CN101724709B - PCR diagnostic kit for porcine infectious pleuropneumonia - Google Patents
PCR diagnostic kit for porcine infectious pleuropneumonia Download PDFInfo
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- CN101724709B CN101724709B CN2010103008187A CN201010300818A CN101724709B CN 101724709 B CN101724709 B CN 101724709B CN 2010103008187 A CN2010103008187 A CN 2010103008187A CN 201010300818 A CN201010300818 A CN 201010300818A CN 101724709 B CN101724709 B CN 101724709B
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Abstract
The invention discloses a PCR diagnostic kit for porcine infectious pleuropneumonia, which comprises 400mu L of proteinase K with the concentration of 20mg/mL, 1000mu L of cracking solution, 1500mu L of TE buffer solution, 250mu L of PCR enzyme, 170mu L of ultra-pure water, 50mu L of MarkerDL2000, 40mu L of primer P1 and primer P2 which are mixed in the same volume and have the same concentration of 20mu M, 20mu L of negative control and 20mu L of positive control. By optimizing the PCR reaction conditions, the invention develops a PCR kit for detecting the actinobacillus of the porcine infectious pleuropneumonia; and by comparing the PCR detection result with the negative control and the positive control, detection conclusions can be obtained, thereby achieving the aim of quickly detecting whether a sample has porcine infectious pleuropneumonia or not. The invention has accurate detection result, quick and sensitive detection process, simple detection mode and good using effect.
Description
Technical field
The present invention relates to a kind of test kit, especially a kind of PCR diagnostic kit for porcine infectious pleuropneumonia.
Background technology
(Porcine Contagious Pleuropneumoniae PCP) is actinobacillus pleuropneumoniae (Actinobacillus Pleuropneumoniae, the porcine respiratory disease that APP) causes to porcine contagious pleuropneumonia; With hemorrhagic, necrotizing pneumonia and chronic fiber disposition pleuropneumonia is characteristic; This disease can cause pig death or poor growth, causes enormous economic loss (Sebunya T N K etc., 1983 for pig industry all over the world; Macinnes J L etc.; 1988, Gram T etc., 2000).Come into vogue in China the eighties, forms popular widely in recent years and the one-tenth ascendant trend.Can be divided into biological I type and two biotypes of biological II type according to whether relying on NAD.Serotype virulencies such as 1,5,9,10 in the biological I type are the strongest.According to statistics, China's popular serotype is types such as 1,2,3,5,7,10, is main with 1,3,7 types, and main serotype lacks cross-immunity.Lack now and a kind ofly can carry out test kit easy, quick, sensitive, that detect accurately the porcine contagious pleuropneumonia of clinical sample.
Summary of the invention
The objective of the invention is: a kind of PCR diagnostic kit for porcine infectious pleuropneumonia is provided, and it can arrive porcine contagious pleuropneumonia in rapid detection, and easy, sensitive, accurate.
The present invention is achieved in that PCR diagnostic kit for porcine infectious pleuropneumonia, and it comprises that 400 μ L concentration are the Proteinase K of 20mg/mL, 1000 μ L lysates; The TE damping fluid of 1500 μ L, the PCR enzyme of 250 μ L, 170 μ L ultrapure waters; The Marker DL2000 of 50 μ L; 40 μ L equal-volume blended primer P1, P2, the concentration of primer P1, P2 is 20 μ M, 20 μ L negative controls and 20 μ L positive controls.
Lysate is for by Tris-HcL50mM, EDTA2mM, NaCl100mM and account for the mixing solutions that the SDS of lysate TV 5% forms.
The TE damping fluid comprises the mixing solutions that Tris-HcL100mM and EDTA1mM form, and the pH value of mixing solutions is 8.0.
The composition of PCR enzyme comprises the Tris-HcL of 10mM, the KCL of 50mM, the MgCL of 1.5mM
2And the Poymerase/uL of 0.05U.
The sequence of primer P1, P2 is respectively P1:5 '-ATGAGGATTTGTTTCTCGGTGGTG-3 ', P2:
5-TATTTCCGTCCGGTTTATTCAGGTC-3。
Negative control is a ultrapure water.
Positive control is reorganization pMD18-T-apxIVA plasmid.
The foundation of PCR method
PCR is reflected at that optimum reaction condition does in the 50 μ L reaction systems; 55 ℃ of annealing temperatures, Taq DNA polymerase1U, primer concentration 20 μ mol/L have all effectively amplified its purpose fragment with primer; Clip size is 600bp, and specific fragment produces (see figure 1) nothing but.The result shows that the PCR detection method of APP sets up successfully.
Sensitivity test
In the PCR reaction, 5CFU/mL～5 * 10
7The APP bacterium of CFU/mL dilution carries out PCR, and the lowest detection amount of APP is the 50CFU (see figure 2).The result shows that the PCR susceptibility of foundation is high.
The specificity test
Carry out the PCR reaction with APP1～10 types, streptococcus aureus, suis, pasteurella multocida, Salmonella typhimurium, haemophilus parasuis, intestinal bacteria.APP1～10 types are all positive, and streptococcus aureus, suis, pasteurella multocida, Salmonella typhimurium, haemophilus parasuis, all negative (see figure 3) of colibacillary amplification.The result shows the PCR high specificity of foundation.
Use the PCR method of setting up, duplicate detection APP DNA sample 3 times, the result is all consistent.
Can know that from experimental result porcine contagious pleuropneumonia pleuropneumoniae PCR detection kit can be carried out the clinical sample of APP fast, sensitive, detection accurately.
Owing to adopted technique scheme; The present invention is through being optimized the PCR reaction conditions; Developed detection porcine contagious pleuropneumonia pleuropneumoniae PCR test kit; Compare through PCR detected result and negative control and positive control, can obtain detecting conclusion, thereby play the purpose whether the rapid detection sample contains porcine contagious pleuropneumonia.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
Accompanying drawing 1 is the PCR test chart of APP;
M:DL2000; 1:APP PCR result;
Accompanying drawing 2 is PCR sensitivity test figure;
M:DL2000; 1～7:5CFU/mL～5 * 10
7The APP PCR result of CFU/mL dilution;
Accompanying drawing 3 is a PCR specificity test chart;
M:DL2000; 1～10:APP, 1～10 type; 11: streptococcus aureus PCR product; 12: suis PCR product; 13: pasteurella multocida PCR product; 14: Salmonella typhimurium PCR product; 15: haemophilus parasuis PCR product; 16: the colibacillus PCR product.
Embodiment
Embodiments of the invention: PCR diagnostic kit for porcine infectious pleuropneumonia, it comprises that 400 μ L concentration are the Proteinase K of 20mg/mL, 1000 μ L lysates, separating liquid is by Tris-HcL50mM, EDTA2mM, NaCl100mM; The TE damping fluid of 1500 μ L, TE damping fluid comprise the mixing solutions that Tris-HcL100mM and EDTA1mM form, and the pH value of mixing solutions is 8.0; The PCR enzyme of 250 μ L, PCR enzyme are the PCR MasterMix of TIANGEN Biotech (Beijing) Co., Ltd. product, and comprise the Tris-HcL of 10mM, the KCL of 50mM, the MgCL of 1.5mM its concrete composition
2, the dNTP Mixture of 25mM and the Poymerase/uL of 0.05U; 170 μ L ultrapure waters, the Marker DL2000 of 50 μ L, 40 μ L equal-volume blended primer P1, P2, the concentration of primer P1, P2 is 20 μ M, and the sequence of primer P1, P2 is respectively P1:
5 '-ATGAGGATTTGTTTCTCGGTGGTG-3 ', P2:5-TATTTCCGTCCGGTTTATTCAGGTC-3; The ultrapure water that adopts 20 μ L is as negative control; Adopt 20 μ L reorganization pMD18-T-apxIVA plasmid as positive control.
Claims (6)
1. PCR diagnostic kit for porcine infectious pleuropneumonia, it is characterized in that: it comprises that 400 μ L concentration are the Proteinase K of 20mg/mL, 1000 μ L lysates; The TE damping fluid of 1500 μ L; The PCR enzyme of 250 μ L, 170 μ L ultrapure waters, the Marker DL2000 of 50 μ L; 40 μ L equal-volume blended primer P1, P2; The concentration of primer P1, P2 is 20 μ M, and the sequence of primer P1, P2 is respectively P1:5 '-ATGAGGATTTGTTTCTCGGTGGTG-3 ', P2:5-TATTTCCGTCCGGTTTATTCAGGTC-3; 20 μ L negative controls and 20 μ L positive controls.
2. PCR diagnostic kit for porcine infectious pleuropneumonia according to claim 1 is characterized in that: lysate is for by Tris-HcL50mM, EDTA2mM, NaCl100mM and account for the mixing solutions that the SDS of lysate TV 5% forms.
3. quick diagnostic reagent kit of dairy cow hepatitis B PCR according to claim 1 is characterized in that: the TE damping fluid comprises the mixing solutions that Tris-HcL100mM and EDTA1mM form, and the pH value of mixing solutions is 8.0.
4. PCR diagnostic kit for porcine infectious pleuropneumonia according to claim 1 is characterized in that: the composition of PCR enzyme comprises the KCL of the Tris-HcL of 10mM, 50mM, the MgCL of 1.5mM
2, the dNTP Mixture of 25mM and the Poymerase/uL of 0.05U.
5. PCR diagnostic kit for porcine infectious pleuropneumonia according to claim 1 is characterized in that: negative control is a ultrapure water.
6. PCR diagnostic kit for porcine infectious pleuropneumonia according to claim 1 is characterized in that: positive control is reorganization pMD18-T-apxIVA plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010103008187A CN101724709B (en) | 2010-01-28 | 2010-01-28 | PCR diagnostic kit for porcine infectious pleuropneumonia |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010103008187A CN101724709B (en) | 2010-01-28 | 2010-01-28 | PCR diagnostic kit for porcine infectious pleuropneumonia |
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| CN101724709A CN101724709A (en) | 2010-06-09 |
| CN101724709B true CN101724709B (en) | 2012-01-18 |
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| CN2010103008187A Expired - Fee Related CN101724709B (en) | 2010-01-28 | 2010-01-28 | PCR diagnostic kit for porcine infectious pleuropneumonia |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220426B (en) * | 2011-05-10 | 2013-03-20 | 贵州省畜牧兽医研究所 | PCR (polymerase chain reaction) detection kit for hacmophilus parasuis |
| CN102925548B (en) * | 2012-08-02 | 2014-12-24 | 四川农业大学 | Actinobacillus pleuropneumoniae LAMP kit and application method thereof |
| CN104313163B (en) * | 2014-10-28 | 2017-04-12 | 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 |
| CN105400881A (en) * | 2015-12-14 | 2016-03-16 | 江苏省农业科学院 | Primer for pleura pneumonia actinobacillus serum 4 type PCR detection and kit therefor |
| CN107937498A (en) * | 2017-12-29 | 2018-04-20 | 江西农业大学 | The Primer composition of auxiliary identification porcine contagious pleuropneumonia ApxI toxin and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358237A (en) * | 2008-09-09 | 2009-02-04 | 中国农业大学 | Test kit of actinobacillus pleuropneumoniae and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358237A (en) * | 2008-09-09 | 2009-02-04 | 中国农业大学 | Test kit of actinobacillus pleuropneumoniae and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王海丽 等.《猪传染性胸膜肺炎PCR诊断方法的建立与应用》.《中国畜牧兽医》.2009,第36卷(第9期),135－138. * |
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