC TC-3’
gapdh-Fw：5’-GGC GCC GAA TTC GTC GAC ATT ATT TAG CAA TTTTTG CG-3’
gapdh-Re：5’-CGC CGC GGA TCC GTA GTT AAA GTT GGT ATTAAC-3’
ef-Fw：5’-ACA AAG GCG TAG GTT CAA TC-3’
ef-Re：5’-CGG CAT CAA GAA TGT CTT TG-3’
tuf-Fw：5’-GTA CAG TTG CTT CAG GAC GTA TC-3’
tuf-Re：5’-ACG TTC GAT TTC ATC ACG TTG-3’
CI 6-s：5’-GTT GAG TCC TTA TAC ACC TGT TAC TCA GTG CCG CAGCTA ACG CAT T-3’
CI7-as：5’-CAG AAA ATT CAT ATT GTC CAC CCG ACT TCA CCC CAATCA TCT ATC C-3’。
Described pcr amplification detection reagent main component is as follows:
PCR damping fluid final concentration is 1 *
Each 10～80 μ M of specificity amplification primer
Deoxidation nucleoside triphosphate mixture 200～1000 μ M
Archaeal dna polymerase 0.5～5 enzyme activity unit/reaction
Surplus is the sterilization pure water.
PCR damping fluid final concentration is 1 *, the meaning is that each component final concentration is identical with 1 * PCR buffer in the damping fluid, promptly described PCR damping fluid is in each component final concentration in pcr amplification reagent, and is composed as follows:
Repone K 500mM
Tris-Cl 100mM
Magnesium chloride 25mM
Polyethylene glycol 6000 0.1%
1,4-DTT 0.1%
Bovine serum albumin 1%.
Above-mentioned percentage concentration is the quality volume percent, and certain material concentration is to contain this material 1g in the 1% expression 100mL solution.
The PCR damping fluid can have different combinations as required, also can have other assembly to replace, but aforesaid combination is comparatively ideal selection.
Described agarose gel electrophoresis reagent main component is as follows:
Tbe buffer liquid final concentration is 5 *
The agarose volume percent is 1 ‰
The ethidium bromide volume percent is 1 ‰
The bromjophenol blue volume percent is 16.67%.
Also can contain the DNA extraction agent in the described pcr amplification detection kit, described DNA extraction agent is one of following: 1. Qiagen DNA extraction agent, 2. Promega DNA extraction agent, 3. TaKaRaDNA extraction agent.The preferred Qiagen DNA of the present invention extraction agent.
Described dna ladder degree standard substance are selected the DNA standard substance of 100bp gradient usually for use, can be one of following formulas: (1) TaKaRa 100bp DNA Ladder Marker, (2) Promega 100bp DNALadder, (3) BioLabs 100bp DNA Ladder; The preferred TaKaRa 100bp of the present invention DNALadder Marker.
Described archaeal dna polymerase is one of following: 1. Ampli Taq archaeal dna polymerase, 2. rTaq archaeal dna polymerase, 3. Platinum Tag archaeal dna polymerase.Working concentration is 0.5～5 enzyme activity unit/reaction (U), U is defined as: the Oncorhynchi sperm DNA of using sensitization is as template/primer, in 74 ℃, 30 minutes, the full Nucleotide of taking in 10nmol is that the activity of acid insolubles is defined as 1 unit of activity (U); Can there be difference in archaeal dna polymerase working concentration and product man, the preferred rTaq archaeal dna polymerase of the present invention.
Described deoxidation nucleoside triphosphate mixture (dNTP) is selected dATP, dTTP, dCTP, dGTP, dUTP combination for use, is preferably one of following: 1. dATP, dCTP, dGTP, the ratio of dTTP amount of substance 1: 1: 1: 1 mixture, 2. dATP, dGTP, dCTP, the ratio of dUTP amount of substance 1: 1: 1: 1 mixture, 3. dATP, dCTP, dGTP, dTTP, the ratio of dUTP amount of substance 4: 4: 4: 4: 1 mixture; Most preferably be 3. dATP, dCTP, dGTP, dTTP, the ratio of dUTP amount of substance 4: 4: 4: 4: 1 combination.
The each component final concentration is as follows in the described pcr amplification detection reagent:
The PCR damping fluid:
Repone K 500mM
Tris-Cl 100mM
Magnesium chloride 25mM
Polyethylene glycol 6000 0.1%
1,4-DTT 0.1%
Bovine serum albumin 1%
8 couples of each 50 μ M of specificity amplification primer
Deoxidation nucleoside triphosphate mixture:
dATP 200μM，dCTP 200μM，dGTP 200μM，dTTP 200μM，dUTP 50μM
0.5 enzyme activity unit/the reaction of rTaq archaeal dna polymerase
Surplus is the sterilization pure water.
The invention still further relates to a kind of 2 type pig streptococcus virulence gene pcr amplification detection methods, described detection method is with the positive contrast of SS2 DNA contrast template, and adopting the DNA of sample to be tested is analyzing samples, and described method steps is as follows:
(1) sample DNA extracts; Described sample DNA is from patients'blood to be measured, ascites, cerebrospinal fluid or postmortem sample; Liver, spleen, ascites, the painstaking effort of sick, dead pig; Fresh or outmoded bacterial cultures etc.;
Take a certain amount of sample of sample to be tested through pre-treatment, extract DNA according to a conventional method:
Sample is a blood, ascites, cerebrospinal fluid or liquid culture: get proteolytic enzyme 40 μ l and manage in 1.5ml, add the 0.4ml sample, mixing adds lysate again, whirlpool mixing 15 seconds, 56 ℃ of water-baths 10 minutes, instantaneous centrifugal, add 400 μ l dehydrated alcohols, whirlpool mixing 15 seconds, instantaneous centrifugal, this mixed solution is carefully moved into the Filter column of 2ml, centrifugal 1 minute of 6000g, abandon filtrate, the careful 500 μ l washing lotions 1 that add are in Filter column, and centrifugal 1 minute of 6000g abandons filtrate, carefully add 500 μ l washing lotions 2 again in Filter column, centrifugal 3 minutes of 20 000g abandon filtrate, and Filter column is moved into new 1.5ml pipe, add 100 μ l lysates or aseptic no DNA enzyme pure water, room temperature left standstill 5 minutes, and centrifugal 1 minute of 6000g gets liquid and directly carries out the PCR operation or place-80 ℃ of preservations.
Sample is parenchymal viscera or tissue block: get PBS 80 μ l and manage in 1.5ml, add tissue block sample 25mg (spleen 10mg), homogenate, add 100 μ l and organize lysate, add 20 μ l Proteinase Ks, whirlpool mixing 3 times, 56 ℃ of water-baths are taken out mixing 2～3 times/hour during organizing dissolving fully (be generally 1～3 hour or spend the night), or put shaking bath.Instantaneous centrifugal, add 200 μ l lysates, whirlpool mixing 15 seconds, 70 ℃ of water-baths 10 minutes, instantaneous centrifugal, add 200 μ l dehydrated alcohols, whirlpool mixing 15 seconds, instantaneous centrifugal, this mixed solution is carefully moved into the Filter column of 2ml, centrifugal 1 minute of 6000g, abandon filtrate, the careful 500 μ l washing lotions 1 that add are in Filter column, and centrifugal 1 minute of 6000g abandons filtrate, carefully add 500 μ l washing lotions 2 again in Filter column, centrifugal 3 minutes of 20000g abandons filtrate, and Filter column is moved into new 1.5ml pipe, add 100 μ l lysates or aseptic no DNA enzyme pure water, room temperature left standstill 5 minutes, and centrifugal 1 minute of 6000g gets liquid and directly carries out the PCR operation or place-80 ℃ of preservations.
Sample is the culture dish bacterium colony: an amount of doubtful bacterium colony of picking is in the 1.5ml of built-in physiological saline 500 μ l pipe, the thorough mixing of whirlpool, centrifugal 3 minutes of 8000rpm, abandon supernatant, physiological saline 1000 μ l ditto wash 2 times again, add sterile pure water 500 μ l, 100 ℃ of water-baths 10 minutes, centrifugal 3 minutes of 8000rpm carefully draws supernatant and directly carries out the PCR operation or place-80 ℃ of preservations.
(2) pcr amplification: get the pcr amplification detection reagent, the DNA that adds SS2 DNA contrast template and analyzing samples carries out amplified reaction respectively;
(3) amplified production is measured: get amplified production, and behind sepharose, reading of data, imaging and saving result under the gel imaging instrument;
(4) interpretation of result:, the positive that is judged as of band occurs in purpose clip size position with reference to gradient dna ladder degree standard substance image;
Described 2 type swine streptococcus (Streptococcus suis Serotype 2, SS2) virulence gene pcr amplification detection kit, comprise SS2 DNA contrast template, pcr amplification detection reagent, agarose gel electrophoresis reagent and 100bp gradient DNA standard substance, described pcr amplification detection reagent mainly comprises PCR damping fluid, specificity amplification primer, deoxidation nucleoside triphosphate mixture, archaeal dna polymerase, sterilization pure water;
Described specificity amplification primer and purpose clip size are:
16S-195(s)：5’-CAG TAT TTA CCG CAT GGT AGA TAT-3’ 328bp
16S-489(as2)：5’-GTA AGA TAC CGT CAA GTG AGA A-3’
cps2J-s：5’-GTT GAG TCC TTA TAC ACC TGT T-3’ 459bp
cps2J-as：5’-CAG AAA ATT CAT ATT GTC CAC C-3’
mrp-Fw：5’-GGT ATA CCT TGC TGG TAC CGT TC-3’ 532bp
mrp-Re：5’-AGT CTC TAC AGC TGT AGC TGG-3’
sly-Fw：5’-AGT TCG CAC TTG ATT TTA AG-3’ 1500bp
sly-Re：5’-AAT ACA TTG CCA GAT TAC TC-3’
gapdh-Fw：5’-GGC GCC GAA TTC GTC GAC ATT ATT TAG CAA TTTTTG CG-3’ 1100bp
gapdh-Re：5’-CGC CGC GGA TCC GTA GTT AAA GTT GGT ATTAAC-3’
ef-Fw：5’-ACA AAG GCG TAG GTT CAA TC-3’ 269bp
ef-Re：5’-CGG CAT CAA GAA TGT CTT TG-3’
tuf-Fw：5’-GTA CAG TTG CTT CAG GAC GTA TC-3’ 205bp
tuf-Re：5’-ACG TTC GAT TTC ATC ACG TTG-3’
CI6-s：5’-GTT GAG TCC TTA TAC ACC TGT TAC TCA GTG CCG CAGCTA ACG CAT T-3’ 620bp
CI7-as：5’-CAG AAA ATT CAT ATT GTC CAC CCG ACT TCA CCC CAATCA TCT ATC C-3’。
Described step (2) PCR cycling condition is set to: 94 ℃ * 5min; By 94 ℃ * 60sec, 58 ℃ * 60sec, 72 ℃ * 90sec, circulate 30 times again; Last 72 ℃ * 5min finishes amplification.
Described detection method step (2) multiplex PCR amplification concrete steps are: each reaction tubes is got 10 * PCR damping fluid, 2.5 μ L, deoxidation nucleoside triphosphate (dNTP) mixture 2 μ L, (primer of same group can be added in the same reaction tubes: 16S for rTaq archaeal dna polymerase 0.25 μ L and grouping specificity amplification primer, cps2J, one group of mrp, sly, one group of gapdh, ef, tuf, one group of CI7) each 0.25 μ L, adding no DNA enzyme sterilization pure water to 23 μ l mixes, prepare simultaneously 3 parts of PCR thin-walled reaction tubess that above-mentioned mixed solution is housed separately, the a dna solution 2 μ l that add step (1) preparation, 1 part adds no DNA enzyme sterilization pure water 2 μ l as negative control, 1 part adds the SS2 dna profiling as positive control in addition, mixing, instantaneous centrifugal, reaction tubes and control tube place the PCR instrument to carry out pcr amplification immediately.EppendorfH-116 or iCycler EN-61010 instrument are recommended the cycling condition setting: 94 ℃ * 5min; By 94 ℃ * 60sec, 58 ℃ * 60sec, 72 ℃ * 90sec, circulate 30 times again; Last 72 ℃ * 5min finishes amplified reaction.
Described detection method step (3) amplified production is measured concrete steps: get pcr amplification product 5 μ L respectively, with 1 μ L bromjophenol blue solution mixing, add in 2% sepharose (the including ethidium bromide) well for preparing in advance; The DNA standard substance that other gets 4 μ L100bp gradients are reference dna segment size (bp), ditto add the sepharose well.Sepharose places the horizontal strip electrophoresis groove that includes 0.5 * tbe buffer liquid, and the application of sample nose end leads to and goes up power supply towards negative pole, electrophoresis is to the dyestuff forward position during approximately apart from sepharose edge 1cm, deenergization takes out gel, reading of data, imaging and saving result under the gel imaging instrument.MiniRun GE-100 type gel-electrophoretic apparatus is recommended the cycling condition setting: voltage 100V, electrophoresis 30～40min.Full-automatic ultraviolet of FR-200A and visible analytical equipment setting: choose the ultra-violet analysis light source, the picture preservation mode is 12 *.
The present invention has set up the multiple PCR method of directly measuring 7 important gene of SS2 from clinical samples or culture with same PCR circulation and reaction conditions simultaneously first, and 78 bacteriums can detect minimum need.Employing can cause that infecting similar meningitis symptom, pneumonia or bacteremic Neisseria meningitidis, streptococcus pneumoniae, beta hemolytic streptococcus etc. with SS2 does contrast, and SS2 specific specificity gene and 5 important virulence genes all do not increase; Wherein with SS2 with streptococcus pneumoniae, the beta hemolytic streptococcus of the Pseudomonas streptococcus specific gene tuf that then can increase.
The round pcr principle: based on duplicating of DNA, its specificity depends on and target sequence two ends complementary Oligonucleolide primers.PCR is by sex change--annealing--extends three primitive reaction steps formations: the 1. sex change of template DNA: template DNA is after being heated to 94 ℃ of left and right sides certain hours, template DNA double-stranded DNA double-stranded or that form through pcr amplification is dissociated, make it to become strand, so that it combines with primer, for the lower whorl reaction is prepared; 2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is reduced to about 55 ℃, and primer combines with the complementary sequence pairing of template DNA strand; 3. the extension of primer: dna profiling--the primer binding substances is under the effect of TaqDNA polysaccharase, with dNTP is reaction raw materials, target sequence is a template, by base pairing and semiconservative replication principle, synthetic one new is extended three processes with template DNA chain complementary semiconservative replication chain recirculation sex change--annealing--, just can obtain more " semiconservative replication chain ", and this new chain can become next round-robin template again.Whenever finish a circulation and need 2～4 minutes, just can will wait to expand the goal gene amplification and amplify millions of times in 2～3 hours.Arrival plateau (Plateau) required cycle index depends on the copy of template in the sample.
Multiple PCR technique principle: on the round pcr basis, with adding in 1 reaction system more than 2 kinds and 2 kinds and a plurality of target sequences two ends complementary Oligonucleolide primers, amplify different goal gene segments simultaneously, to detect whether contain a plurality of goal gene in the sample simultaneously.
The present invention is according to above-mentioned principle, the synthetic streptococcus (tuf) of design, SS2 kind (16S rRNA), hemolysin (sly), muramidase-released protein (mrp), capsular polysaccharide (cps2J), extracellular protein factor (ef) and the isogenic Auele Specific Primer of Glycerose 3-phosphate dehydrogenase (gapdh), goal of the invention is to provide a kind of gene detecting kit and gene tester that can quick and precisely detect 2 type swine streptococcus and relevant virulence factor, thereby whether quick diagnosis patient carries 2 type swine streptococcus and can identify simultaneously that entrained bacterium contains several relevant virulence factor genes, and tentatively judges its virulence in view of the above.
Advantage applies of the present invention exists:
1. filled up the association area blank
The present domestic SS2 laboratory diagnosis reagent listing of still not having brings very big inconvenience for laboratory diagnosis and the research of SS2, and the present invention has filled up this blank, is more conducive to the carrying out of SS2 laboratory diagnosis and research.
2. early diagnosis is shortened than the time greatly with Immunological Method
Immunology diagnosis mainly is antigen antibody reaction, be applied to clinically produce after the antibody in vivo usually, and there be long " window phase " in the immunology detection of many pathogenic agent, is unfavorable for the early diagnosis to disease.The paired sera antibody of still needing in addition raises and can get rid of previous infection more than 4 times and point out recent infection, and this often need infect and can gather the 2nd part of serum after 1 month, is unfavorable for early diagnosis, and does not still have commercially available SS2 immunology detection reagent at present.PCR method directly detects the DNA of pathogenic agent, can shorten " window phase " greatly, and its highly sensitive detection obviously also helps the early diagnosis of disease.For making a definite diagnosis early and treatment is provided convenience.
3. the sample diversity is bigger, quicker than compatibility with traditional Bacteria Identification method
Can directly detect multiple sample, sample is compatible big.Even outmoded sample still can detect.And traditional Bacteria Identification needs to carry out bacterium separation and Culture, dyeing microscopic examination, biochemical identification etc. with fresh specimens, and whole process needs 2～3 day time, is unfavorable for the early diagnosis to disease, and can't be to outmoded or preserve improperly that sample detects.
4. fast and convenient, with present other SS2 PCR detection method ratio, more convenient, quick
Polygenic amplification, particularly the multiplex PCR of goal gene detects more than 3 kinds, often has the problems that need gradation between different primer systems because of desired cycling condition difference, therefore prolongs experimental period or needs 2 above PCR reaction instrument or reactive system.Though existing at present utilization multiplex PCR detects the research report of SS2 virulence associated gene, but 2～3 goal gene of 1 property mensuration SS2 at most simultaneously, used PCR reaction and cycling condition are not quite similar, and need repeatedly increase, and are difficult to satisfy the emergent needs that detect.
The present invention has set up the directly multi-PCR detection method of 7 important gene of while 1 property mensuration SS2 from various clinical samples or culture first.Compare with present other SS2 PCR detection method, detection method provided by the invention saves time, easy, to the SS2 dna sample as long as can detect in 2 hours, and can be according to contained virulence gene preliminary judgement virulence situation.
5. highly sensitive, as long as there are 78 bacterium to pick in the sample
The present invention is through optimizing the optimization of PCR condition and reactive system, and through primer to permutation and combination, choose best primer to pairing,, adopt the viable count method that the SS2 positive is carried out the susceptibility of enumeration with the method for inspection with reference to strain to improve the susceptibility of experiment to greatest extent.Experiment shows as long as contain 78 SS2 bacterium and can pick in the sample, and to compare susceptibility higher with other method.
6. high specificity: relevant and irrelevant bacterial strain results of comparison is satisfied
The present invention is to causing that infecting similar meningitis symptom, pneumonia or bacteremic Neisseria meningitidis, streptococcus pneumoniae, beta hemolytic streptococcus etc. with SS2 sets up all do not increase 5 important virulence genes of SS2 specific specificity gene and SS2 of relevant bacterial strain control experiment, result; To suis such as streptococcus pneumoniae, the beta hemolytic streptococcus streptococcus specific gene tuf that then can increase; All can amplify the thalline betaglobulin encoding gene of 650bp purpose band in the detection architecture to all DNA of bacteria, then all do not amplify any dna segment to adding pure water or other non-streptococcic DNA of bacteria, show this detection method susceptibility height, high specificity.
7. differentiate pathogenic bacterium fast, and carry out virulence and identify, prediction prognosis and disease early warning
7 important gene of 1 property of the present invention mensuration SS2, according to whether amplifying the streptococcus specific gene, SS2 specific specificity gene and 5 important SS2 virulence associated genes, can be to whether being that hammer belongs to and infecting, there is not SS2 infection to make differential diagnosis, and virulence to the SS2 infectious bacteria, the prognosis of invasiveness tendency and disease is made fast, early prediction, help the early diagnosis and the differential diagnosis of clinical suspected case, and help the early warning of disease popularity and hazard rating, be expected to be used for the people and infect that SS2 is emergent to be detected, disease surveillance, epidemiology survey and clinical early stage express laboratory diagnosis.
(4) description of drawings
Fig. 1 is the amplified production gel electrophoresis figure of sensitivity test in the SS2 gene tester; Swimming lane 1:DNA template blank; Swimming lane 2～4:SS2 is with reference to strain (A group primer separately); Swimming lane 5～10: SS2 is followed successively by 780,78,7.8,0.78,0.078 with reference to the CFU of strain in every reaction system; Swimming lane M:100bp DNA Ladder (100～1000bp); Swimming lane 10～12: experiment strain SS205001, SS205002, SS205003; Swimming lane 13～16: negative control bacterial strain S.pne, S.vi, S. β-hemo, N.m; 3 pairs of primers of swimming lane 1,5～16:A group are all merged into 1 pipe;
Fig. 2 is (tuf, ef+CI) the amplified production gel electrophoresis figure (amplimer adopts C group primer tuf, ef and CI) of different strains C group gene in the test of SS2 gene tester specificity; Swimming lane 1,2,3: streptococcus control strain S.pne, S.vi and S. β-hemo; Swimming lane 4～6: negative control bacterial strain S.epi, N.m and V.cho; Swimming lane 7: blank; Swimming lane 8～10: experiment strain SS205004; Swimming lane 10: pure culture bacterium; Swimming lane 11 is a samples of CSF, and swimming lane 12 is a blood preparation; Swimming lane M:100bp DNALadder (100～1500bp);
Fig. 3 is the gel electrophoresis figure of following 1 the property amplification different genes group of same reaction conditions; Swimming lane 1,3, the 5:SS2 positive are with reference to strain; Swimming lane 2,4,6: two-way nutrient solution (experiment strain SS205004); Swimming lane 7: Neisseria meningitidis (N.m); Swimming lane 8:DNA blank; Swimming lane M:100bp DNA Ladder (100～1500bp); Swimming lane 1,2:B group primer amplification segment (sly, gapdh); Swimming lane 3,4:A group primer segment (16SrRNA, cps2J, mrp); Swimming lane 5,6:C organize primer segment (ef, tuf).
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
The SS2 virulence gene detection kit of being correlated with comprises following component:
(1) Qiagen DNA extraction agent
(2) multiplex PCR augmentation detection reagent:, be that solvent forms with distilled water by 10 * PCR damping fluid, deoxidation nucleoside triphosphate (dNTP) mixture, archaeal dna polymerase and specific amplification primer etc.
Totally 8 pairs of described specificity amplification primers, its primer title, sequence amplification product size and be in charge of and see Table 1.
Table 1: primer sequence and grouping
| Group × | The primer title | Sequence (5 '-3 ') | Product size (bp) |
| A | 16S-195(s) 16S-489(as2) | CAG TAT TTA CCG CAT GGT AGA TAT GTA AGA TAC CGT CAA GTG AGA A | 328 |
| cps2J-s cps2J-as | GTT GAG TCC TTA TAC ACC TGT T CAG AAA ATT CAT ATT GTC CAC C | 459 | |
| mrp-Fw mrp-Re | GGT ATA CCT TGC TGG TAC CGT TC AGT CTC TAC AGC TGTAGC TGG | 532 | |
| B | sly-Fw sly-Re | AGT TCG CAC TTG ATT TTA AG AAT ACA TTG CCA GAT TAC TC | 1500 |
| gapdh-Fw gapdh-Re | GGC GCC GAA TTC GTC GAC ATT ATT TAG CAA TTT TTG CG CGC CGC GGA TCC GTA GTT AAA GTT GGT ATT AAC | 1100 | |
| C | ef-Fw ef-Re | ACAAAGGCGTAG GTTCAATC CGG CAT CAA GAA TGT CTT TG | 269 |
| tuf-Fw tuf-Re | GTA CAG TTG CTT CAG GAC GTA TC ACG TTC GAT TTC ATC ACG TTG | 205 | |
| CI6-s CI7-as | GTT GAG TCC TTA TAC ACC TGT TAC TCA GTG CCG CAG CTA ACG CAT T CAG AAA ATT CAT ATT GTC CAC CCG ACT TCA CCC CAA TCA TCT ATC C | 620 |
Described multiplex PCR augmentation detection reagent is that solvent forms with distilled water by following component:
10 * PCR damping fluid content volume per-cent 10%
The deoxidation nucleoside triphosphate:
dATP 200μM，dCTP 200μM，dGTP 200μM，dTTP 200μM，dUTP 50μM
Each 50 μ M of every primer of Auele Specific Primer pipe 1 (100 *) content
Each 50 μ M of every primer of Auele Specific Primer pipe 2 (100 *) content
Each 50 μ M of every primer of Auele Specific Primer pipe 3 (100 *) content
Surplus is the sterilization pure water of no DNA enzyme.
(3) agarose gel electrophoresis reagent: mainly by compositions such as 5 * tbe buffer liquid, agarose, ethidium bromide (1000 *, content volume per-cent is 1 ‰) and bromjophenol blues (6 *, content volume per-cent is 16.67%).
(4) the DNA standard substance of 100bp gradient: TaKaRa 100bp DNA Ladder Marker
Each composition in the above-mentioned PCR detection reagent is hybridly prepared into sufficient amount by proportioning, is used for the pcr amplification detection reagent of using when following multiplex PCR increases.
Use the mentioned reagent box, it is as follows to detect step:
(1) sample DNA extracts
Take a certain amount of sample of patient to be measured through pre-treatment, extract DNA according to a conventional method:
Sample is a blood, and (pharyngeal secretory product) is wiped away in cerebrospinal fluid or pharynx: get proteolytic enzyme 40 μ l and manage in 1.5ml, add the 0.4ml sample, mixing, add lysate again, whirlpool mixing 15 seconds, 56 ℃ of water-baths 10 minutes, instantaneous centrifugal, add 400 μ l dehydrated alcohols, whirlpool mixing 15 seconds, instantaneous centrifugal, this mixed solution is carefully moved into the Filter column of 2ml, centrifugal 1 minute of 6000g abandons filtrate, carefully adds 500 μ l washing lotions 1 in Filter column, centrifugal 1 minute of 6000g, abandon filtrate, carefully add 500 μ l washing lotions 2 again in Filter column, centrifugal 3 minutes of 20 000g, abandon filtrate, Filter column is moved into new 1.5ml pipe, add 100 μ l lysates or aseptic no DNA enzyme pure water, room temperature left standstill 5 minutes, centrifugal 1 minute of 6000g gets liquid and directly carries out the PCR operation or place-80 ℃ of preservations.
Sample is the culture dish bacterium colony: an amount of doubtful bacterium colony thalline of picking or bacterium liquid are in the 1.5ml of built-in physiological saline 500 μ l pipe, the thorough mixing of whirlpool, centrifugal 3 minutes of 8000rpm, abandon supernatant, physiological saline 1000 μ l ditto wash 2 times again, add sterile pure water 500 μ l, 100 ℃ of water-baths 10 minutes, centrifugal 3 minutes of 8000rpm carefully draws supernatant and directly carries out the PCR operation or place-80 ℃ of preservations.
(2) multiplex PCR amplification
Get 10 * PCR damping fluid, 2.5 μ l, deoxidation nucleoside triphosphate (dNTP) mixture (each 200mM) 2 μ l, rTaq archaeal dna polymerase 0.25 μ l and each 0.25 μ l of grouping specificity amplification primer (primer of same group can be added in the same reaction tubes), adding no DNA enzyme sterilization pure water to 23 μ l mixes, prepare 3 parts of PCR thin-walled reaction tubess that above-mentioned mixed solution is housed simultaneously, the a dna solution 2 μ l that add step (1) preparation, 1 part adds no DNA enzyme sterilization pure water 2 μ l as negative control, 1 part adds the SS2DNA template as positive control in addition, mixing, instantaneous centrifugal, reaction tubes and control tube place EppendorfH-116 type grads PCR instrument to carry out pcr amplification immediately.Cycling condition is provided with: 94 ℃ * 5min; By 94 ℃ * 60sec, 58 ℃ * 60sec, 72 ℃ * 90sec, circulate 30 times again; Last 72 ℃ * 5min finishes amplified reaction.
(3) amplified production is measured
Get pcr amplification product 5 μ l respectively,, add in 2% sepharose (the including ethidium bromide) well for preparing in advance with 1 μ l bromjophenol blue solution mixing; The DNA standard substance that other gets 4 μ l 100bp gradients are reference dna segment size (bp), ditto add the sepharose well.Sepharose places the horizontal strip electrophoresis groove of the MiniRun GE-100 type gel-electrophoretic apparatus that includes 0.5 * tbe buffer liquid, the application of sample nose end is towards negative pole, voltage 100V, electrophoresis 30～40min, to the dyestuff forward position during approximately apart from sepharose edge 1cm, deenergization takes out gel, reading of data, imaging and saving result under full-automatic ultraviolet of FR-200A and visible analyser.Reading of data, imaging and saving result under the gel imaging instrument.
(4) interpretation of result
According to electrophoresis result,, occur being judged to of band in purpose segment size (bp is referring to table 1) position and increase positive with reference to the DNA standard substance position of 100bp gradient.
Clinical application: (sample collector and experimenter separate to adopt double-blind method, experimenter's unknown sample clinical settings), use the mentioned reagent box, the utilization aforesaid method respectively to 9 the example clinical doubtful SS2 meningitis cases cerebrospinal fluid (FCS) or blood or culture (thalline or enrichment liquid), the general contactee's's (eating at the same table) of 4 examples pharynx is wiped away and is carried out SS2 and relevant virulence gene detection, the result has 5 examples to detect SS2 kind opposite sex gene (16S rRNA) in clinical suspected case, and laboratory diagnosis is that SS2 infects.5 virulence genes of 4 examples (cps2J, mrp, sly, gapdh, ef) total positives wherein, 1 routine protein clostridium gene (cps) feminine gender.In general contactee, all do not detect SS2 specific gene (referring to table 2).
Table 2:SS2 and relevant virulence gene detect the clinical application result
| Goal gene | Doubtful SS2 meningitis case | General contactee | |||||||||||
| 1 FCS | 2 thalline | 3 thalline | 4 bacterium liquid | 5 blood | 6 FCS | 7 blood | 8 thalline | 9 blood | 1 pharynx is wiped away | 2 pharynxs are wiped away | 3 pharynxs are wiped away | 4 pharynxs are wiped away | |
| 16S | + | + | + | + | + | + | - | + | - | - | - | - | - |
| cps2J | + | + | + | - | + | + | - | + | - | - | - | - | - |
| mrp | + | + | + | + | + | + | - | + | - | - | - | - | - |
| sly | + | + | + | + | + | + | - | + | - | - | - | - | - |
| gapdh | + | + | + | + | + | + | - | + | - | - | - | - | - |
| ef | + | + | + | + | + | + | - | + | - | - | - | - | - |
| tuf | + | + | + | + | + | + | - | + | - | - | - | - | - |
| CI | + | + | + | + | + | + | - | + | - | + | + | + | + |
Wherein 4 routine isolated strains are scraped and are got lawn and prepare bacterium liquid, identify with API rapid ID32 STREP V2.0 type automatic bacterial assessing instrument (Biom é rieux, France), the results are shown in Table 3.
Table 3: full-automatic biochemical Bacteria Identification result
| Strain number | Biochemical spectrum | SS2 ID value (%) | The SS2T value | SS2 result judges |
| SS205001 | 33072561421 | 99.9 | 0.68 | Very good qualification result |
| SS205002 | 33072561110 | 99.9 | 0.93 | Fabulous qualification result |
| SS205003 | 33072561110 | 99.9 | 1.00 | Fabulous qualification result |
| SS205004 | 33072561110 | 99.9 | 1.00 | Fabulous qualification result |
Automatic bacterial biochemical identification results suggest meets 2 type swine streptococcus.
Embodiment 2:
Relevant virulence gene detection kit, component that comprises such as embodiment 1, also comprise: SS2 DNA contrast template (DNA 〉=100ng/ μ L), the source: streptococcus suis 2-type (Streptococcus suis serotype 2) Denmark is with reference to strain (professor Fang Weihuan of Zhejiang University is so kind as to give), and ordinary method extracting DNA obtains.
The test of PCR specificity: the positive adopts streptococcus suis 2-type (Streptococcus suisserotype 2) Denmark with reference to strain (being the used SS2 DNA of this preferred SS2 virulence gene detection kit contrast template) with reference to strain; Feminine gender adopts with reference to strain: A) streptococcus: streptococcus pneumoniae (Streptococcus pneumoniae, S.pne), and beta hemolytic streptococcus (β-hemolytic streptococcus, S. β-hemo), Streptococcus viridans (Streptococcus viridans, S.vi); B) other Pseudomonas: Neisseria meningitidis (Neisseriameningitides, N.m), staphylococcus epidermidis (Staphylococcus epidermidis, S.epi) and vibrio cholerae (Vibrio cholerae, V.cho) etc., aforementioned bacterium source: separate and preservation by Zhejiang Center For Disease Control and Prevention culture presevation chamber.(sampling personnel, experimenter and interpretation of result personnel are separately to adopt 3 blind methods, the practical significance of experimenter and interpretation of result personnel unknown sample numbering, the actually operating that the unknown experimenter of interpretation of result personnel carries out sample), use the mentioned reagent box, carry out pcr amplification by the preferred method of the present invention, check PCR specificity.The result shows that the negative control bacterial strain of setting up there is no the specific band that amplifies SS2; Beta hemolytic streptococcus S. β-hemo, Streptococcus viridans S.vi and streptococcus pneumoniae S.pne respective strap Neisseria meningitidis N.m, staphylococcus epidermidis S.epi occur on the pulsating 200bp of streptococcus specific gene tuf position and any band does not then appear in vibrio cholerae V.cho.Illustrate that detection specificity is strong; All experimental strains and negative control bacterial strain all can amplify the internal reference band, illustrate that the PCR reaction system is reasonable, restraining effect (referring to Fig. 1～3) do not occur.
Embodiment 3～7:
Embodiment 3～7th, adopt the viable count method that the SS2 positive is carried out enumeration with reference to strain: the bacteria suspension of getting 0.5 Maxwell unit, do a series of 10 times of dilutions, then quantitative diluent is carried out flat board and cultivate,, calculate the viable count in the culture according to the colony number of turning out.Be diluted to 1 bacterium from 1,000 ten thousand continuous 10 times, use the mentioned reagent box, carry out pcr amplification, to detect the detectivity of used kit sample by the preferred method of the present invention.The result shows that when 8 target gene fragment all specific band occurs its PCR susceptibility is the 78CFU/ reaction.When dna profiling was lower than 7.8 CFU, some band was not obvious (referring to table 4, Fig. 1).
Table 4:PCR sensitivity test
*
| Goal gene (bp) | Bacteria suspension extent of dilution and contained CFU | ||||
| 1∶10 -3(780) | 1∶10 -4(78) | 1∶10 -5(7.8) | 1∶10 -6(0.78) | 1∶10 -7(0.078) | |
| 16S rRNA(328) | + | + | - | - | - |
| cps-2J(450) | + | + | + | + | - |
| mrp(532) | + | + | - | - | - |
| The result judges | + | + | - | - | - |
*Annotate: bacterial strain adopts the SS2 positive with reference to strain
Embodiment 8:
The epidemiology field monitoring is used: (spot sampling personnel and experimenter separate to adopt double-blind method, the epidemiology background of experimenter's unknown sample), Application Example 1 described test kit, the 100 routine specific professional populations pharynxs of using aforesaid method respectively the SS2 monitoring point to be gathered are wiped away and are directly carried out SS2 and relevant virulence gene detection, and with isolated culture relatively, the result shows that all 100 routine specific professional populations pharynxs are wiped away and directly carries out SS2 and relevant virulence gene and detect all negatively that and isolated culture detects and also all do not isolate SS2.
Sequence table .ST25
SEQUENCE LISTING
＜110〉Zhejiang Center For Disease Control and Prevention
＜120〉2 type pig streptococcus virulence gene pcr amplification detection kit and detection methods
<130>
<160>16
<170>PatentIn version 3.2
<210>1
<211>24
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
cagtatttac cgcatggtag atat 24
<210>2
<211>22
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
gtaagatacc gtcaagtgag aa 22
<210>3
<211>22
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>3
gttgagtcct tatacacctg tt 22
<210>4
<211>22
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>4
cagaaaattc atattgtcca cc 22
<210>5
<211>23
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>5
ggtatacctt gctggtaccg ttc 23
<210>6
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>6
agtctctaca gctgtagctg g 21
<210>7
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>7
agttcgcact tgattttaag 20
<210>8
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>8
aatacattgc cagattactc 20
<210>9
<211>38
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>9
ggcgccgaat tcgtcgacat tatttagcaa tttttgcg 38
<210>10
<211>33
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>10
cgccgcggat ccgtagttaa agttggtatt aac 33
<210>11
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>11
acaaaggcgt aggttcaatc 20
<210>12
<211>20
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>12
cggcatcaag aatgtcttt g 20
<210>13
<211>23
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>13
gtacagttgc ttcaggacgt atc 23
<210>14
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>14
acgttcgatt tcatcacgtt g 21
<210>15
<211>46
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>15
gttgagtcct tatacacctg ttactcagtg ccgcagctaa cgcatt 46
<210>16
<211>46
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>16
cagaaaattc atattgtcca cccgacttca ccccaatcat ctatcc 46
Claims (10)
1. type swine streptococcus (Streptococcus suis Serotype 2, SS2) virulence gene pcr amplification detection kit, comprise SS2 DNA contrast template, pcr amplification detection reagent, agarose gel electrophoresis reagent and dna ladder degree standard substance, described pcr amplification detection reagent mainly comprises PCR damping fluid, specificity amplification primer, deoxidation nucleoside triphosphate mixture, archaeal dna polymerase and sterilization pure water; It is characterized in that: described specificity amplification primer is:
16S-195(s)：5’-CAG TAT TTA CCG CAT GGT AGA TAT-3’
16S-489(as2)：5’-GTA AGA TAC CGT CAA GTG AGA A-3’
cps2J-s：5’-GTT GAG TCC TTA TAC ACC TGT T-3’
cps2J-as：5’-CAG AAA ATT CAT ATT GTC CAC C-3’
mrp-Fw：5’-GGT ATA CCT TGC TGG TAC CGT TC-3’
mrp-Re：5’-AGT CTC TAC AGC TGT AGC TGG-3’
sly-Fw：5’-AGT TCG CAC TTG ATT TTA AG-3’
sly-Re：5’-AAT ACA TTG CCA GAT TAC TC-3’
gapdh-Fw：5’-GGC GCC GAA TTC GTC GAC ATT ATT TAG CAA TTT
TTG CG-3’
gapdh-Re：5’-CGC CGC GGA TCC GTA GTT AAA GTT GGT ATT
AAC-3’
ef-Fw：5’-ACA AAG GCG TAG GTT CAA TC-3’
ef-Re：5’-CGG CAT CAA GAA TGT CTT TG-3’
tuf-Fw：5’-GTA CAG TTG CTT CAG GAC GTA TC-3’
tuf-Re：5’-ACG TTC GAT TTC ATC ACG TTG-3’
CI6-s：5’-GTT GAG TCC TTA TAC ACC TGT TAC TCA GTG CCG
CAG CTA ACG CAT T-3’
CI7-as：5’-CAG AAA ATT CAT ATT GTC CAC CCG ACT TCA CCC
CAA TCA TCT ATC C-3’。
2. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that described pcr amplification detection reagent main component is as follows:
PCR damping fluid final concentration is 1 *
Each 10～80 μ M of specificity amplification primer
Deoxidation nucleoside triphosphate mixture 200～1000 μ M
Archaeal dna polymerase 0.5～5 enzyme activity unit/reaction
Surplus is the sterilization pure water.
3. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that described PCR damping fluid in each component final concentration in pcr amplification reagent, and are composed as follows:
Repone K 500mM
Tris-Cl 100mM
Magnesium chloride 25mM
Polyethylene glycol 6000 0.1%
1,4-DTT 0.1%
Bovine serum albumin 1%.
4. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that described agarose gel electrophoresis reagent main component is as follows:
Tbe buffer liquid final concentration is 5 *
The agarose volume percent is 1 ‰
The ethidium bromide volume percent is 1 ‰
The bromjophenol blue volume percent is 16.67%.
5. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1, it is characterized in that also containing the DNA extraction agent in the described pcr amplification detection kit, described DNA extraction agent is one of following: 1. Qiagen DNA extraction agent, 2. Promega DNA extraction agent, 3. TaKaRa DNA extraction agent.
6. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that described archaeal dna polymerase is one of following: 1. Ampli Taq archaeal dna polymerase, 2. rTaqDNA polysaccharase, 3. Platinum Tag archaeal dna polymerase.
7. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that described deoxidation nucleoside triphosphate mixture is one of following:
1. dATP, dCTP, dGTP, the ratio of dTTP amount of substance 1: 1: 1: 1 mixture
2. dATP, dGTP, dCTP, the ratio of dUTP amount of substance 1: 1: 1: 1 mixture
3. dATP, dCTP, dGTP, dTTP, the ratio of dUTP amount of substance 4: 4: 4: 4: 1 mixture.
8. 2 type pig streptococcus virulence gene pcr amplification detection kit as claimed in claim 1 is characterized in that the each component final concentration is as follows in the described pcr amplification detection reagent:
The PCR damping fluid:
Repone K 500mM
Tris-Cl 100mM
Magnesium chloride 25mM
Polyethylene glycol 6000 0.1%
1,4-DTT 0.1%
Bovine serum albumin 1%
8 couples of each 50 μ M of specificity amplification primer
Deoxidation nucleoside triphosphate mixture:
dATP 200μM，dCTP 200μM，dGTP 200μM，dTTP 200μM，dUTP 50μM
0.5 enzyme activity unit/the reaction of rTaq archaeal dna polymerase
Surplus is the sterilization pure water.
9. type pig streptococcus virulence gene pcr amplification detection method, described detection method are with the positive contrast of SS2DNA contrast template, and adopting the DNA of sample to be tested is analyzing samples, and described method steps is as follows:
(1) sample DNA extracts;
(2) pcr amplification: get the pcr amplification detection reagent, the DNA that adds SS2 DNA contrast template and analyzing samples carries out amplified reaction respectively;
(3) amplified production is measured: get amplified production, and behind sepharose, reading of data, imaging and saving result under the gel imaging instrument;
(4) interpretation of result:, the positive that is judged as of band occurs in purpose clip size position with reference to gradient dna ladder degree standard substance image;
Described 2 type swine streptococcus (Streptococcus suis Serotype 2, SS2) virulence gene pcr amplification detection kit, comprise SS2 DNA contrast template, pcr amplification detection reagent, agarose gel electrophoresis reagent and 100bp gradient DNA standard substance, described pcr amplification detection reagent mainly comprises PCR damping fluid, specificity amplification primer, deoxidation nucleoside triphosphate mixture, archaeal dna polymerase, sterilization pure water;
Described specificity amplification primer and purpose clip size are:
16S-195(s)：5’-CAG TAT TTA CCG CAT GGT AGA TAT-3’328bp
16S-489(as2)：5’-GTA AGA TAC CGT CAA GTG AGA A-3’
cps2J-s：5’-GTT GAG TCC TTA TAC ACC TGT T-3’ 459bp
cps2J-as：5’-CAG AAA ATT CAT ATT GTC CAC C-3’
mrp-Fw：5’-GGT ATA CCT TGC TGG TAC CGT TC-3’ 532bp
mrp-Re：5’-AGT CTC TAC AGC TGT AGC TGG-3’
sly-Fw：5’-AGT TCG CAC TTG ATT TTA AG-3’ 1500bp
sly-Re：5’-AAT ACA TTG CCA GAT TAC TC-3’
gapdh-Fw：5’-GGC GCC GAA TTC GTC GAC ATT ATT TAG CAA TTT
TTG CG-3’ 1100bp
gapdh-Re：5’-CGC CGC GGA TCC GTA GTT AAA GTT GGT ATT
AAC-3’
ef-Fw：5’-ACA AAG GCG TAG GTT CAA TC-3’ 269bp
ef-Re：5’-CGG CAT CAA GAA TGT CTT TG-3’
tuf-Fw：5’-GTA CAG TTG CTT CAG GAC GTA TC-3’ 205bp
tuf-Re：5’-ACG TTC GAT TTC ATC ACG TTG-3’
CI6-s：5’-GTT GAG TCC TTA TAC ACC TGT TAC TCA GTG CCG
CAG CTA ACG CAT T-3’ 620bp
CI7-as：5’-CAG AAA ATT CAT ATT GTC CAC CCG ACT TCA CCC
CAA TCA TCT ATC C-3’。
10. method as claimed in claim 9 is characterized in that: described step (2) PCR cycling condition is set to: 94 ℃ * 5min; By 94 ℃ * 60sec, 58 ℃ * 60sec, 72 ℃ * 90sec, circulate 30 times again; Last 72 ℃ * 5min finishes amplification.
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| CNA2007100674875A CN101020929A (en) | 2007-03-14 | 2007-03-14 | Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene |
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|---|---|---|---|
| CNA2007100674875A CN101020929A (en) | 2007-03-14 | 2007-03-14 | Kit and process for PCR amplification detecting type 2 pig streptococcus virulence gene |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101812518A (en) * | 2010-03-08 | 2010-08-25 | 上海交通大学 | Multiple PCR detection kit for virulence factors of streptococcus suis and detection method thereof |
| CN101440400B (en) * | 2008-12-04 | 2011-04-06 | 浙江省疾病预防控制中心 | Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene |
| CN105063172A (en) * | 2015-07-30 | 2015-11-18 | 山东省滨州畜牧兽医研究院 | Method for rapidly screening and identifying streptococcus suis |
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| CN101440400B (en) * | 2008-12-04 | 2011-04-06 | 浙江省疾病预防控制中心 | Fluorescent detection kit and method for Streptococcus suis 2 type nucleic acid containing 89K pathogenicity island gene |
| CN101812518A (en) * | 2010-03-08 | 2010-08-25 | 上海交通大学 | Multiple PCR detection kit for virulence factors of streptococcus suis and detection method thereof |
| CN105063172A (en) * | 2015-07-30 | 2015-11-18 | 山东省滨州畜牧兽医研究院 | Method for rapidly screening and identifying streptococcus suis |
| CN105063172B (en) * | 2015-07-30 | 2018-01-26 | 山东省滨州畜牧兽医研究院 | A kind of method of quick Screening and Identification Streptococcus suis |
| CN105648101A (en) * | 2016-03-28 | 2016-06-08 | 上海市动物疫病预防控制中心 | Liquid-phase chip kit for screening seven virulence genes of streptococcus suis |
| CN105779625A (en) * | 2016-04-21 | 2016-07-20 | 韶关学院 | Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis |
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| CN111534611A (en) * | 2019-11-12 | 2020-08-14 | 广州微芯生物科技有限公司 | Fluorescent quantitative PCR method for detecting toxigenic streptococcus suis and corresponding kit |
| CN113151309A (en) * | 2021-04-01 | 2021-07-23 | 华中农业大学 | Human high-risk zoonosis type streptococcus suis specific sequence and application |
| CN113151309B (en) * | 2021-04-01 | 2023-10-03 | 华中农业大学 | Streptococcus suis specific sequence with high risk of human beings and livestock and application thereof |
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