CN106755523A - A kind of kit of Direct PCR detection Porcine contagious pleuropneumonia cause of disease and its application - Google Patents
A kind of kit of Direct PCR detection Porcine contagious pleuropneumonia cause of disease and its application Download PDFInfo
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 35
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 208000012353 Contagious Pleuropneumonia Diseases 0.000 title claims abstract description 21
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Abstract
Kit and its application of Porcine contagious pleuropneumonia cause of disease are detected the present invention relates to a kind of kit and its application, more particularly to a kind of Direct PCR, belongs to biological technical field.Kit of the invention includes following reagent：Lysate, PCR amplifing reagents, positive control and negative control.The kit can fast and accurately detect Porcine contagious pleuropneumonia cause of disease.Using when, can to Porcine contagious pleuropneumonia infection single sample detection, also can to the detection of mixed infection in clinical case, can be used for Porcine contagious pleuropneumonia cause of disease generaI investigation, Molecule Epidemiology Investigation and vaccine screening and monitor.
Description
Technical field
The present invention relates to a kind of kit and its application, more particularly to a kind of Direct PCR detection pig contagiousness pleura
The kit of pneumonia cause of disease and its application, belong to biological technical field.
Background technology
Pig contagious infection pleuropneumonia, also known as necrotizing pleuropneumonia, is drawn by Actinobacillus pleuropneumoniae (APP)
A kind of Acute respiratory infectious disease for rising, be with acute hemorrhagic fibrinous pneumonia and chronic fibro disposition gangrenosum acne pleurisy
Principal character.Acute person's case fatality rate is high, and chronic person was often resistant to.The pig at pig contagious infection pleuropneumonia various ages
Infection, generally the pig with 2-5 monthly ages, body weight as 30-60kg is multiple.Actinobacillus pleuropneumoniae is that have height host specific to pig
Property respiratory tract parasitic animal and plant, acute infection can not only be seen in lung pathologies change and blood, and also have big in nose liquid
Bacterium exists amount.Swinery scale is bigger, and initiation potential is also bigger.This disease has obvious seasonality, is sent out in the 4-5 months and the 9-11 months more
It is raw.Sick pig and the pig that carries disease germs are this sick major source of infection, have pathological change pig without clinical symptom, or become without pathology without clinical symptom
Change the feminine gender pig that carries disease germs more typical.Such as secondary or concurrent other diseases, clinical symptom is often caused to be aggravated and death rate rising.The disease is near
Occur in the trend for rising year by year in China over year, it has also become one of disease of harm pig industry most serious, give China's pig industry
Cause serious economic loss.In recent years, the pig contagious infection pleuropneumonia as caused by Actinobacillus has turned into influence
A kind of significant bacterial disease of pig industry development.Carrying out treatment using antibiotic in time after morbidity can significantly reduce animal dead
Rate, while being properly added antibiotic in process of production also can to a certain extent reduce the sick incidence.But with a large amount of
Antimicrobial particularly broad spectrum antibiotic widely using in pig industry, the drug resistance problems of bacteria medicine are dashed forward increasingly
Go out, at the same time also there is research data to show that the antibiotic of sub- inhibition concentration can influence the metabolic process of bacterium and change bacterium
Virulence or bacterium to adaptability of environment etc..The pig contagious infection pleuropneumonia reported at present has 15 serum
Type, each serotype has specificity, and its serotype specificity depends on blooming polysaccharide and thalline lipopolysaccharides.It is fast according to nicotinoyl amine gland
15 serotypes can be divided into two bions by the dependence of nicotinamide adenine dinucleotide.Biological I type is NADH
Rely on bacterial strain, including serotype 1-12 types and 15 types；The growth of biological II type is independent of NADH, but needs
Product is with assisting growth, including serotype 13,14 before wanting other specific purine or purine.The wherein type of serotype 1 and 5 can be divided into again
Two hypotypes of A and B, i.e. serotype 1A, 1B and 5A, 5B.Additionally, also having the Actinobacillus that some are separated to according to present classification
System can't divide its serotype.Pathogenicity has and does not exist cross protection between obvious difference, serotype between different serotypes
Power.Actinobacillus is widely distributed, and there is prevalence countries in the world.With between various countries, introduced pigs are on the increase, serotype
Tend to complicated.There is multiple serotypes prevalence simultaneously in some countries, or even different blood are there is likely to be in same pig farm
Clear type.China has now been found that and is separated to serotype have the various serotypes such as 1,2,3,4,5,7,8 and 15, Major Epidemic serum
Type has 1,3,5 and 7 types.At present, commonly using bacterium Serotype Identification method mainly has blood coagulation tests, ELISA method, PCR methods etc., wherein
Hemagglutination Method and PCR methods are the most commonly used.But being extracted to purifying and DNA from bacteria distribution culture at least needs can just make for 3-4 days
Preliminary Identification, not only wastes time and energy, as a result also inaccurate, and sensitiveness is poor, easily produces the result of false positive, is unfavorable for supporting
Grow family and scale enterprise takes corresponding serogroup vaccine and medicine timely to be treated.
The content of the invention
The purpose of the present invention is directed to the defect of prior art presence, a kind of Direct PCR detection pig contagion of proposition
Property pleuropneumonia cause of disease kit and its application, detection is rapid, and sensitivity is good.
The present invention solves technical problem by the following technical programs：A kind of Direct PCR detects pig contagiousness pleura lung
The kit of scorching cause of disease, comprising following reagent：The μ l of 5 μ l, PCR amplifing reagent of lysate 20, positive control and negative control.
The lysate of mentioned reagent contains 2~4M isothiocyanates, 7~10M ethylenediamine tetra-acetic acids, 15~20M ten
The PVP of sarkosyl and 3~6% volume ratios；The PCR amplifing reagents contain 5 × PCR
Buffer, 2.0M dNTP, 10pM forward primer, 10pM reverse primers, high-fidelity Taq enzyme 5U/ μ l, 20mM MgCl2With it is pure
Water.The forward primer is APPF：CAAGCTATCAAATAAGGCGAAACTA, the reverse primer is APPR：
AGTATCCGTATCAGAAGAAG。
The kit that the present invention is provided can fast and accurately detect Porcine contagious pleuropneumonia cause of disease.Can be to pig
The single sample detection of contagiousness pleuropneumonia infection, also can be used for pig to the detection of mixed infection in clinical case
The generaI investigation of contagiousness pleuropneumonia cause of disease, Molecule Epidemiology Investigation and vaccine screening and monitoring.
The present invention further provides a kind of Direct PCR detection Porcine contagious pleuropneumonia cause of disease kit should
With comprising the following steps：
The collection of the first step, sample, is divided into pathological material of disease and Nasopharyngeal swabs collection, and the pathological material of disease collection is collection under aseptic condition
Pathological material of disease 0.1mg~100mg, is fitted into standby in the centrifuge tube without RNase pollution, and the Nasopharyngeal swabs collection is collection nasopharynx liquid,
It is fitted into standby in the centrifuge tube without RNase pollution；
Second step, sample dissociation, take the μ l~100 μ l of liquid sample 10 or grinding after solid-like sample 0.1mg~
100mg, plus 400~600 μ l lysates, and concussion is mixed；
3rd step, Direct PCR reaction, reaction system is 25 μ l, takes the μ l of sample 5 after cracking, adds PCR amplifing reagents 20
μ l, are sufficiently mixed uniform rear of short duration centrifugation, and PCR amplification programs are 95 DEG C of 5min；Then 30 circulations：94 DEG C of 1min, 55 DEG C
1min, 72 DEG C of 1min S；Last 72 DEG C of 5min；
4th step, the detection of PCR primer, take 5 μ l amplified productions and 2 μ l Loading Buffer are mixed, and are added to containing bromination
In the Ago-Gel of second ingot, while adding control and DNA Marker (100bp), voltage is 150V, electrophoresis 30min, then
Result is observed in gel imaging instrument.
Reaction system in 3rd step of the above method is 25 μ l, and μ l, the 10pmol/ μ l of sample 5 after cracking are just
To 2 μ l, 5 × PCR Buffer of reverse primer, 5 μ l, 2.0mM/L dNTPs 5 μ l, the 5U/ μ l of primer 2 μ l, 10pmol/ μ l
High-fidelity Taq enzyme 0.5 μ l, 20mM MgCl23.5 μ l, the μ l of pure water 2, are sufficiently mixed uniform rear of short duration centrifugation.Amplification length is
250bp.Gel strength in 4th step during agarose gel electrophoresis is 1.0%, wherein containing 1% gel fuel.
The method of the present invention is built upon on molecular biology mechanism, negative and positive control is provided in detection, greatly
The big degree of accuracy that improve detection, reduces the occurrence probability of false positive, and specificity is relatively strong, time saving and energy saving, and the kit will be detected
Time foreshortens to 2h, substantially increases operating efficiency.The invention is particularly suited to the big of Porcine contagious pleuropneumonia infection
The quick diagnosis of clinical sample are measured, can be for Porcine contagious pleuropneumonia be prevented and treated and treatment provides science in clinical and scientific research
Foundation, and it is significant to improving detection, monitoring, the vaccine development etc. of Porcine contagious pleuropneumonia cause of disease.
Brief description of the drawings
Fig. 1 is the testing result figure of RT-PCR products, and the Marker in figure is DM 2000bp, is followed successively by from top to bottom
2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, it will be seen from figure 1 that APP template PCR amplifications fragments are about
250bp, it was demonstrated that amplified fragments are purpose fragment, meet desired design size, and 1 is positive control, and 2 is sample, is pleuropneumonia
Actinobacillus amplified fragments；3 is negative control.
Fig. 2 is kit sensitivity Detection result.
Specific embodiment
The present invention is further illustrated by the following examples, and the percentage composition in text is such as not particularly illustrated and refers both to weight
Percentage.
Embodiment 1
The Direct PCR of the present embodiment detects the kit of Porcine contagious pleuropneumonia cause of disease, and composition includes：
(1) lysate：2-4M isothiocyanates, 7-10M ethylenediamine tetra-acetic acids, the acid of 15-20M lauryl creatines, 3-6%
(volume ratio) PVP.
(2) PCR amplifing reagents：5 × PCR Buffer, 2.0M dNTP, 10pM forward primers, 10pM reverse primers, Gao Bao
True Taq enzyme (5U/ μ l), 20mM MgCl2, pure water.
The concrete composition of each composition：
Reaction system is 25 μ l, μ l, APPF (5 '-CAAGCTATCAAATAAGGCGAAACTA-3 ') of sample 5 after cracking
μ l, 5 × PCR Buffer 5 of (10pmol/ μ l) 2 μ l, APPR (3 '-AGTATCCGTATCAGAAGAAG-5 ') (10pmol/ μ l) 2
The μ l of μ l, 2.0mM/L dNTPs 5, the μ l of high-fidelity Taq enzyme 0.5 (5U/ μ l), 20mM MgCl23.5 μ l, the μ l of pure water 2, fully
Of short duration centrifugation after well mixed.
One pair of which specific primer is as follows：APPF：CAAGCTATCAAATAAGGCGAAACTA, APPR：
AGTATCCGTATCAGAAGAAG。
Kit is used for direct detection Porcine contagious pleuropneumonia cause of disease, is comprised the following steps：
(1) collection of sample：
1. pathological material of disease：Aseptic collection pathological material of disease 0.1mg-100mg, is fitted into standby in the centrifuge tube without RNase pollution.
2. Nasopharyngeal swabs：Nasopharynx liquid is gathered with Nasopharyngeal swabs, is fitted into standby in the centrifuge tube without RNase pollution.
(2) sample dissociation
1. the animal tissue of solid-like is first carried out into milled processed, liquid animal tissue is directly used in cracking；
2. the solid-like animal tissue 0.1mg-100mg after liquid 10 μ l-100 μ l or grinding is taken, plus 400-600 μ l split
Solution liquid, concussion is mixed；
(3) Direct PCR
Reaction system is 25 μ l, takes the μ l of sample 5 after cracking, adds the μ l of PCR amplifing reagents 20, is sufficiently mixed uniform rear short
Temporarily centrifugation.PCR amplification programs are：95℃5min；Then 30 circulations：94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min S；Finally
72℃5min。
(4) detection of RT-PCR products
Take 5 μ l amplified productions and 2 μ l Loading Buffer mixed, be added in containing EB 1.0% Ago-Gel,
Control and DNA Marker (100bp) are added simultaneously, and voltage is 150V, electrophoresis 30min, and knot is then observed in gel imaging instrument
Really, as a result as shown in figure 1,1 be positive control, 2 is sample, and 3 is negative control.If amplifying 250bp bands, positive product are illustrated
It is the positive, is otherwise feminine gender.
Direct PCR template DNA is quantified, 10 times of dilutions, are 1pg, 10 successively-1pg,10-2Pg, 10-3Pg, 10-4Pg,
10-5Pg, 10-6Pg, 10-7pg.Direct PCR amplification is carried out respectively, determines its sensitiveness.Then knot is observed in gel imaging instrument
Really, as a result as shown in Fig. 2 its sensitiveness is up to 10-3pg。
In addition to above-mentioned implementation, the present invention can also have other embodiment.All use equivalents or equivalent transformation are formed
Technical scheme, all fall within the protection domain of application claims.
Sequence table
Porcine contagious pleuropneumonia Actinobacillus（Actinobacillus pleuropneumoniae, APP）
GGACTGGTACATAGACGAGATTCTGTCATGCAGAATGGTTCTATCAGTATGGGATAGTATTAGAAAAATTAGG
ACATTTTCTCCAAGCATCTAAAGCATATGAGCAAGCTAAATCTCTTTCTATGAAAGAGAATTTATCTGATCTCTATT
TCCGTTTAGGGTTTGTAAATGAAAATCAAGGACATGATAATGAAATTGATCTTGAAGTAGCTAAACAGGCTTATGGC
TTAGCAATTCAAGCGGATCGAAAACTTAGAGCAAAGGATTTTGGCATTGGTGTATTTCATGAACATCGTAGAGATTG
GGGAAGAGCTATTATTGCGTATAAAGCTCAATTAGAAATTACGCCTAATAATCCAGAGTTATTATATCGTTTAGGCT
TTGCTTATGATCGCAATTATCAGTTTGGACAAGCTGAAAATATATATAAGGAAGCTCTATCGTTAAAACAAAAACCA
GAATGGCGTTCCCGATTAGGA
APPF（5′ - CAAGCTATCAAATAAGGCGAAACTA -3′）Sense primer
APPR（3′ - AGTATCCGTATCAGAAGAAG -5′）Anti-sense primer
Claims (7)
1. a kind of Direct PCR detects the kit of Porcine contagious pleuropneumonia cause of disease, comprising following reagent：Lysate, PCR
Amplifing reagent, positive control and negative control.
2. Direct PCR detects the kit of Porcine contagious pleuropneumonia cause of disease according to claim 1, and its feature exists
In：The lysate contains 2~4M isothiocyanates, 7~10M ethylenediamine tetra-acetic acids, 15~20M lauryl creatines acid and 3
The PVP of~6% volume ratio；The PCR amplifing reagents contain 5 × PCR Buffer, 2.0M dNTP, 10pM
Forward primer, 10pM reverse primers, high-fidelity Taq enzyme 5U/ μ l, 20mM MgCl2And pure water.
3. Direct PCR detects the kit of Porcine contagious pleuropneumonia cause of disease according to claim 2, and its feature exists
In：The forward primer is APPF：CAAGCTATCAAATAAGGCGAAACTA, the reverse primer is APPR：
AGTATCCGTATCAGAAGAAG。
4. a kind of application of the kit of Direct PCR detection Porcine contagious pleuropneumonia cause of disease, comprises the following steps：
The collection of the first step, sample, is divided into pathological material of disease and Nasopharyngeal swabs collection, and the pathological material of disease is gathered to gather pathological material of disease under aseptic condition
0.1mg~100mg, is fitted into standby in the centrifuge tube without RNase pollution, and the Nasopharyngeal swabs collection is collection nasopharynx liquid, is loaded
It is standby in centrifuge tube without RNase pollution；
Second step, sample dissociation, take the solid-like sample 0.1mg~100mg after the μ l~100 μ l of liquid sample 10 or grinding,
Plus 400~600 μ l lysates, concussion is mixed；
3rd step, Direct PCR reaction, reaction system is 25 μ l, takes the μ l of sample 5 after cracking, adds the μ l of PCR amplifing reagents 20,
It is sufficiently mixed uniform rear 4000rpm to be centrifuged 10 seconds, positive control and negative control take 5 μ l and add 20 μ l PCR amplification examinations respectively
Agent, PCR amplification programs are 95 DEG C of 5min；Then 30 circulations：94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of last 72 DEG C of 5min of 1min；
4th step, the detection of PCR primer, take 5 μ l amplified productions and 2 μ l LoadingBuffer are mixed, and are added to containing ethidium bromide
Ago-Gel in, while add DNAMarker (100bp), voltage is 150V, electrophoresis 30min, then in gel imaging instrument
Middle observation result, is the positive when sample amplification goes out 260bp bands, i.e. sample, and positive control amplifies 260bp bands, negative right
According to no band.
5. the application of the kit of Direct PCR detection Porcine contagious pleuropneumonia cause of disease according to claim 4, it is special
Levy and be：Reaction system in 3rd step is 25 μ l, the μ l of forward primer 2 of μ l, the 10pmol/ μ l of sample 5 after cracking,
The high-fidelity Taq enzyme of μ l, 2.0mM/L dNTPs5 μ l, the 5U/ μ l of 2 μ l, 5 × PCR Buffer of reverse primer 5 of 10pmol/ μ l
0.5 μ l, 20mM MgCl23.5 μ l, the μ l of pure water 2, are sufficiently mixed uniform rear centrifugation.
6. the application of the kit of Porcine contagious pleuropneumonia cause of disease Direct PCR detection is used for according to claim 5,
It is characterized in that：Amplification length is 250bp.
7. the application of the kit of Direct PCR detection Porcine contagious pleuropneumonia cause of disease according to claim 4, it is special
Levy and be：Gel strength in 4th step during agarose gel electrophoresis is 1.0%, wherein containing 1% gel fuel.
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Citations (3)
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|---|---|---|---|---|
| EP0733708A2 (en) * | 1995-03-24 | 1996-09-25 | Laboratorios Hipra, S.A. | Transferrin-binding protein 1 (Tbp1) gene of Actinobacillus pleuropneumoniae, its use in vaccines for pleuropneumonia and as diagnostic reagents |
| CN1259522A (en) * | 1998-10-22 | 2000-07-12 | 辉瑞产品公司 | Novel protain from lobar pneumonia Actinomyces |
| CN104313163A (en) * | 2014-10-28 | 2015-01-28 | 河南省农业科学院畜牧兽医研究所 | Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 |
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2017
- 2017-02-20 CN CN201710089474.1A patent/CN106755523A/en active Pending
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|---|---|---|---|---|
| EP0733708A2 (en) * | 1995-03-24 | 1996-09-25 | Laboratorios Hipra, S.A. | Transferrin-binding protein 1 (Tbp1) gene of Actinobacillus pleuropneumoniae, its use in vaccines for pleuropneumonia and as diagnostic reagents |
| CN1259522A (en) * | 1998-10-22 | 2000-07-12 | 辉瑞产品公司 | Novel protain from lobar pneumonia Actinomyces |
| CN104313163A (en) * | 2014-10-28 | 2015-01-28 | 河南省农业科学院畜牧兽医研究所 | Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 |
Non-Patent Citations (1)
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| LAWRENCE,M.L. ET AL.: ""Actinobacillus pleuropneumoniae serotype 2 capsular polysaccharide biosynthesis gene cluster, partial sequence，AY357726.1"", 《GENBANK》 * |
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