CN103667510B - Streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit - Google Patents
Streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit Download PDFInfo
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- CN103667510B CN103667510B CN201310742629.9A CN201310742629A CN103667510B CN 103667510 B CN103667510 B CN 103667510B CN 201310742629 A CN201310742629 A CN 201310742629A CN 103667510 B CN103667510 B CN 103667510B
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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The invention relates to a kit, in particular to a streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit. The kit comprises 18 mu. L of PCR buffer solution, 30 mu. L of ultrapure water, 3 mu. L of MarkerDl2000, 3 mu. L of 15pmol/mu. L upper primer, 3 mu. L of 15pmol/mu. L lower primer, 3mu. L of polymerase, 18mu. L of mass concentration10 percent SDS (Sodium Dodecyl Sulfate), 15 mu. L of TE buffer solution, 5 mu. L of a negative control part and 5 mu. L of a positive control part. The streptococcus suis 7 type PCR detection kit has the beneficial effects that the detection kit has the advantage of high detection speed of a PCR detection technology; moreover, the kit is cost in cost, adopts special primers, can satisfy the detection of a gene segment PCR product of 0.00984ng at the minimum, and is high in detection sensitivity.
Description
Technical field
The present invention relates to a kind of test kit, more specifically a kind of swine streptococcus 7 type PCR detection kit.
Background technology
Rounded or the oval of swine streptococcus (Streptococcus suis, S. suis) swine streptococcus, often in catenation, different in size, be gram-positive cocci.Blood agar substratum grows, and periphery of bacterial colonies forms zone of hemolysis.Have now found that its kantigen serotype has more than 35 kinds.Virulence factor has pod membrane, muramidase-released protein (MRP), extracellular factor (EF), hemolysin.Pod membrane can protect bacterium, and opposing is engulfed.The existence of muramidase-released protein, extracellular factor improves the virulence of bacterial strain.This bacterium resistibility is not strong, and to dry, damp and hot all more responsive, conventional Herb of Common violet all can be killed, and infects oneself through becoming one of subject matter of whole world pig industry.It can cause the meningitis of pig, septicemia, endocarditis, pneumonia and sacroiliitis etc., also can cause the meningitis of people even dead, at present, oneself finds 35 kinds of serotypes, i.e. 1 ~ 34 type and 1/2 type, and streptococcus suis 2 type is that the whole world is by the serotype be the most frequently separated to.And in Denmark, except 2 type bacterial strains, 7 type bacterial strains are also by one of serotype of being often separated to.Along with the frequent discovery of 7 type bacterial strains in the sick pig in the whole world, the research of its each side characteristic is also just seemed very necessary.Since summer has set in 2006, some areas of the province such as Zhejiang, Jiangxi, Anhui, Jiangsu, Hunan of China, popular is the swine disease epidemic situation of principal character with high heat, and rapid spread.China Animal Disease Control And Prevention Center's doctor's diagnosis room formally assert that at the end of 2006 9 months the main pathogen of " nameless high-fever disease " is for variation PRRS virus, but has report to show that swine streptococcus is a kind of important bacterial pathogen.Therefore, in hyperpyrexia, whether there is swine streptococcus 7 type and 7 type roles also merit attention, but not a kind of conveniently for detecting the test kit of swine streptococcus 7 type at present, and this is deficiency of the prior art.
Summary of the invention
Goal of the invention of the present invention is for deficiency of the prior art, provides a kind of quick, accurate, easy swine streptococcus 7 type PCR detection kit.
The present invention is achieved by the following technical solutions:
A kind of swine streptococcus 7 type PCR detection kit, it is characterized in that: described test kit comprises PCR damping fluid, 30 μ L ultrapure waters, the 3 μ LMarker DL2000 of 18 μ L, primer on 3 μ L15pmol/ μ L, 3 μ L concentration are the lower primer of 15pmol/ μ L, 3 μ L polysaccharases, 18 μ L mass concentrations are the SDS of 10%, the TE damping fluid of 15 μ L, 5 μ L negative controls, 5 μ L positive controls.
Above-mentioned PCR damping fluid is by 25 mmol/L MgCl
23 μ L, 10 × PCR buffer 10 μ L, 10mmol/L dNTPx2 μ L.
The sequence number of above-mentioned upper primer is 5 '-AGCTCTAACACGAAATARRRR-3 ', and the sequence number of lower primer is 5 '-GTCAAACACCCTGGATAGCCG-3 '.
Above-mentioned negative control is the DNA fragmentation of non-swine streptococcus 7 type.
Above-mentioned positive control is the PCR primer of standard swine streptococcus 7 type.
In above-mentioned upper primer, R is degenerated primer.
Reactions steps: the first step 50 min 50 DEG C, 1 circulation; Second step 94 DEG C of 3 min, 1 circulation; 3rd step 94 DEG C 1 min, 52 DEG C of 1 min, 72 DEG C of 2 min, 35 circulations, the 4th step 72 DEG C extends 7min, 1 circulation
Get the above-mentioned reaction product of 5 μ L and carry out 1.2% agarose gel electrophoresis inspection, with Marker DL2000 as a reference, TAE damping fluid is electrophoretic buffer, with 5V/cm electrophoresis 1h, and gel imaging system observations;
Reclaim object band, 16 DEG C of 1.5 h is connected to PMD19-T Easy Vector, transformed competence colibacillus DH5 α, the LB solid medium be spread evenly across containing Amp, IPTG and X-gal is dull and stereotyped, and cultivate 16h for 37 DEG C, picking white colony is cultivated in a small amount, extract plasmid and do pcr amplification qualification, checked order by positive bacteria liquid, and adopt non-swine streptococcus 7 type DNA fragmentation as negative control, standard swine streptococcus 7 type DNA fragmentation is as positive control.
The invention has the beneficial effects as follows: the present invention has the fast advantage of the detection of PCR detection technique, and this test kit cost low, have employed special primer, can meet the minimum detection for 0.00984ng fragment PCR product, detection sensitivity is high.
Accompanying drawing explanation
Fig. 1 is sensitivity experiments result figure of the present invention;
1-98.4ng fragment PCR product, 2-9.84 ng fragment PCR product, 3-0.984ng fragment PCR product, 4-0.0984ng fragment PCR product, 5-0.00984ng fragment PCR product, 6-0.000984ng fragment PCR product.
Fig. 2 is the results in electrophoresis figure of the present invention
1-Marker DL2000,2-PCR product (250bp), 3-reference culture PCR primer (250bp), 4-negative control.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described, so that those skilled in the art better can understand the present invention, but therefore do not limit the present invention.
Embodiment 1
A kind of swine streptococcus 7 type PCR detection kit, it is characterized in that: described test kit comprises PCR damping fluid, 30 μ L ultrapure waters, the 3 μ LMarker DL2000 of 18 μ L, primer on 3 μ L15pmol/ μ L, 3 μ L concentration are the lower primer of 15pmol/ μ L, 3 μ L polysaccharases, 18 μ L mass concentrations are the SDS of 10%, the TE damping fluid of 15 μ L, 5 μ L negative controls, 5 μ L positive controls.
Above-mentioned PCR damping fluid is by 25 mmol/L MgCl
23 μ L, 10 × PCR buffer 10 μ L, 10mmol/L dNTPx2 μ L.
The sequence number of above-mentioned upper primer is 5 '-AGCTCTAACACGAAATARRRR-3 ', and the sequence number of lower primer is 5 '-GTCAAACACCCTGGATAGCCG-3 '.
Above-mentioned negative control is the DNA fragmentation of non-swine streptococcus 7 type.
Above-mentioned positive control is the PCR primer of standard swine streptococcus 7 type.
In above-mentioned upper primer, R is degenerated primer.
Claims (3)
1. a swine streptococcus 7 type PCR detection kit, it is characterized in that: described test kit comprises PCR damping fluid, 30 μ L ultrapure waters, the 3 μ LMarker DL2000 of 18 μ L, primer on 3 μ L15pmol/ μ L, 3 μ L concentration are the lower primer of 15pmol/ μ L, 3 μ L polysaccharases, 18 μ L mass concentrations are the SDS of 10%, the TE damping fluid of 15 μ L, 5 μ L negative controls, 5 μ L positive controls, the sequence of described upper primer is 5 '-AGCTCTAACACGAAATARRRR-3 ', and the sequence of lower primer is 5-GTCAAACACCCTGGATAGCCG-3 '.
2. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: described negative control is the DNA fragmentation of non-swine streptococcus 7 type.
3. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: described positive control is the PCR primer of standard swine streptococcus 7 type.
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| CN201310742629.9A CN103667510B (en) | 2013-12-30 | 2013-12-30 | Streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit |
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| CN107400733A (en) * | 2017-09-27 | 2017-11-28 | 广东省农业科学院动物卫生研究所 | Detect intersection isothermal duplication primer sets, kit and the application of Streptococcus suis |
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Non-Patent Citations (3)
| Title |
|---|
| Rapid PCR test for Streptococcus suis serotype 7;Smith H E等;《FEMS Microbiology Letters》;19991231;第178卷;摘要、第266页右栏第2.4节、第269页右栏第3.4节，图2 * |
| 张文东等.猪链球菌PCR检测技术研究进展.《动物医学进展》.2006,第27卷(第6期), * |
| 郑升博登.多重PCR方法检测猪链球菌主要致病血清型及其毒力因子.《上海交通大学学报(农业科学版)》.2010,第28卷(第1期), * |
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