CN103667510A - Streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit - Google Patents

Streptococcus suis 7 type PCR (Polymerase Chain Reaction) detection kit Download PDF

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CN103667510A
CN103667510A CN201310742629.9A CN201310742629A CN103667510A CN 103667510 A CN103667510 A CN 103667510A CN 201310742629 A CN201310742629 A CN 201310742629A CN 103667510 A CN103667510 A CN 103667510A
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detection kit
pcr
swine streptococcus
primer
detection
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CN103667510B (en
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杨灵芝
陈申秒
王风莲
周成成
杨琴
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SHANDONG BINZHOU BOLAIWEI BIOTECH CO Ltd
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SHANDONG BINZHOU BOLAIWEI BIOTECH CO Ltd
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14Streptococcus; Staphylococcus

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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

A kind of swine streptococcus 7 type PCR detection kit
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, is often catenation, different in size, is gram-positive cocci.On blood agar substratum, grow, 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 be protected bacterium, and opposing is engulfed.The existence of muramidase-released protein, extracellular factor has improved 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 that people's meningitis is 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 being the most frequently separated to.Er Denmark, except 2 type bacterial strains, one of serotype that 7 type bacterial strains are also 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 to very necessary.Since summer has set in 2006, some areas that economize in the Zhejiang of China, Jiangxi, Anhui, Jiangsu, Hunan etc., popularly take the swine disease epidemic situation that high heat is principal character 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 variation PRRS virus, but has report to show that swine streptococcus is a kind of important bacterial pathogen.Whether therefore, exist swine streptococcus 7 types and 7 type roles also to merit attention in hyperpyrexia, but do not have at present a kind ofly conveniently for detection of the test kit of swine streptococcus 7 types, 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, and a kind of quick, accurate, easy swine streptococcus 7 type PCR detection kit are provided.
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, the SDS that 18 μ L mass concentrations are 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 types.
Above-mentioned positive control is the PCR product of standard swine streptococcus 7 types.
In above-mentioned upper primer, R is degenerated primer.
Reactions steps: 50 ℃ of the first step 50 min, 1 circulation; 94 ℃ of 3 min of second step, 1 circulation; The 3rd 94 1 of step min, 52 ℃ of 1 min, 72 ℃ of 2 min, 35 circulations, the 4th 72 ℃ of steps are extended 7min, 1 circulation
Get the above-mentioned reaction product of 5 μ L and carry out 1.2% agarose gel electrophoresis check, with Marker DL2000 as a reference, TAE damping fluid is electrophoretic buffer, with 5V/cm electrophoresis 1h, gel imaging system observations;
Reclaim object band, 16 ℃ of 1.5 h is connected to PMD19-T Easy Vector, transformed competence colibacillus DH5 α, evenly coat the LB solid medium flat board containing Amp, IPTG and X-gal, cultivate 16h for 37 ℃, picking white colony is cultivated in a small amount, extract plasmid and do pcr amplification evaluation, by the order-checking of positive bacteria liquid, and adopt non-swine streptococcus 7 type DNA fragmentations as negative control, standard swine streptococcus 7 type DNA fragmentations are as positive control.
The invention has the beneficial effects as follows: the present invention has the fast advantage of detection of PCR detection technique, and this test kit cost low, adopted special primer, can meet minimum is the detection of 0.00984ng gene fragment PCR product, detection sensitivity is high.
Accompanying drawing explanation
Fig. 1 is sensitivity experiments result figure of the present invention;
1-98.4ng gene fragment PCR product, 2-9.84 ng gene fragment PCR product, 3-0.984ng gene fragment PCR product, 4-0.0984ng gene fragment PCR product, 5-0.00984ng gene fragment PCR product, 6-0.000984ng gene 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 product (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 can better 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, the SDS that 18 μ L mass concentrations are 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 types.
Above-mentioned positive control is the PCR product of standard swine streptococcus 7 types.
In above-mentioned upper primer, R is degenerated primer.

Claims (6)

1. 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, the SDS that 18 μ L mass concentrations are 10%, the TE damping fluid of 15 μ L, 5 μ L negative controls, 5 μ L positive controls.
2. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: described PCR damping fluid is by 25 mmol/L MgCl 23 μ L, 10 * PCR buffer, 10 μ L, 10mmol/L dNTPx2 μ L.
3. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: the sequence number of described upper primer is 5 '-AGCTCTAACACGAAATARRRR-3 ', and the sequence number of lower primer is 5 '-GTCAAACACCCTGGATAGCCG-3 '.
4. 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 types.
5. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: described positive control is the PCR product of standard swine streptococcus 7 types.
6. a kind of swine streptococcus 7 type PCR detection kit according to claim 1, is characterized in that: in described upper primer, R is degenerated primer.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SMITH H E等: "Rapid PCR test for Streptococcus suis serotype 7", 《FEMS MICROBIOLOGY LETTERS》 *
张文东等: "猪链球菌PCR检测技术研究进展", 《动物医学进展》 *
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)》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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