CN102154525B - Perlsucht PCR (polymerase chain reaction) quick diagnosis reagent kit - Google Patents
Perlsucht PCR (polymerase chain reaction) quick diagnosis reagent kit Download PDFInfo
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Abstract
The invention discloses a perlsucht PCR (polymerase chain reaction) quick diagnosis reagent kit comprises 800mu L of protease K with the concentration of 10mg/mL, 3.2mL of lysis solution, 6mL of TE (traffic engineering) buffer solution, 250mu L of PCR enzyme, 170mu L of ultrapure water, 200mu L of Marker DL (delay line) 2000, 20mu L of upper primer with the concentration of 20mu M, 20mu L of lower primer with the concentration of 20mu M, 40mu L of negative control and 40mu L of positive control. The kit is used for optimizing a reaction condition by a normal PCR method, is researched for detecting the perlsucht according to the gene segment design primer of the mycobacterium bovis IS6110, can be used for detecting the mycobacterium bovis, is suitable for the detection, the diagnosis and the epidemiological survey of the perlsucht, and has the characteristics of being fast, high in sensitivity, high in specificity and the like.
Description
Technical field
The present invention relates to a kind of test kit, especially a kind of bovine tuberculosis PCR quick diagnosis reagent kit.
Background technology
At present, tuberculosis is spreading trend in the world, and there are tuberculosis 2OOO ten thousand people in the whole world, and the existing tuberculosis patient of China is about 6,000,000, and annual dead about 280,000, China is one of countries of 22 high burdens of tuberculosis in the world.Data according to statistics, our province have tuberculosis patient about 200,000, and children account for 1/3.
Tuberculosis is to be caused by bacillus tuberculosis typus humanus, mycobacterium tuberculosis var bovis, avain tuberculosis mycobacterium, and tuberculosis can also make more than 50 kind of Mammals and 25 kinds of birds infect except infected person, and ox is the most responsive animal.Prapes is mainly caused by mycobacterium tuberculosis var bovis; be a kind of chronic infectious disease of infecting both domestic animals and human, cattle infected tuberculosis is through very slow, because ill organ difference; each is inconsistent for clinical symptom; mainly be divided into following three kinds: first kind is pulmonary tuberculosis, and characteristics are long-term intractable dry cough, and nose liquid is mucus, purulence; ill ox fatigue; become thin anaemia, expiratory dyspnea.Second kind: tuberculosis of breast, the lymphonodi mammarici enlargement, the breast district is ill, and it is painless not have heat, is characteristics so that limitation or diffuse hard tubercle to take place, and breast surface is uneven, and lactation amount reduces, and milk is thinning, mastatrophy when serious, lactation stops.The third: tuberculosis of intestine, becoming thin and lasting diarrhea, or diarrhoea and constipation alternately appear as characteristics, and ight soil is the gruel shape, is mixed with mucus or purulent secretion, and flavor is stench, poor growth, the death of becoming thin at last.In addition, tubercule bacillus can also encroach on other organs, so tuberculosis of testis, tuberculosis of uterus, lymphoid tuberculosis, tuberculosis of serous membrane and pulmonary tuberculosis can take place.
Bovine tuberculosis brings massive losses for the cattle-raising of China at generation and the popular ubiquity of China.The lO% of people's tuberculosis is above to be caused by Mycobacterium bovis, because ox and the mankind's relation (milk, beef and farm cattle etc.) is more close than other animals, the generation of prapes and popular and human tuberculosis's relation of being proportionate, want detuberculization, at first be to eliminate bovine tuberculosis, eliminate and control the bovine tuberculosis key is to diagnose this disease.It is long that traditional mycobacterium tuberculosis var bovis detection method consumes the time, generally is 1～2 month, and conventional cockade reaction detection method constantly has report to have nonspecific reaction.Now market lacks and a kind ofly can carry out easy, quick, sensitive, the test kit of detection accurately to the bovine tuberculosis of clinical sample.
Summary of the invention
The objective of the invention is: a kind of bovine tuberculosis PCR quick diagnosis reagent kit is provided, and it can the rapid detection bovine tuberculosis, and easy, sensitive, accurate, high specificity.
The present invention is the bovine tuberculosis PCR quick diagnosis reagent kit of realizing like this, it comprises that 800 μ L concentration are the Proteinase K of 10mg/mL, 3.2mL lysate, the TE damping fluid of 6mL, the PCR enzyme of 250 μ L, 170 μ L ultrapure waters, 1mL clean-out system, the Marker DL2000 of 200 μ L, 20 μ L concentration are the last primer of 20 μ M, 20 μ L concentration are the following primer of 20 μ M, 40 μ L negative controls, 40 μ L positive controls and 20 adsorption columns.
Lysate is for by the NaCL of EDTA, the 2.5M of Tris-HCL, the 1mM of 10mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms.
The TE damping fluid comprises the mixing solutions that the EDTA of the Tris-HCL of 10mM and 1mM forms, and the pH value of mixing solutions is 7.8.
The composition of PCR enzyme comprises the Tris-HCL(PH8.3 of 10mM), the KCL of 50mM, the MgCL of 1.5mM
2And 0.05U Polymerase/ μ L.
Last primer is 5 '-GATGGCGAACTCAAGGAGCACA-3 ', following primer is 5 '-ACTGAGATCCCCTATCCGTATGGT-3 '.
Negative control is ultrapure water.
Positive control is standard mycobacterium tuberculosis var bovis dna profiling.
Clean-out system is that volume percent is 70% ethanol.
Adopt adsorption column to collect dna profiling.
The foundation of bovine tuberculosis PCR diagnostic method
Materials and methods
1 reference strain
Mycobacterium bovis reference strain (being numbered 93006(4)), actinobacillus pleuropneumoniae, intestinal bacteria, clostridieum welchii are available from Beijing pharmaceutical biological product calibrating institute.Pasteurellosis bacillus, segmental bronchus sepsis bordetella bacilli, riemerella anatipestifer, Salmonellas are by Guizhou Farming Animal Science and Veterinary Research Institute's livestock and poultry pestilence research department isolation identification.
2 main agents
2.1 primer (concentration is 20 μ M, with the preparation of TE solution)
According to the gene fragment design primer of mycobacterium tuberculosis var bovis IS6110, pcr amplification goes out the specific fragment that a length is the 478bp size, and the accession number of this sequence on Genbank is X57835.2.Primer sequence is as follows:
Last primer 5 '-GATGGCGAACTCAAGGAGCACA-3 '
Following primer 5 '-ACTGAGATCCCCTATCCGTATGGT-3 '
Synthetic by Shanghai Jierui Biology Engineering Co., Ltd.
2.2 TE buffered soln (10mM Tris-HCL, 1mM EDTA), Proteinase K (concentration 10mg/mL, with TE solution preparation), lysate (10mM Tris-HCL, 1mM EDTA, 2.5M NaCL and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms), ultrapure water, 70% ethanol, physiological saline, agarose, dehydrated alcohol, 3.8% Trisodium Citrate, 1 * TBE electrophoretic buffer, ethidium bromide (EB) or Goldview nucleic acid dye are prepared by project team.Marker DL2000, PCR enzyme (10mM Tris-HCL (PH8.3), 50mM KCL, 1.5mM MgCL
2, 0.05U Polymerase/ μ L) available from sky root biochemical technology [Beijing] company limited.
2.3 the collection of sample
The aseptic collection fresh milk ,-20 ℃ of preservations are standby.As antithrombotics aseptic collection whole blood ,-20 ℃ of preservations are standby with Trisodium Citrate or EDTA.
The extraction of 3 templates
3.1 the extraction of the cultivation template of Mycobacterium bovis
Mycobacterium bovis is inoculated on the modified Russell medium, cultivated 15～30 days for 37 ℃, can be observed beige, coarse, protuberance, opaque, the edge is irregular, the bacterium colony that is the particle tubercle, collect thalline in the 1.5mL centrifuge tube, add TE buffered soln 200 μ L, Proteinase K 40 μ L, lysate 160 μ L, abundant mixing, 60 ℃ of water-bath effects 15～20 minutes add dehydrated alcohol 200 μ L, adopt adsorption column to collect dna profiling, clean-out system is 70% ethanol.
.2The extracting method of actinobacillus pleuropneumoniae, intestinal bacteria, clostridieum welchii, pasteurellosis bacillus, segmental bronchus sepsis bordetella bacilli, riemerella anatipestifer, Salmonellas dna profiling is identical with the extracting method of mycobacterium tuberculosis var bovis dna profiling.
.3The extraction of sample template
Get milk (or whole blood) 1mL in the 1.5mL centrifuge tube, the centrifugal 2-5 of 10000r/min minute, abandon supernatant liquor, precipitation adopts physiological saline to clean 2 times, abandons supernatant liquor.Add TE buffered soln 200 μ L, Proteinase K 40 μ L, lysate 160 μ L, abundant mixing, 60 ℃ of water-bath effects 15-20 minute add dehydrated alcohol 200 μ L, adopt adsorption column to collect dna profiling, and clean-out system is 70% ethanol.
The pcr amplification of 4 mycobacterium bovis specific fragments
Adopt the DNA that extracts to touch plate, utilize synthetic primer under the effect of PCR enzyme, to increase.Reaction system and condition are as follows: the PCR enzyme is 12.5 μ L, and standard bacillus tuberculosis bovis DNA touches plate 2 μ L, and sample DNA is touched plate 5 μ L, and each 0.75 μ L of primer adds ddH up and down
2O to 25 μ L, under Tgradient96 amplification instrument, carry out 30 following circulations: 94 ℃ of sex change 5 minutes, 94 ℃ 40 seconds, 63 ℃ 40 seconds, 72 ℃ 60 seconds, 30 circulations, 72 ℃ were extended 7 minutes.After reaction finishes amplified production is got 7 μ L and carry out the detection of 1% agarose gel electrophoresis.Electrophoresis detection shows about the 478bp place, a PCR object tape occurs, and its result as shown in Figure 1, and is very approaching with the result who estimates.
The specific assay of 5 PCR methods amplification
Respectively with mycobacterium tuberculosis var bovis, actinobacillus pleuropneumoniae, intestinal bacteria, clostridieum welchii pasteurellosis bacillus, segmental bronchus sepsis bordetella bacilli, riemerella anatipestifer, Salmonellas DNA for touching plate, carry out pcr amplification, except amplified band appears in mycobacterium tuberculosis var bovis, other bacterium does not see specificity 478bp fragment amplification.The result as shown in Figure 2.
The mensuration of the susceptibility of 6 PCR methods amplification
Adopt TU-1800spc ultraviolet-visible photometer, the content of mycobacterium tuberculosis var bovis standard DNA template is measured, record OD
260Mean value is 0.040nm, and the result is as calculated: the content of dna profiling is 0.304 μ g/ μ L.Get standard prapes dna profiling, dilute by 10 times of dilution methods, the template weaker concn is followed successively by: 10-
1, 10-
2, 10-
310-
8, get 2 μ L and increase, then mycobacterium tuberculosis var bovis standard DNA concentration is followed successively by 304ng, 30. 4ng, 3.04ng, 304pg, 30. 4pg, 3.04pg, 304fg, 30.4fg, 3.04fg.Test-results shows: the minimum content of the mycobacterium tuberculosis var bovis standard DNA template that can detect is 30.4pg.The result as shown in Figure 3.
Owing to adopted technique scheme, the present invention adopts conventional PCR, reaction conditions is optimized, gene fragment design primer according to mycobacterium tuberculosis var bovis IS6110, developed the PCR test kit for detection of bovine tuberculosis, this test kit can detect mycobacterium tuberculosis var bovis, is applicable to detection, diagnosis and the epidemiology survey of bovine tuberculosis.The present invention adopts conventional PCR method, can directly detect mycobacterium tuberculosis var bovis from clinical samples such as milk, whole blood, and sensitivity is about 30.4pg, and the time that needs is 6 hours only, has quick, characteristics such as susceptibility is high, 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
Accompanying drawing 1 is mycobacterium bovis specific fragment test chart;
M:Marker DL2000; 1: standard female; 2: standard positive; 3: the positive sample that is detected;
Accompanying drawing 2 is PCR specificity test chart;
M:Marker DL2000; 1: mycobacterium tuberculosis var bovis; 2: intestinal bacteria; 3: Salmonellas; 4: pasteurellosis bacillus; 5: actinobacillus pleuropneumoniae; 6: segmental bronchus sepsis bordetella bacilli; 7: clostridieum welchii; 8: Listeria monocytogenes;
Accompanying drawing 3 is PCR sensitivity test figure;
M:Marker DL2000; The 1:DNA template is 304ng; The 2:DNA template is 30.4ng; The 3:DNA template is 3.04ng; The 4:DNA template is 304pg; The 5:DNA template is 30.4pg; The 6:DNA template is 3.04pg; The 7:DNA template is 304fg; The 8:DNA template is 30.4fg; The 9:DNA template is 3.04fg.
Embodiment
Embodiments of the invention: bovine tuberculosis PCR quick diagnosis reagent kit, it comprises that 800 μ L concentration are the Proteinase K of 10mg/mL, 3.2mL lysate, lysate is for by the NaCL of EDTA, the 2.5M of Tris-HCL, the 1mM of 10mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms; The TE damping fluid of 6mL, TE damping fluid comprise the mixing solutions that the EDTA of the Tris-HCL of 10mM and 1mM forms, and the pH value of mixing solutions is 7.8; 250 μ L's
PCR enzyme, PCR enzyme are the PCR MasterMix of TIANGEN Biotech (Beijing) Co., Ltd. product, its concrete KCL, MgCL of 1.5mM that forms the Tris-HCL (PH8.3), the 50mM that comprise 10mM
2And 0.05U Polymerase/ μ L; 170 μ L ultrapure waters, the Marker DL2000 of 200 μ L, 20 μ L concentration are the last primer of 20 μ M, and 20 μ L concentration are the following primer of 20 μ M, and last primer is 5-GATGGCGAACTCAAGGAGCACA-3, and following primer is 5-ACTGAGATCCCCTATCCGTATGGT-3; Adopt the ultrapure water of 40 μ L as negative control; Adopt 40 μ L standard mycobacterium tuberculosis var bovis dna profilings as positive control; Adopting volume percent is that 70% ethanol is as clean-out system; Adopt adsorption column to collect dna profiling.
Claims (1)
1. bovine tuberculosis PCR quick diagnosis reagent kit, it is characterized in that: it comprises that 800 μ L concentration are the Proteinase K of 10mg/mL, 3.2mL lysate, the TE damping fluid of 6mL, the PCR enzyme of 250 μ L, 170 μ L ultrapure waters, 1mL clean-out system, the Marker DL2000 of 200 μ L, 20 μ L concentration are the last primer of 20 μ M, 20 μ L concentration are the following primer of 20 μ M, 40 μ L negative controls, 40 μ L positive controls and 20 adsorption columns; Last primer is 5 '-GATGGCGAACTCAAGGAGCACA-3 ', following primer is 5 '-ACTGAGATCCCCTATCCGTATGGT-3 '; Lysate is for by the NaCL of EDTA, the 2.5M of Tris-HCL, the 1mM of 10mM and account for the mixing solutions that the SDS of lysate cumulative volume 5% forms; The TE damping fluid comprises the mixing solutions that the EDTA of the Tris-HCL of 10mM and 1mM forms, and the pH value of mixing solutions is 7.8; The composition of PCR enzyme comprises MgCL2 and the 0.05U Polymerase/ μ L of KCL, 1.5mM of Tris-HCL, the 50mM of 10mM; Negative control is ultrapure water; Positive control is standard mycobacterium tuberculosis var bovis dna profiling; Clean-out system is that volume percent is 70% ethanol; Adopt adsorption column to collect dna profiling.
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| KR101403507B1 (en) * | 2013-03-21 | 2014-06-09 | 주식회사 현일바이오 | Methods for Selectively Detecting Mycobacterium tuberculosis complex and Nontuberculous mycobacteria and Kits Using the Same |
| CN106119395A (en) * | 2016-08-25 | 2016-11-16 | 甘肃省动物疫病预防控制中心 | Anthrax, brucellosis, tuberculosis triple PCR detection primer to and test kit |
| CN109055582A (en) * | 2018-07-28 | 2018-12-21 | 贵州省畜牧兽医研究所 | A kind of Mycoplasma bovis disease PCR quick diagnosis reagent kit |
| CN111518929A (en) * | 2020-03-23 | 2020-08-11 | 贵州省畜牧兽医研究所 | Triple PCR rapid diagnosis kit for mycoplasma bovis, mycobacterium bovis and pasteurella multocida |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101168777A (en) * | 2007-10-31 | 2008-04-30 | 中国农业大学 | Method and kit for detecting Chinese holstein cattle produced milk property |
| CN101603098A (en) * | 2009-04-07 | 2009-12-16 | 厦门出入境检验检疫局检验检疫技术中心 | Multiple fluorescence PCR detects primer, probe, test kit and the detection method thereof of shrimp white spot syndrome virus and prawn infectious hypodermal and hematopoietic necrosis disease poison simultaneously |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168777A (en) * | 2007-10-31 | 2008-04-30 | 中国农业大学 | Method and kit for detecting Chinese holstein cattle produced milk property |
| CN101603098A (en) * | 2009-04-07 | 2009-12-16 | 厦门出入境检验检疫局检验检疫技术中心 | Multiple fluorescence PCR detects primer, probe, test kit and the detection method thereof of shrimp white spot syndrome virus and prawn infectious hypodermal and hematopoietic necrosis disease poison simultaneously |
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