CN100560735C - A kind of pathogenic bacteria multiple PCR detection kit and detection method thereof - Google Patents
A kind of pathogenic bacteria multiple PCR detection kit and detection method thereof Download PDFInfo
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- CN100560735C CN100560735C CNB2005100122423A CN200510012242A CN100560735C CN 100560735 C CN100560735 C CN 100560735C CN B2005100122423 A CNB2005100122423 A CN B2005100122423A CN 200510012242 A CN200510012242 A CN 200510012242A CN 100560735 C CN100560735 C CN 100560735C
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Abstract
The present invention be directed to mammitis of cow The main pathogenic fungi streptococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae and intestinal bacteria 16S-23S rRNA interval, synthetic 4 pairs of corresponding specific amplification primers are used the multi-PRC reaction system mammitis of cow pathogenic bacteria are detected.The length of amplified fragments is respectively streptococcus agalactiae 162bp, streptococcus dysgalactiae 264bp, streptococcus aureus 439bp and intestinal bacteria 544bp.This multiple PCR detection kit and detection method high specificity, sensitivity.Can simultaneously four kinds of pathogenic bacterias be detected and identify, thereby reach the workload of reduction PCR diagnosis and the purpose of cost.
Description
Technical field
The invention belongs to the molecular Biological Detection field, be specifically related to four kinds of pathogenic bacteria multiple PCR detection kits of a kind of mammitis of cow and detection method thereof.
Background technology
Mammitis of cow (Bovine Mastitis) is the inflammation of cow mammary gland tissue, is mainly due to the mammary tissue pathogenic infection microorganism, is common disease, the frequently-occurring disease of milk cow.The pathogenic bacterium that cause mammitis of cow are about kind more than 150, based on streptococcus aureus, suis and intestinal bacteria, account for more than 90% of whole mammitis of cow case.Can be divided into recessive type and clinic mastitis according to clinical manifestation.The sickness rate of China's milk cow clinic mastitis is about 33.4%, causes that milk yield descends, sick breast is discarded, and suppurate in severe patient breast district, gangrene, atrophy, causes forever to lose milking capacity and be eliminated, and accounts for total 10% of ox of eliminating; Average the positive rate of latent mammitis milk cow is 73.9%, causes the decline of milk yield and the downgrade of dairy products, and the direct economic loss that causes every year is up to 1,200,000,000 yuans.
At present, China still lacks mammitis of cow pathogenic bacteria responsive fast detection and authenticate technology, and conventional microbiology detects and authentication method is time-consuming, effort, and be difficult to detect fast and accurately.Therefore, special, the sensitivity of research and industrialization development mammitis of cow pathogenic bacteria and rapid detection and authenticate technology, will improve the level of preventing and treating of China's mammitis of cow to integral body, improve the economic benefit of dairy, promote the development of dairy, play the sound assurance effect for society provides more high quality safety milk.
Summary of the invention
Purpose of the present invention aims to provide a kind of quick, responsive, special four kinds of pathogenic bacteria detection kit of mammitis of cow and detection method thereof.
The invention provides a kind of pathogenic bacteria PCR detection kit, it is characterized in that it contains following primer:
(1) primer of detection streptococcus aureus (Staphylococcus aureus):
Upstream primer 5 ' AAG GAT ATA TTC GGA ACA TCT 3 '
Downstream primer 5 ' TAA GTC AAA CGT TAA CAT ACG 3 '
(2) primer of detection streptococcus agalactiae (Streptococcus agalactiae):
Upstream primer 5 ' GCT AGG CTC CAT TGA ATC 3 '
Downstream primer 5 ' TTAACC TAG TTT CTT TAA AAC TAG AA 3 '
(3) primer of detection streptococcus dysgalactiae (Strpetococcus dysgalactiae):
Upstream primer 5 ' GAA CAC GTT AGG GTC GTC 3 '
Downstream primer 5 ' AGT ATA TCT TAA CTA GAA AAA CTA TTG 3 '
(4) primer of detection intestinal bacteria (Escherichia coli):
Upstream primer 5 ' AAG AAG CTT GCT TCT TTG CTG 3 '
Downstream primer 5 ' GAG CCC GGG GAT TTC ACA T 3 '.
Preferred test kit of the present invention, wherein each component final concentration is: primer 0.5 μ mol/L, Mg that primer 3.0 μ mol/L, the primer 1.25 μ mol/L that detect streptococcus agalactiae, the primer 1.5 μ mol/L that detect streptococcus dysgalactiae, the detection intestinal bacteria that detect streptococcus aureus are
2+1.0mmol/L, dNTP 0.175mmol/L, 10 * PCR reaction buffer, 4 μ L, TaqDNA polysaccharase 2U, supply volume to 40 μ L with the sterilization deionized water.
The present invention also provides a kind of detection method, and it comprises the steps:
(1) template DNA is added in the test kit of the present invention;
(2) hatch on the pcr amplification instrument, the PCR loop parameter is:
95 ℃ of pre-sex change 5min;
95 ℃ of sex change 1min, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, 36 circulations;
72 ℃ are extended 7min eventually, and 4 ℃ of 10min finish pcr amplification.
(3) amplified production is carried out electrophoresis detection.
Detection method of the present invention, wherein the specific band of amplified fragments is respectively streptococcus aureus 439bp, streptococcus agalactiae 162bp, streptococcus dysgalactiae 264bp, intestinal bacteria 544bp behind the electrophoresis.
The application of test kit of the present invention in detecting milk cow streptococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, intestinal bacteria.
Four kinds of pathogenic bacteria multiple PCR detection kits of mammitis of cow of the present invention and detection method, be at mammitis of cow The main pathogenic fungi streptococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae and intestinal bacteria 16S-23S rRNA interval, design 4 pairs of pcr amplification primers, use the multi-PRC reaction system four kinds of pathogenic bacterias of mammitis of cow are detected.The length of amplified fragments is respectively streptococcus agalactiae 162bp, streptococcus dysgalactiae 264bp, streptococcus aureus 439bp and intestinal bacteria 544bp.Adopting method of the present invention to can be used for the mammitis of cow pathogenic bacteria detects.
Product advantage applies of the present invention exists:
1. multiple PCR method has good specificity, and it has extensive applicability to the same bacterioid of different strains.
2. the substance PCR method of multiple PCR method and each pathogenic bacteria is the same has quite high susceptibility, can detect the dna profiling of trace.
3. detection kit of the present invention, market application foreground is wide.
Description of drawings
Fig. 1: test kit detected result figure of the present invention.
M:DL2000; 1: streptococcus agalactiae; 2: streptococcus dysgalactiae; 3: streptococcus aureus; 4: intestinal bacteria; 5: streptococcus agalactiae+streptococcus dysgalactiae; 6: streptococcus agalactiae+streptococcus aureus; 7: streptococcus agalactiae+intestinal bacteria; 8: streptococcus dysgalactiae+streptococcus aureus; 9: streptococcus dysgalactiae+intestinal bacteria; 10: streptococcus aureus+intestinal bacteria; 11: streptococcus agalactiae+streptococcus dysgalactiae+streptococcus aureus; 12: streptococcus agalactiae+streptococcus dysgalactiae+intestinal bacteria; 13: streptococcus agalactiae+streptococcus aureus+intestinal bacteria; 14: streptococcus dysgalactiae+streptococcus aureus+intestinal bacteria; 15: streptococcus agalactiae+streptococcus dysgalactiae+streptococcus aureus+intestinal bacteria.
Embodiment
(1) gathers pathological material of disease, pathogenic bacteria cultivation
Clean cow breast with warm water earlier when the milk sample is gathered and clean, use the tincture of iodine cotton balls wiping nipple of 75% cotton ball soaked in alcohol or 5% again, squeeze go each breast district the 1st～3 milk, then milk is clamp-oned sterilization in advance in vitro, the milk amount is the 5mL/ pipe, performs mark, and 4 ℃ of preservations are to be checked.The new fresh milk sample of gathering is shaken up, every part of milk sample is drawn 0.1mL and is inoculated in 10% sheep blood agar substratum respectively, puts 37 ℃ of thermostat containers and cultivates 48h, observes colonial morphology, the different single colony inoculation of picking form is put 37 ℃ and is cultivated 48h in the sheep blood agar culture-medium.The picking purifying is cultivated colony inoculation 37 ℃ of violent joltings in the THB liquid nutrient medium and is cultivated 18h.
(2) preparation of pathogenic bacteria masterplate DNA (boiling method)
Get the centrifugal 3min of 1mL bacterium liquid 8000rpm/min (4 ℃); Abandon supernatant, and residual droplets is exhausted.Add 500 μ L TE (pH8.0) and boil 10min for 100 ℃; Put into mixture of ice and water immediately, the centrifugal 3min of cooling back 10000rpm/min (4 ℃); Get the masterplate of supernatant as the PCR reaction ,-20 ℃ of preservations are standby.
(3) multi-PRC reaction
With the DNA that extracts pathological material of disease, add in the detection kit, in PCR reaction instrument, carry out PCR.
Test kit 40 μ L reaction systems: wherein each component final concentration is: primer 0.5 μ mol/L, Mg that primer 3.0 μ mol/L, the primer 1.25 μ mol/L that detect streptococcus agalactiae, the primer 1.5 μ mol/L that detect streptococcus dysgalactiae, the detection intestinal bacteria that detect streptococcus aureus are
2+1.0mmol/L, dNTP 0.175mmol/L, 10 * PCR reaction buffer, 4 μ L, TaqDNA polysaccharase 2U, supply volume to 40 μ L with the sterilization deionized water.
Each 2 μ L of each pathogenic bacteria dna profiling.
Hatch on the pcr amplification instrument, the PCR loop parameter is:
95 ℃ of pre-sex change 5min;
95 ℃ of sex change 1min, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, 36 circulations;
72 ℃ are extended 7min eventually, and 4 ℃ of 10min finish pcr amplification.
(4) agarose electrophoretic analysis PCR product
1 * TAE damping fluid is mixed with 2% sepharose solution, and after the microwave oven heating was dissolved agarose fully, adding ethidium bromide (5mg/mL EB) was 0.5 μ g/mL to final concentration, encapsulating; Get electrophoresis sample solution (0.25% tetrabromophenol sulfonphthalein, the blue or green FF of 0.25% dimethylbenzene, the 40% sucrose) mixing for the treatment of electrophoretic DNA sample and 1/6 volume in right amount then, sample to be checked is added in the sample groove, carry out the constant voltage electrophoresis with 5V/cm.When sample electrophoresis to the appropriate location, with long-wave ultra violet lamp observe, the record result, with ultraviolet gel imaging system (Bio-Rad, Gel Doc 2000) the preservation (see figure 1) of taking pictures.The length of amplified fragments is respectively streptococcus agalactiae 162bp, streptococcus dysgalactiae 264bp, streptococcus aureus 439bp and intestinal bacteria 544bp behind the electrophoresis.Carry out detection and the evaluation of pathogenic bacteria according to having or not above fragment.
Claims (2)
1, a kind of pathogenic bacteria PCR detection kit is characterized in that it contains following primer:
(1) primer of detection streptococcus aureus:
Upstream primer 5 ' AAG GAT ATA TTC GGA ACA TCT 3 '
Downstream primer 5 ' TAA GTC AAA CGT TAA CAT ACG 3 '
(2) primer of detection streptococcus agalactiae:
Upstream primer 5 ' GCT AGG CTC CAT TGA ATC 3 '
Downstream primer 5 ' TTA ACC TAG TTT CTT TAA AAC TAG AA 3 '
(3) primer of detection streptococcus dysgalactiae:
Upstream primer 5 ' GAA CAC GTT AGG GTC GTC 3 '
Downstream primer 5 ' AGT ATA TCT TAA CTA GAA AAA CTA TTG 3 '
(4) detect colibacillary primer:
Upstream primer 5 ' AAG AAG CTT GCT TCT TTG CTG 3 '
Downstream primer 5 ' GAG CCC GGG GAT TTC ACA T 3 '.
2, test kit according to claim 1, it is characterized in that wherein each component final concentration is: the primer 3.0 μ mol/L, the primer 1.25 μ mol/L that detect streptococcus agalactiae, the primer 1.5 μ mol/L that detect streptococcus dysgalactiae, detection colibacillary primer 0.5 μ mol/L, the Mg that detect streptococcus aureus
2+1.0mmol/L, dNTP 0.175mmol/L, 10 * PCR reaction buffer, 4 μ L, Taq archaeal dna polymerase 2U, supply volume to 40 μ L with the sterilization deionized water.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942522A (en) * | 2010-10-14 | 2011-01-12 | 扬州大学 | Assay kit for identifying dairy cow mastitis resistance-related molecular marker |
Families Citing this family (8)
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| CN101307356A (en) * | 2008-04-29 | 2008-11-19 | 广州华峰生物科技有限公司 | Rapid diagnosis kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof |
| CN101597649B (en) * | 2009-06-03 | 2011-07-27 | 范红结 | PCR detection method for streptococcus equi subsp zooepidemicus and kit used thereby |
| CN102242202B (en) * | 2011-01-28 | 2013-05-01 | 通威股份有限公司 | Method and kit for detecting streptococcus agalactiae |
| CN102766680B (en) * | 2012-03-15 | 2014-01-01 | 深圳市生科源技术有限公司 | Staphylococcus aureus and hemolytic streptococcus detection kit and detection method |
| CN102851390B (en) * | 2012-10-17 | 2014-11-05 | 西北农林科技大学 | Preparation method of milk goat mastitis pathogen multi-PCR (polymerase chain reaction) detection kit |
| CN105199948A (en) * | 2015-09-29 | 2015-12-30 | 窦晓鸣 | Quick detection kit and method of Escherichia coli |
| CN106801103B (en) * | 2017-02-28 | 2021-05-14 | 中国水产科学研究院南海水产研究所 | Detection primer group, detection kit and multiplex PCR detection method for streptococcus agalactiae |
| CN110734990A (en) * | 2019-11-08 | 2020-01-31 | 南京农业大学 | Detection reagent, kit and application thereof |
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Non-Patent Citations (2)
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| 奶牛乳房炎四种主要病原菌PCR诊断方法的研究. 孙冰.黑龙江八一农垦大学硕士学位论文. 2004 |
| 奶牛乳房炎四种主要病原菌PCR诊断方法的研究. 孙冰.黑龙江八一农垦大学硕士学位论文. 2004 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942522A (en) * | 2010-10-14 | 2011-01-12 | 扬州大学 | Assay kit for identifying dairy cow mastitis resistance-related molecular marker |
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