CN102220426A - PCR (polymerase chain reaction) detection kit for hacmophilus parasuis - Google Patents
PCR (polymerase chain reaction) detection kit for hacmophilus parasuis Download PDFInfo
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Abstract
The invention discloses a PCR detection kit for hacmophilus parasuis. The kit comprises 8-12.5 microliters of PCR premix buffer, 25 microliters of ultrapure water, 2-3 microliters of Marker DL2000, 0.75-2.0 microliters of two primers with a concentration of 12pmol per microliter, 2 microliters of negative control and 2 microliters of positive control. In the invention, the conventional PCR method is employed to replace a biochemical test and a serological surveillance which are labor and time consuming, and the PCR detection kit for detecting hacmophilus parasuis is developed. Able to detect hacmophilus parasuis, the kit is applicable to detection, diagnosis and epidemiological survey of hacmophilus parasuis. Errors caused by human judgments can be avoided, and harms on pigs are also reduced. With the kit, hacmophilus parasuis can be detected within 24 hours, which greatly shortens the detection time of hacmophilus parasuis. Thus, the kit of the invention is characterized by speediness, high sensitivity, strong specificity and the like.
Description
Technical field
The present invention relates to a kind of test kit, especially a kind of haemophilus parasuis PCR detection kit.
Background technology
(Haemophilus parasuis is a kind of Gram-negative, non-hemolytic, NAD dependency bacterium HPS) to haemophilus parasuis, and 15 serotypes are arranged.HPS can cause fiber disposition polyserositis, polyarthritis, pleuritis and the meningitis of pig.This bacterium just has been considered to the pathogenic agent of pig Glasser disease since 1910.HPS is present in the upper respiratory tract of health pig, mainly infects piggy clinically, the weanling pig in 4 ages in age～6 weeks week particularly, and Clinical symptoms is an acute sepsis, often 2 d death after infection.This disease can cause great financial loss to pig industry equally.There is the health of serious harm swinery in the swinery of haemophilus parasuis disease some bases of raising pigs, self-employed pig raiser and peasant household in China.Now market lacks and a kind ofly can carry out easy, quick, sensitive, the test kit of detection accurately to the haemophilus parasuis of clinical sample.
Summary of the invention
The objective of the invention is: a kind of haemophilus parasuis PCR detection kit is provided, and it can the rapid detection haemophilus parasuis, and easy, sensitive, accurate, high specificity.
The present invention is achieved in that haemophilus parasuis PCR detection kit, it comprises the PCR premix damping fluid of 8～12.5 μ L, 25 μ L ultrapure waters, the Marker DL2000 of 2～3 μ L, 0.75～2.0 μ L concentration are that last primer and 0.75～2.0 μ L concentration of 12pmol/ μ L is the following primer of 12pmol/ μ L, 25 μ L concentration are the Proteinase K of 20mg/ml, and 20～50 μ L mass concentrations are 10% SDS, TE damping fluid, 2 μ L negative controls and 2 μ L positive controls.
PCR premix damping fluid is by the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul, the Tris-HCl of 25mmol/ μ L and 0.2 μ L Taq enzyme are formed, and its pH value is pH8.3.
The sequence number of upstream primer is LPF 5 '-GTGATGAGGAAGGGTGGTGT-3 ' or LPF 5 '-GCTAACTCCGTGCCAGCAG-3 ', and the sequence number of downstream primer is LPR 5 '-GGCTTCGTCACCCTCTGT-3 ' or LPR 5 '-GTCATCATGGCCCTTACGAG-3 '.
Negative control is the dna segment of non-haemophilus parasuis.
Positive control is the PCR product of standard haemophilus parasuis.
The TE damping fluid comprises the mixing solutions that Tris-HCL10mM and EDTA1mM form, and the pH value of mixing solutions is 7.0～8.0.
The foundation of haemophilus parasuis PCR diagnostic method
1, materials and methods
1.1 material
1.1.1 bacterial strain
Bacillus coli, Salmonellas, actinobacillus pleuropneumoniae, Pseudomonas aeruginosa, suis separates preservation with staphylococcus etc. by this laboratory.
1.1.2 enzyme and reagent
TaqDNA polysaccharase, dNTP (mix), Marker2000 are available from Huamei Bio-Engrg Co., and permanent in biotech firm, and ProteinaseK, RNaseA are available from Shanghai China Shun biotechnology company limited. and common agents such as sodium acetate, glacial acetic acid, ethanol are homemade analytical pure.Calf serum, horse serum etc. are bought because of biotech firm by perseverance.
1.1.2 substratum
Contain 0.005%VB
1The factor, 0.0025% NADH and 5% horse serum plain agar are dull and stereotyped or with like factor Chocolate Agar nutrition flat board (promptly improveing the Chocolate Agar nutritional medium); The TSA substratum, the TSB liquid nutrient medium.
1.2 method
1.2.1 direct microscopy
Get liver, spleen contact, gramstaining.Haemophilus parasuis belongs to the Gram-negative bacillus pumilis, and form is changeable.
1.2.2 the cultivation of bacterial strain
Cultivate and use nutrient agar medium, add the NAD of 50 mL/L calf serums and 30 mg/l～50 mg/L, preserve at 4 ℃ the back about 37 ℃ of cultivation 24 h.Or the HPS strain isolated is inoculated in (improvement Chocolate Agar nutrition base) on the Chocolate Agar nutrition flat board that contains the V factor and NAD, cultivate 18～24h in the thermostat container.
1.2.3 the extraction of DNA
Method one:
A little bacterium colony of picking adds the TE damping fluid and is diluted to little turbid in the 1.5ml centrifuge tube, get 1ml centrifugal 10 seconds of the 9000r/min that packs in the centrifuge tube, abandon supernatant, in the bacterial precipitation pipe, add 20ulDB liquid, 160ullysozyme and 20ulRNaseA, thoroughly vibration suspends, and 37 ℃ of temperature were bathed 45 minutes, during put upside down centrifuge tube back and forth for several times.Add 400ulDL and 25ul proteopepsis enzyme K(ProteinaseK) leniently put upside down the thorough mixing of centrifuge tube rapidly back and forth.Put 65 ℃ of temperature and bathed 45 minutes, centrifugal 1 minute, get supernatant liquor.Add the 200ul Virahol, after acutely putting upside down centrifuge tube solution being mixed, pipette 600ul to adsorption column, centrifugal 30 seconds.Discard the liquid in the collection tube, adsorption column is put into same collection tube.All move in the adsorption column centrifugal 30 seconds with remaining.Discard the liquid in the collection tube, adsorption column is put into same collection tube.Add 500ulW1 liquid, leave standstill one minute after, centrifugal 30 seconds.Adsorption column is put into the another one collection tube.Add 500ulW1 liquid, centrifugal 15 seconds.Discard the liquid in the collection tube, again adsorption column is put into same collection tube.Centrifugal 1 minute.Adsorption column is moved in the clean 1.5ml centrifuge tube.Add 100 ulT1 liquid in adsorption film central authorities, 65 ℃ leave standstill 5 minutes after, centrifugal 1 minute.4 ℃ of preservations of 1.5ml centrifuge tube (DNA) are standby.
Method two:
Several bacterium colonies of picking are suspended in the small test tube that contains 100 μ L ultrapure waters, the concussion mixing heats 5 min or boils 6 min with 95 ℃ in PCR instrument and is placed on cooled on ice 2 min, centrifugal 1 min of 12 000 r/min, get the rough dna profiling of supernatant as PCR reaction, it is standby to put-20 ℃ of preservations.
1.2.4 the PCR selection of primers, design is with synthetic
The Hps 16 S rRNA gene orders of announcing according to GenBank design two pairs of Auele Specific Primers, or search the single PCR method of abroad having set up at pathogenic bacteria, selection specificity susceptibility is good, specimen preparation simple, pcr amplification product is easy to distinguish, the PCR condition is close, the clinical data reference is arranged, adopt the primer sequence of wherein having delivered, send by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic.Upstream primer 1:LPF 5 '-GTGATGAGGAAGGGTGGTGT-3 '; Downstream primer 1:LPR 5 '-GGCTTCGTCACCCTCTGT-3 ', the expection expanding fragment length is 822 bp.The HPS amplification length is 822 bp.
The a pair of primer that designs voluntarily by Haemophilus parasuis bacterial strain Bakos B26 16S ribosomal RNA gene (AY362910) is: upstream primer 2:LPF 5 '-GCTAACTCCGTGCCAGCAG-3 ', downstream primer 2:LPR 5 '-GTCATCATGGCCCTTACGAG-3 ', the expection expanding fragment length is 650 bp.And analyze relatively each parameter and the homology of primer with DNAstar software and BLAST.
1.2.5 pcr amplification
PCR reaction conditions after the optimization is as follows: adopt 25 μ L reaction systems.Be PCR premix damping fluid 8～12.5 μ L(MgCl
2Concentration is 3 mmol/ul, 500pmol/ul dNTP 2.0 μ L, 25 mmol/ μ L Tris-HCl and Taq enzyme 0.2 μ L, the pH value is 8.3), concentration is each 0.75～2.0 μ L of primer 2 bar of 12pmol/ μ L, and bacterium liquid template is 0.5～2.0 μ L, ddH
2The O(ultrapure water) adds to 25.0 μ L.Carry out PCR reaction by following parameter: 95 ℃ of pre-sex change 5 min, 94 ℃ of sex change 30s then, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations, last 72 ℃ are extended 10 min eventually.Product is standby in-20 ℃ of preservations.
1.2.6 the detection of PCR product
Get 2～5 μ L amplified productions and add point sample Confucius, with 100 V voltages, 2% sepharose carries out electrophoresis on (contain 1% ethidium bromide, 2 μ L, then change Goldview into).Electrophoresis liquid is with 1 * TAE damping fluid.Under ultraviolet lamp, observe amplification after 1 hour, and Polaroid taking pictures.
1.2.7 the application of PCR detection method
Adopt ordinary method to collect pathological material of disease, cause of disease is carried out separation and Culture, use the PCR method of having set up again 350 part cases in the morbidity swinery are detected.
2 experiments
2.1 the optimum combination of haemophilus parasuis PCR detection method, as shown in Figure 1.
2.2 the sensitivity test of PCR method, as shown in Figure 2.
2.3 the PCR specific detection, as shown in Figure 3.
2.4 the PCR detection method is to the detection of the local strain in haemophilus parasuis Guizhou, as shown in Figure 4.
3 results
3.1 bacterium that animal pseudomonas aeruginosa strains that this test is preserved with this laboratory and pig are common such as intestinal bacteria, porcine contagious pleuropneumonia, swine streptococcus, swine red cell disease pathogen etc. are PCR for template, only the wealthy band that increases of specific DNA appears in haemophilus parasuis, has proved the specificity of this method.By product is carried out purifying, conversion, clone, gene sequencing, further prove the PCR specificity.By the extraction of DNA, ultraviolet spectrophotometer is measured, and the average OD260 value of breadboard standard DNA template is 0.01022nm, with dna profiling content is after 0.05111 μ g/ μ L dilutes by 10 times of dilution methods, to get 2 μ L and increase, and then haemophilus parasuis DNA concentration is followed successively by 103.2ng, 10.32ng, 1.032ng, 103.2pg, 10.32pg, 1.032pg, 103.2fg, 10.32fg, 1.032fg the haemophilus parasuis dna profiling minimum content that can detect is 1.032pg.The sensitivity of mycetocyte is about 200.The PCR annealing temperature is 49～60 ℃; Template DNA concentration: 0.75～2.0 μ L.The template DNA that this method is used, the DNA standard model is relative with product less with class methods with MasterMix using dosage ratio.This method is for the diagnosis of haemophilus parasuis disease, has special, responsive, outstanding advantage such as recall rate is high, quick, easy, easy automatization.
3.2 observing according to medical history, clinical symptom and characteristic pathology, on the basis of pathological material of disease collection, bacterium separation and Culture, biochemical identification (micro-culture tube is provided because of biology by perseverance), indirect hemagglutination test, set up PCR Lab detection method at Guizhou Province's haemophilus parasuis, and adopting this method that morbidity swinery 350 parts are suspected to be that case carries out PCR and detect, the PCR recall rate is 8%-49%.Carry out indirect hemagglutination inhibition test and PCR method simultaneous test at representative serum and 90 parts of pathological material of disease of Guiyang Nanming District three pig farms and the collection of Ben Sheng Wengan County simultaneously, the result shows: the indirect hemagglutination inhibition test positive rate is 30.5%.The PCR method recall rate is 36%.PCR method not only has the automatization characteristics, and the error of avoiding the artificial judgement of serological method to bring more can reduce the injury that pig is caused if adopt the nasopharynx cotton to wipe away paper collection pathological material of disease.
3.3 being it, the advantage of this PCR can carry out differential diagnosis to the similar disease of several epidemiological features, symptom or diseased organ, to improve checkability, reduction expense.Show by test: the laboratory PCR method has been substituted time-consuming biochemical test and serology monitoring, just can detect haemophilus parasuis in 24 hours, has shortened the detection time of haemophilus parasuis.
Owing to adopted technique scheme, the present invention is optimized the PCR reaction conditions, adopt conventional PCR method to replace time-consuming biochemical test and serology monitoring, developed be used to detect haemophilus parasuis the PCR test kit, this test kit can detect haemophilus parasuis, be applicable to the detection of haemophilus parasuis, diagnosis and epidemiology survey, can avoid the artificial error of bringing of judging, more can reduce the injury that pig is caused, in 24 hours, just detect haemophilus parasuis, shortened the detection time of haemophilus parasuis, had fast, the susceptibility height, characteristics such as high specificity.Detected result of the present invention is accurate, and quick, sensitive, and detection mode is simple, and result of use is good.
Description of drawings
M:Marker DL2000; 1, PCR product (650 bp); 2, reference culture PCR product (650 bp); 3: negative control;
1:103.2ng gene segment PCR product; 2:10.32ng gene segment: PCR product; 3:1.032ng gene segment PCR product; 4:103.2 pg gene segment PCR product; 5:10.32pg gene segment PCR product;
M:Marker DL2000; 1: bacillus coli; 2: Salmonellas; 3: actinobacillus pleuropneumoniae; 4: suis; 5: staphylococcus; 6: the swine red cell disease; 7: the animal Pseudomonas aeruginosa; 8: pasteurellosis bacillus; 9: Leptospira; 10: sterile saline; 11:PCR product (650bP);
Accompanying drawing 4 is a haemophilus parasuis Guizhou local strain 16SrRNA gene segment PCR detected result
M:Marker DL2000; 1: strain isolated HPS1 PCR product (650 bp); 2: strain isolated HPS1 PCR product (650 bp); 3: strain isolated HPS2 PCR product; 4: strain isolated HPS3 PCR product; 5: strain isolated HPS4 PCR product; 6: negative sample; 7: reference culture PCR product (650 bp); 8: the sterilization distilled water.
Embodiment
Embodiments of the invention 1: haemophilus parasuis PCR detection kit, it comprises 10 μ LPCR premix damping fluids, PCR premix damping fluid is by the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul, the Tris-HCl of 25 mmol/ μ L and 0.2 μ L Taq enzyme are formed, and its pH value is pH8.3; 25 μ L ultrapure waters; The Marker DL2000 of 2 μ L; 1 μ L concentration is that last primer and the 1 μ L concentration of 12pmol/ μ L is the following primer of 12pmol/ μ L, the sequence number of upstream primer is LPF 5 '-GTGATGAGGAAGGGTGGTGT-3 ', and the sequence number of downstream primer is LPR 5 '-GGCTTCGTCACCCTCTGT-3 '; 25 μ L concentration are the Proteinase K of 20mg/ml, and 30 μ L mass concentrations are 10% SDS, and 500 μ L TE damping fluids, TE damping fluid comprise the mixing solutions that Tris-HCL10mM and EDTA1mM form, and the pH value of mixing solutions is 7.3; The dna segment that adopts the non-haemophilus parasuis of 2 μ L is as negative control; The PCR product that adopts 2 μ L standard haemophilus parasuises is as positive control.
Embodiments of the invention 2: haemophilus parasuis PCR detection kit, it comprises 8 μ LPCR premix damping fluids, PCR premix damping fluid is by the MgCl of 3mmol/ul
2, the dNTP2.0 μ L of 500pmol/ul, the Tris-HCl of 25 mmol/ μ L and 0.2 μ L Taq enzyme are formed, and its pH value is pH8.3; 25 μ L ultrapure waters; The Marker DL2000 of 3 μ L; 0.75 μ L concentration is that last primer and the 0.75 μ L concentration of 12pmol/ μ L is the following primer of 12pmol/ μ L, the sequence number of upstream primer is LPF 5 '-GCTAACTCCGTGCCAGCAG-3 ', and the sequence number of downstream primer is LPR 5 '-GTCATCATGGCCCTTACGAG-3 '; 25 μ L concentration are the Proteinase K of 20mg/ml, and 20 μ L mass concentrations are 10% SDS, and 500 μ L TE damping fluids, TE damping fluid comprise the mixing solutions that Tris-HCL10mM and EDTA1mM form, and the pH value of mixing solutions is 7.0; The dna segment that adopts the non-haemophilus parasuis of 2 μ L is as negative control; The PCR product that adopts 2 μ L standard haemophilus parasuises is as positive control.
Embodiments of the invention 3: haemophilus parasuis PCR detection kit, it comprises 12.5 μ LPCR premix damping fluids, PCR premix damping fluid is by the MgCl of 3mmol/ul
2, the dNTP2.0 μ L of 500pmol/ul, the Tris-HCl of 25mmol/ μ L and 0.2 μ L Taq enzyme are formed, and its pH value is pH8.3; 25 μ L ultrapure waters; The Marker DL2000 of 3 μ L; 2 μ L concentration are that last primer and the 2 μ L concentration of 12pmol/ μ L are the following primer of 12pmol/ μ L, the sequence number of upstream primer is LPF 5 '-GCTAACTCCGTGCCAGCAG-3 ', and the sequence number of downstream primer is LPR 5 '-GTCATCATGGCCCTTACGAG-3 '; 25 μ L concentration are the Proteinase K of 20mg/ml, and 50 μ L mass concentrations are 10% SDS, and 500 μ L TE damping fluids, TE damping fluid comprise the mixing solutions that Tris-HCL10mM and EDTA1mM form, and the pH value of mixing solutions is 8.0; The dna segment that adopts the non-haemophilus parasuis of 2 μ L is as negative control; The PCR product that adopts 2 μ L standard haemophilus parasuises is as positive control.
Claims (6)
1. haemophilus parasuis PCR detection kit, it is characterized in that: it comprises the PCR premix damping fluid of 8～12.5 μ L, 25 μ L ultrapure waters, the Marker DL2000 of 2～3 μ L, 0.75～2.0 μ L concentration are that last primer and 0.75～2.0 μ L concentration of 12pmol/ μ L is the following primer of 12pmol/ μ L, 25 μ L concentration are the Proteinase K of 20mg/ml, and 20～50 μ L mass concentrations are 10% SDS, the TE damping fluid of 500 μ L, 2 μ L negative controls and 2 μ L positive controls.
2. haemophilus parasuis PCR detection kit according to claim 1 is characterized in that: PCR premix damping fluid is by the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul, the Tris-HCl of 25mmol/ μ L and 0.2 μ L Taq enzyme are formed, and its pH value is pH8.3.
3. haemophilus parasuis PCR detection kit according to claim 1, it is characterized in that: the sequence number of going up primer is LPF 5 '-GTGATGAGGAAGGGTGGTGT-3 ' or LPF 5 '-GCTAACTCCGTGCCAGCAG-3 ', and the sequence number of following primer is LPR 5 '-GGCTTCGTCACCCTCTGT-3 ' or LPR 5 '-GTCATCATGGCCCTTACGAG-3 '.
4. haemophilus parasuis PCR detection kit according to claim 1 is characterized in that: negative control is the dna segment of non-haemophilus parasuis.
5. haemophilus parasuis PCR detection kit according to claim 1 is characterized in that: positive control is the PCR product of standard haemophilus parasuis.
6. haemophilus parasuis PCR detection kit according to claim 1 is characterized in that: the TE damping fluid comprises the mixing solutions that Tris-HCL10mM and EDTA1mM form, and the pH value of mixing solutions is 7.0～8.0.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776283A (en) * | 2012-07-05 | 2012-11-14 | 福建省农业科学院畜牧兽医研究所 | Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis |
| CN103789422A (en) * | 2014-01-16 | 2014-05-14 | 中国农业科学院哈尔滨兽医研究所 | Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis |
| CN104388340A (en) * | 2014-10-28 | 2015-03-04 | 中国动物卫生与流行病学中心 | Blood serum 5 type haemophilus parasuis and application thereof |
| CN104561283A (en) * | 2014-12-24 | 2015-04-29 | 广东海大畜牧兽医研究院有限公司 | Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis |
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| CN101724709A (en) * | 2010-01-28 | 2010-06-09 | 贵州省畜牧兽医研究所 | PCR diagnostic kit for porcine infectious pleuropneumonia |
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| CN101724709A (en) * | 2010-01-28 | 2010-06-09 | 贵州省畜牧兽医研究所 | PCR diagnostic kit for porcine infectious pleuropneumonia |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776283A (en) * | 2012-07-05 | 2012-11-14 | 福建省农业科学院畜牧兽医研究所 | Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis |
| CN103789422A (en) * | 2014-01-16 | 2014-05-14 | 中国农业科学院哈尔滨兽医研究所 | Primer group using vtaA9 gene as target spot and application of primer group to identification and diagnosis of haemophilus parasuis |
| CN103789422B (en) * | 2014-01-16 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | A kind of with vtaA9 gene be target spot primer sets and haemophilus parasuis differentiate and diagnosis in application |
| CN104388340A (en) * | 2014-10-28 | 2015-03-04 | 中国动物卫生与流行病学中心 | Blood serum 5 type haemophilus parasuis and application thereof |
| CN104561283A (en) * | 2014-12-24 | 2015-04-29 | 广东海大畜牧兽医研究院有限公司 | Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis |
| CN104561283B (en) * | 2014-12-24 | 2017-01-18 | 广东海大畜牧兽医研究院有限公司 | Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis |
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