CN107022629A - A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application - Google Patents
A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application Download PDFInfo
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- C12Q—MEASURING 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
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Abstract
The invention discloses a kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application, the detection primer group includes sense primer sip F 1 and anti-sense primer sip R 1, the sense primer isp F 1 and anti-sense primer isp R 1 that detect streptococcus dysgalactiae isp genes, the sense primer pauA F 1 and anti-sense primer pauA R 1 of streptococcus uberis pauA genes, the sense primer nuc F 1 and anti-sense primer nuc R 1 for detecting staphylococcus aureus nuc genes of detection Streptococcusagalactiae sip genes；Above-mentioned application is that the primer sets are applied in mastitis for milk cows pathogen multi-PCR detection method；This method detection time is short, cost is low, specific high and result judgement is simple.
Description
Technical field
The present invention relates to a kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application, belong to microorganism
Technical field.
Background technology
Milk is one of most ancient natural drink, is described as " white blood ", containing abundant mineral matter, with very high
Nutritive value.With the development of China's economic, the living standard of people is improved constantly, and the consumption demand to dairy products increases very
It hurry up.Mastitis for milk cows is one of principal disease of the Chinese milk cow production development of restriction, not only reduces the output of milk and causes economy
Loss, and it is greatly reduced the quality of breast.
Mastitis for milk cows (Bouine Mastitis) is the inflammation of the breast as caused by the various causes of disease, is the characteristics of main
Physicochemical property occurs for milk and pathological change etc. occurs for bacteriology change, breast tissue.The morbidity of China's mastitis for milk cows is very high,
It is reported that the recall rate of most of China Dairy Cattle mammitis is between 40%~65%.Causing the cause of disease of mammitis has carefully
The kind more than 150 such as bacterium, virus, fungi, Mycoplasma, main cause of disease is that streptococcus, staphylococcus aureus, Escherichia coli and branch are former
Body.The original Streptococcusagalactiae of wherein main spreading venereal diseases (Streptococcus agalactiae), staphylococcus aureus
(Staphylococcus aureus) and Mycoplasma (Mycoplasma), Environmental cause of disease has Escherichia coli (Escherichia
Coli), streptococcus uberis (Streptococcus uberis), streptococcus dysgalactiae (Streptococcus
Dysgalactiae), streptococcus pneumonia (Streptococcus pneumoniae), mycobacterium (Mycobacteria), wax
Sample bacillus (Bacillus cereus), pasteurella multocida (Pasteurellamultocida), Pseudomonas aeruginosa
(Pseudomonas aeruginosa), salmonella (Salmonella), bacteroid (Bacteroides), fungi
(Fungus) etc..
Mastitis for milk cows is one of maximum disease of cattle farm harm, seriously hinders the sound development of dairy.At present
Bacterium infection is still topmost in the complicated pathogenic factor of mastitis for milk cows.Traditional authentication method is mainly by thin
The classical ways such as bacterium separation identification, biochemical identification, but classical way is cumbersome, time-consuming, recall rate is low, and the bacterium is accurately examined
Survey and disease treats very unfavorable in time.It is born to later 1980s Polymerization chain reaction technology, greatly promotes
The development of molecular biology science and technology, and promote the tremendous development of the horizontal detection technique of nucleic acid of pathogenic microorganism, as most heavy
One of detection technique wanted.The round pcr of existing detection mastitis for milk cows pathogen has PCR, multiplex PCR, real time fluorescent quantitative
PCR and LAMP methods etc..Wherein multiplex PCR, which has, can detect a variety of cause of diseases, simple to operate, the characteristics of cost is low simultaneously.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the invention is to provide a kind of mastitis for milk cows pathogen
Multiple PCR detection primer group, using the primer sets can detect simultaneously Streptococcusagalactiae, streptococcus dysgalactiae, streptococcus uberis and
4 kinds of pathogens of staphylococcus aureus, detection efficiency is greatly improved.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：
A kind of multiple PCR detection primer group of mastitis for milk cows pathogen, including detection Streptococcusagalactiae sip genes are upper
Trip primer sip-F-1 and anti-sense primer sip-R-1, the sense primer isp-F-1 of detection streptococcus dysgalactiae isp genes and downstream are drawn
Thing isp-R-1, the sense primer pauA-F-1 of streptococcus uberis pauA genes and anti-sense primer pauA-R-1, the golden yellow Portugal of detection
The sense primer nuc-F-1 and anti-sense primer nuc-R-1 of grape coccus nuc genes；The nucleotides sequence of primer sets is classified as：
Preferably, it is 605bp that obtained purpose band size is expanded by primer sip-F-1 and sip-R-1；By drawing
The purpose band size that thing isp-F-1 and isp-R-1 amplification are obtained is 385bp；Expanded by primer pauA-F-1 and pauA-R-1
It is 273bp to increase obtained purpose band size；Expanding obtained purpose band size by primer nuc-F-1 and nuc-R-1 is
155bp。
Second object of the present invention is answering for the multiple PCR detection primer group for providing above-mentioned mastitis for milk cows pathogen
With by the primer sets applied to PCR detection method, this method detection time is short, cost is low, specific high and result judgement letter
It is single.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：
A kind of application of the multiple PCR detection primer group of above-mentioned mastitis for milk cows pathogen, for by the primer sets application
In mastitis for milk cows pathogen multi-PCR detection method.
Preferably, the step of mastitis for milk cows pathogen multi-PCR detection method includes：
1) sample DNA is extracted, enters performing PCR amplification；50 μ L PCR detection architectures are：
Taq enzyme, 3.75U,
10 × PCR buffer solutions, 5 μ L,
MgCl2, concentration is 1.5mmol/L,
DNTP, concentration is 200 μm of ol/L,
Sip-F-1 and sip-R-1 primers, respective concentration is 0.05 μm of ol/L,
Isp-F-1 and isp-R-1 primers, respective concentration is 0.06 μm of ol/L,
PauA-F-1 and pauA-R-1 primers, respective concentration is 0.16 μm of ol/L,
Nuc-F-1 and nuc-R-1 primers, respective concentration is 0.16 μm of ol/L,
Sample DNA templates 50ng,
ddH2O is supplied to 50 μ L；
2) electrophoresis detection amplified production is condensed by agarose.
Preferably, step 1) in, PCR amplification condition is：95℃5min；95 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 90s,
35 circulations；72 DEG C, 10min.
Preferably, step 3) in, obtained electrophoretogram is compared with positive, negative control, if in electrophoretogram
There is amplified band in 605bp positions, then illustrate to contain Streptococcusagalactiae in sample；If existed in electrophoretogram in 385bp positions
Amplified band, then illustrate to contain streptococcus dysgalactiae in sample；If electrophoretogram has amplified band in 273bp positions, illustrate
Contain streptococcus uberis in sample；If electrophoretogram has amplified band in 155bp positions, illustrate in sample containing golden yellow
Staphylococcus；If there is not above-mentioned band, illustrate not containing corresponding bacterium in sample.
Compared with prior art, the beneficial effects of the present invention are：
1st, the multiple PCR detection primer group for the mastitis for milk cows pathogen that the present invention is provided, can be using the primer sets simultaneously
Streptococcusagalactiae, streptococcus dysgalactiae, 4 kinds of pathogens of streptococcus uberis and staphylococcus aureus are detected, detection efficiency is carried significantly
It is high；
2nd, primer sets are applied in multi-PCR detection method by the present invention, and the detection method can quickly, efficiently, accurately
Whether detect measuring samples is Streptococcusagalactiae, streptococcus dysgalactiae, streptococcus uberis, the list of staphylococcus aureus pathogenic bacteria
One or mixed infection；In addition, this detection method can also be used for milk cattle infected Streptococcusagalactiae, streptococcus dysgalactiae, streptococcus uberis,
The epidemiology survey of staphylococcus aureus and curative effect monitoring, and science can be provided to the treatment of clinical, subclinical mammitis
Foundation, and to improving detection, the monitoring level of mammitis, ensure that milk and its dairy foodstuff are significant safely.
Brief description of the drawings
Fig. 1 is the specific evaluation test result of embodiment 2；
Fig. 2 is the sensitivity assessment result of the test of the Streptococcusagalactiae of embodiment 3；
Fig. 3 is the sensitivity assessment result of the test of the streptococcus dysgalactiae of embodiment 3；
Fig. 4 is the sensitivity assessment result of the test of the streptococcus uberis of embodiment 3；
Fig. 5 is the sensitivity assessment result of the test of the staphylococcus aureus of embodiment 3.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further：
Embodiment 1：The structure of pMD-18T-pauA recombinant vectors
Streptococcusagalactiae sip genes (accession number HQ878436), the streptococcus dysgalactiae isp genes announced according to GenBank
(accession number CP002215), streptococcus uberis pauA genes (accession number KT006567.1), staphylococcus aureus nuc genes
(accession number DQ399678) designs primer, obtains：The sense primer sip-F-2 and downstream for expanding Streptococcusagalactiae sip genes draw
Thing sip-R-2, the sense primer isp-F-2 and anti-sense primer isp-R-2 that expand streptococcus dysgalactiae isp genes, amplification breast chain
The sense primer pauA-F-2 and anti-sense primer pauA-R-2 of coccus pauA genes, amplification staphylococcus aureus nuc genes
Sense primer nuc-F-2 and anti-sense primer nuc-R-2；The nucleotides sequence of primer is classified as：
Then using genomic DNA as template, sip, isp, pauA and nuc full length sequence, purpose fragment connection pMD- are expanded
18T carriers, are identified through PCR, and positive colony delivers to Invitrogen's sequencing, and sequence is compared through Blast,
PMD-18T-sip is 99% with sip gene orders similitude, and sequence is as shown in SEQ ID No.17；PMD-18T-isp and isp
Gene order similitude is 99%, and sequence is as shown in SEQ ID No.18；PMD-18T-pauA and pauA gene order similitudes
For 100%, sequence is as shown in SEQ ID No.19；PMD-18T-nuc is 99%, sequence such as SEQ with nuc gene orders similitude
Shown in ID No.20.Illustrate to have successfully been obtained sip, isp, pauA and nuc gene, the gene of acquisition is used for follow-up test.
Embodiment 2：Specific evaluation test
To the Streptococcusagalactiae for being clinically separated and being preserved by this laboratory, streptococcus dysgalactiae, streptococcus uberis, golden yellow Portugal
Grape coccus, Escherichia coli, salmonella, streptococcus pneumonia and bacillus cereus carry out pcr amplification reaction,
50 μ L PCR detection architectures are：
Taq enzyme, 3.75U,
10 × PCR buffer solutions, 5 μ L,
MgCl2, concentration is 1.5mmol/L,
DNTP, concentration is 200 μm of ol/L,
Sip-F-1 and sip-R-1 primers, respective concentration is 0.05 μm of ol/L,
Isp-F-1 and isp-R-1 primers, respective concentration is 0.06 μm of ol/L,
PauA-F-1 and pauA-R-1 primers, respective concentration is 0.16 μm of ol/L,
Nuc-F-1 and nuc-R-1 primers, respective concentration is 0.16 μm of ol/L,
Sample DNA templates 50ng,
ddH2O is supplied to 50 μ L；
PCR condition is：95℃5min；95 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 90s, 35 circulations；72 DEG C, 10min.
The nucleotides sequence of primer is classified as：
Four kinds of recombinant vectors and sterile ddH in embodiment 1 are set2O is respectively positive and negative control.1.5% concentration
Agarose gel electrophoresis detection pcr amplification product.
Specific evaluation test result is as shown in figure 1, swimming lane M in figure：DL1,000 DNA Marker, swimming lane 1, which contains, to be whether there is
Streptococcus lactis, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus, swimming lane 2-11 are respectively Streptococcusagalactiae, stop breast
Streptococcus, streptococcus uberis, staphylococcus aureus, salmonella, Escherichia coli, bacillus cereus, streptococcus pneumonia, the moon
Property control and positive control.It can be seen that in addition to respective strap and positive control occurs in correspondence bacterium, the expansion of remaining sample
Increase result and be showed no single purpose band, show that this method specificity is good.
Embodiment 3：Sensitivity assessment is tested
Extract constructed recombinant vector, sterile ddH in embodiment 12O is with 10 times of gradient dilutions, and it is 10 to take copy number8、107、
106、105、104、103、1027 concentration gradients be respectively template, sterile ddH is set2O is negative control.PCR reaction systems
It is identical in embodiment 2.The agarose gel electrophoresis detection pcr amplification product of 1.5% concentration.
The sensitivity assessment result of the test of Streptococcusagalactiae is as shown in Fig. 2 swimming lane 1-7 is respectively in figure：Copy number is
108、107、106、105、104、103、102Recombinant vector, swimming lane 8 be sterile ddH2O negative controls, swimming lane 9 is that four kinds of restructuring are carried
Body positive control, swimming lane M：DL1,000 DNA Marker.It can be seen that PCR detection is limited to 105Copy number.
Streptococcus dysgalactiae sensitivity assessment result of the test is shown in that swimming lane 1-7 is respectively in Fig. 3, figure：Copy number is 108、107、
106、105、104、103、102Recombinant vector, swimming lane 8 be sterile ddH2O negative controls, swimming lane 9 is four kinds of recombinant vector positives
Control, swimming lane M：DL1,000 DNA Marker.It can be seen that PCR detection is limited to 104Copy number.
Streptococcus uberis sensitivity assessment result of the test is shown in that swimming lane 1-7 is respectively in Fig. 4, figure：Copy number is 108、107、
106、105、104、103、102Recombinant vector, swimming lane 8 be sterile ddH2O negative controls, swimming lane 9 is four kinds of recombinant vector positives
Control, swimming lane M：DL1,000 DNA Marker.It can be seen that PCR detection is limited to 105Copy number.
Staphylococcus aureus sensitivity assessment result of the test is shown in that swimming lane 1-7 is respectively in Fig. 5, figure：Copy number is 108、
107、106、105、104、103、102Recombinant vector, swimming lane 8 be sterile ddH2O negative controls, swimming lane 9 is four kinds of recombinant vectors
Positive control, swimming lane M：DL1,000 DNA Marker.It can be seen that PCR detection is limited to 105Copy number.
Proved by above-mentioned implementation, a kind of cow breast pathogen multi-PCR detection method set up has good spy
Different in nature and preferable sensitivity, this method has extreme high reliability.
Therefore, this research is established for Streptococcusagalactiae, streptococcus dysgalactiae, streptococcus uberis and staphylococcus aureus
Multiplex PCR, can fast and accurately simultaneously this four kinds of encountered pathogenic bacterias are detected, be mastitis for milk cows
Integrated control research provide technical support.
For those skilled in the art, technical scheme that can be as described above and design, make other each
It is kind corresponding to change and deform, and all these change and deformation should all belong to the protection model of the claims in the present invention
Within enclosing.
SEQUENCE LISTING
<110>Guangdong Haida Husbandry and Veterinary Institute Co., Ltd.
<120>A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application
<130> 2017
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
ctcaatacaa tttcggaagg 20
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<212> DNA
<213>It is artificial synthesized
<400> 2
actccataag ttgacgcta 19
<210> 3
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 3
gtcagtcgca aagttcaac 19
<210> 4
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 4
cttatcccgt cgttatgg 18
<210> 5
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 5
tccacccact acctattt 18
<210> 6
<211> 18
<212> DNA
<213>It is artificial synthesized
<400> 6
gagtttcacc gagttctt 18
<210> 7
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<213>It is artificial synthesized
<400> 7
ataaagaacc tgcgacat 18
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<213>It is artificial synthesized
<400> 8
acttgcttca ggaccata 18
<210> 9
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 9
atggaaatga ataaaaaggt acta 24
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<212> DNA
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<400> 10
ttagttaaag gatacgtgaa cg 22
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ttttttaccg accacatagg tc 22
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<213>It is artificial synthesized
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aataaaaaca agttaagagt g 21
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tttgggaata tttggttgtg c 21
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ggatacttca tttaaggttt at 22
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gttatgacag actacttatt aag 23
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<212> DNA
<213>It is artificial synthesized
<400> 16
ccagcgttgt cttcgctcc 19
<210> 17
<211> 1302
<212> DNA
<213>It is artificial synthesized
<400> 17
atggaaatga ataaaaaggt actattgaca tcgacaatgg cagcttcgct attatcagtc 60
gcaagtgttc aagcacaaga aacagatacg acgtggacag cacgtactgt ttcagaggta 120
aaggctgatt tggtaaagca agacaataaa tcatcatata ctgtgaaata tggtgataca 180
ctaagcgtta tttcagaagc aatgtcaatt gatatgaatg tcttagcaaa aattaataac 240
attgcagata tcaatcttat ttatcctgag acaacactga cagtaactta cgatcagaag 300
agtcatactg ccacttcaat gaaaatagaa acaccagcaa caaatgctgc tggtcaaaca 360
acagctactg tcgatttgaa aaccaatcaa gtttctgttg cagaccaaaa agtttctctc 420
aatacaattt cggaaggtat gacaccagaa gcagcaacaa cgattgtttc gccaatgaag 480
acatattctt ctgcgccagc tttgaaatca aaagaagtat tagcacaagg gcaagctgtt 540
agtcaagcag cagctaatga acaggtatca ccagctcctg tgaagtcgat tacttcagaa 600
gttccagcag ctaaagagga agttaaacca actcagacgt cagtcagtca gtcaacaaca 660
gtatcaccag cttctgttgc cgctgaaaca ccagctccag tagctaaagt agcaccggta 720
agaactgtag cagcccctag agtggcaagt gttaaagtag tcactcctaa agtagaaact 780
ggtgcatcac cagagcatgt atcagctcca gcagttcctg tgactacgac ttcaacagct 840
acagacagta agttacaagc gactgaagtt aagagcgttc cggtagcaca aaaagctcca 900
acagcaacac cggtagcaca accagcttca acaacaaatg cagtagctgc acatcctgaa 960
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acaaatagta tttatggacc tgctaatact tggaatgcaa tgccagatcg tggtggcgtt 1260
actgccaacc actatgacca cgttcacgta tcctttaact aa 1302
<210> 18
<211> 1494
<212> DNA
<213>It is artificial synthesized
<400> 18
tgcatgcctg caggtcgacg attaataaaa acaagttatt aagagtgatg atgttactaa 60
tccttttagc tccaactgct ggaagtgtga cagtattagc tcaggatgct ttgcctgaag 120
tcaatcaagc aactgcgaca agcactgatg acgtcacaag tactgactcg gactctcaga 180
aagaaaaata ccctaatgtg acgacagaag atcaaactaa cccagtttca tcagacgata 240
tttctgccaa atctgatgtt cttaatcctg cagagcctac tcgtccagaa cctgaagacc 300
aaacagatca gtcaacaaca cctaatgata ctattcctgg tcaagatcaa ggacacgaca 360
agactcctag cagtcaagag ccatctaaaa cacctcagga aagccctgtt cccgttagcc 420
ctgaaccaca agttccggca actgtgacac ctcttccgtg gtcacctttt gcctctgatt 480
taaatttgga gataccagta cttccatctg ttaatactta tgcggcctat gtggagcatt 540
ggagtggtaa agatgcttat acgcataatc tcttatcccg tcgttatggc atcaaagcag 600
agcaaattga tggttatttg aaatcaacag gcatttctta tgataaaact cgtatcaatg 660
gtgagaaatt gttgcaatgg gagaaaaaaa gtggactaga tgtccgtgcc attgttgcta 720
ttgccatgtt agaaagttct ttgggaacca aaggtgttgc taccttactt ggtgccaata 780
tgtttggtta tgcggccttt gacatagacc caacacaggc gagtaaattt aacgatgatg 840
tggctatcat taaaatgact caagaaacca tcatcaagaa taaaaatact aattttgccc 900
ttcaagatgc aaaagctgct aagttctcac taggacagtt gaactttgcg actgacggtg 960
gagtttactt tactgacatc actggaagtg gtaaacgtcg tgcccaaatc atggaagata 1020
tggatgaatg gattgatgcc catggaggca ctccagctat cccagcagaa ttgaaaattc 1080
aatcttcttc gagctttgca gaagtgccaa caggctacaa actctctaag agttacgatg 1140
ttttgagtta tcaagcatct agttacgctt ggggtcaatg tacttggtat gtttataacc 1200
gcgccaaaga actgggctac caattcgatc cattcatggg aaatggtggg gactggaaat 1260
acaaggcagg ttatgctctt tctaagacac caaaagttgg gtatgcagta tcttttgcgc 1320
caggtcaagc tggtgctgat gggatgtacg gccatgtctc tattgtggaa gatgtcagaa 1380
aagacggctc tatccttatc tcagaatcta actgtatagg cttaggaaaa gtatcctaca 1440
gaacattcac tgcccaacaa gcagaccaat tgacctatgt ggtcggtaaa aaaa 1494
<210> 19
<211> 840
<212> DNA
<213>It is artificial synthesized
<400> 19
tgggaatatt tggttgtgct actcaaccat caaaggttgc agcaataacc ggttatgatt 60
ccgactacta cgctagatat attgatcccg atgaaaataa aataacattt gccataaatg 120
ttgatggttt tgtcgaaggt agtaatcaag aaatccttat tagaggaatt catcatgttt 180
taacagatca aaaccaaaag attgttacaa aggccgagtt gttagacgct attagacatc 240
aaatggttct tctacaattg gattattcct atgaactagt cgactttgcg cctgatgcac 300
aattattaac acgagatcga cggcttttat ttgccaatcg aaattttgag gaatccgtat 360
cacttgaaga tactattcaa gaataccttt taaaagggca tgttattctc agaaaacggg 420
ttgaagaacc tatcactcat cctactgaga ctgctaatat tgagtataaa gttcaattcg 480
cgactaaaga tggggaattc cacccactac ctatttttgt agactacgga gaaaaacata 540
ttggagaaaa attaacctct gacgagtttc gaaaaattgc agaagaaaag cttttgcaac 600
gctaccctga ctatatgatt gatcaaaaag aatatactat cattaaacac aattctcttg 660
gtcaacttcc aagatattat tcttatccag atgatttcag ctacgaaatt caagataggc 720
aacgtatcat ggctaaggac ccaaagtccg gaaaagaact cggtgaaact caaagtattg 780
ataatgtttt tgagaaatac cttattacca aaaaaagtta taaaccttaa atgaagtatc 840
<210> 20
<211> 675
<212> DNA
<213>It is artificial synthesized
<400> 20
tttatgacag actacttatt aagtgctggc atatgtatgg caatcgtttc aatattactt 60
atagggatgg ctatcagtaa tgtttcgaaa gggcaatacg caaagaggtt tttctatttc 120
gctactagtt gtttagtgtt aactttagtt gtagtttcaa gtctaagtag ctcagcaaat 180
gcatcacaaa cagataatgg cgtaaataga agtggttctg aagatccaac agtatatagt 240
gcaacttcaa ctaaaaaatt acataaagaa cctgcgacat taattaaagc gattgatggt 300
gatacggtta aattaatgta caaaggtcaa ccaatgacat tcagactatt attggttgat 360
acacctgaaa caaagcatcc taaaaaaggt gtagagaaat atggtcctga agcaagtgca 420
tttacgaaaa aaatggtaga aaatgcaaag aaaattgaag tcgagtttga caaaggtcaa 480
agaactgata aatatggacg tggcttagcg tatatttatg ctgatggaaa aatggtaaac 540
gaagctttag ttcgtcaagg cttggctaaa gttgcttatg tttataaacc taacaataca 600
catgaacaac ttttaagaaa aagtgaagca caagcaaaaa aagagaaatt aaatatttgg 660
agcgaagaca acgct 675
Claims (6)
1. a kind of multiple PCR detection primer group of mastitis for milk cows pathogen, it is characterised in that including detection Streptococcusagalactiae
The sense primer sip-F-1 and anti-sense primer sip-R-1 of sip genes, the sense primer isp- for detecting streptococcus dysgalactiae isp genes
F-1 and anti-sense primer isp-R-1, the sense primer pauA-F-1 of streptococcus uberis pauA genes and anti-sense primer pauA-R-1,
Detect the sense primer nuc-F-1 and anti-sense primer nuc-R-1 of staphylococcus aureus nuc genes；The nucleosides of the primer sets
Acid sequence is：
2. the multiple PCR detection primer group of mastitis for milk cows pathogen as claimed in claim 1, it is characterised in that by drawing
The purpose band size that thing sip-F-1 and sip-R-1 amplification are obtained is 605bp；Expanded by primer isp-F-1 and isp-R-1
Obtained purpose band size is 385bp；Expanding obtained purpose band size by primer pauA-F-1 and pauA-R-1 is
273bp；It is 155bp that obtained purpose band size is expanded by primer nuc-F-1 and nuc-R-1.
3. a kind of application of the multiple PCR detection primer group of mastitis for milk cows pathogen as claimed in claim 1, its feature exists
In：The primer sets are applied in mastitis for milk cows pathogen multi-PCR detection method.
4. the application of the multiple PCR detection primer group of mastitis for milk cows pathogen as claimed in claim 3, it is characterised in that：
The step of mastitis for milk cows pathogen multi-PCR detection method, includes：
1) sample DNA is extracted, enters performing PCR amplification；50 μ L PCR detection architectures are：
Taq enzyme, 3.75U,
10 × PCR buffer solutions, 5 μ L,
MgCl2, concentration is 1.5mmol/L,
DNTP, concentration is 200 μm of ol/L,
Sip-F-1 and sip-R-1 primers, respective concentration is 0.05 μm of ol/L,
Isp-F-1 and isp-R-1 primers, respective concentration is 0.06 μm of ol/L,
PauA-F-1 and pauA-R-1 primers, respective concentration is 0.16 μm of ol/L,
Nuc-F-1 and nuc-R-1 primers, respective concentration is 0.16 μm of ol/L,
Sample DNA templates 50ng,
ddH2O is supplied to 50 μ L；
2) electrophoresis detection amplified production is condensed by agarose.
5. the application of the multiple PCR detection primer group of mastitis for milk cows pathogen as claimed in claim 4, it is characterised in that：
Step 1) in, PCR amplification condition is：95℃5min；95 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 90s, 35 circulations；72 DEG C,
10min。
6. the application of the multiple PCR detection primer group of mastitis for milk cows pathogen as claimed in claim 4, it is characterised in that：
Step 3) in, obtained electrophoretogram is compared with positive, negative control, if there is amplification bar in electrophoretogram in 605bp positions
Band, then illustrate to contain Streptococcusagalactiae in sample；If there is amplified band in 385bp positions in electrophoretogram, illustrate sample
In contain streptococcus dysgalactiae；If electrophoretogram has amplified band in 273bp positions, illustrate to contain breast hammer in sample
Bacterium；If electrophoretogram has amplified band in 155bp positions, illustrate to contain staphylococcus aureus in sample；If no
There is above-mentioned band, then illustrate not containing corresponding bacterium in sample.
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| CN114959083B (en) * | 2022-06-29 | 2023-08-22 | 西北农林科技大学 | A kind of triple PCR detection primer set and kit |
| CN116640867A (en) * | 2023-06-27 | 2023-08-25 | 四川农业大学 | A primer pair and probe combination for RAA-LFD detection of Streptococcus dysgalactiae and its application |
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Application publication date: 20170808 |