CN103409520A - PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof - Google Patents
PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof Download PDFInfo
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Abstract
A PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as a preparation method and a using method thereof, and belongs to the field of gene detection of anthropozoonosis pathogens. The PCR kit comprises a PCR liquid reactant, DNA polymerase, positive quality control standard substances and negative quality control standard substances; the PCR liquid reactant comprises four pairs of primers for identifying Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis and a PCR buffer solution containing dNTPs, Mg<2+> and double distilled water. The PCR kit has high practicability, can be used for directly detecting the type of Brucella in a contaminated sample, has high detection speed, high sensitivity (10<2>cfu/ml), high specificity and good repeatability, is safe, reliable, quick, simple and convenient, can be used for qualitative detection of the type of Brucella in the sample, and can replace traditional etiological and serological methods.
Description
Technical field
The present invention relates to the technique of gene detection field of infectious diseases common to human beings and animals pathogenic agent, be specifically related to a kind of four kinds of brucellar PCR test kits of cattle and sheep pig dog and preparation method thereof and using method of detecting simultaneously.
Background technology
Brucella is a kind of born of the same parents' endophyte, is gram-negative coccobacillus, ox, sheep, pig, dog, horse, deer, camel, people's susceptible.The brucella invasiveness is very strong, is mainly by digestive tract infection, complete mucocutaneously also can be subject to brucellar intrusion, causes brucellosis.
Brucellosis (Brucellosis), claim again the Mediterranean Sea remittent fever, and brucellosis is popular infectious diseases common to human beings and animals in a kind of world wide.The patient of world's brucellosis approximately has 500～6,000,000 people, annual new patient approximately has 500,000, the population that China is threatened by brucellosis approximately has 3.5 hundred million, this disease not only can cause huge financial loss, and in the disease popularity area, can cause crowd's high incidence, therefore quarantine and the prevention and control of brucellosis are seemed to particularly important.
Brucella is comprised of 9 kinds, is respectively B. abortus, brucella melitensis, pig kind brucella, kind of dog brucella, sheep epididymis kind brucella, sarin mouse kind brucella, vole type brucella, fin type brucella and whale type brucella.
At present, brucellar detection be take serological method as main, but do not seek unity of standard, serological method detects the level that can only reach Brucella, can not distinguish is the brucellosis which kind of brucella kind type causes, and there is cross reaction, had a strong impact on diagnosis, epidemiology survey and the Epidemic disease prevention of brucellosis.
Take the PCR test kit as the more traditional serological method of basic brucella detection method more applicable, and for the PCR detection method of brucella kind and Biotype Determination among continuous development and improvement.Therefore, set up a kind of brucellar multi-PCR detection method that can differentiate simultaneously these four kinds of cattle and sheep pig dog, have very important significance for quarantine and the prevention and control of brucellosis.
Summary of the invention
In order to solve the existing problem that can't distinguish simultaneously the kind type that causes infection with serological method detection brucella, the invention provides a kind of four kinds of brucellar PCR test kits of cattle and sheep pig dog and preparation method thereof and using method of detecting simultaneously.
The present invention is that the technical scheme that adopts of technical solution problem is as follows:
Detect simultaneously four kinds of brucellar PCR test kits of cattle and sheep pig dog, this PCR test kit is comprised of PCR reaction solution, archaeal dna polymerase, positive quality control standard substance and negative quality control standard product;
Described PCR reaction solution comprises identifies ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog, is respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
Described positive quality control standard substance comprise B. abortus positive quality control standard substance, brucella melitensis positive quality control standard substance, pig kind brucella positive quality control standard substance and kind of dog brucella positive quality control standard substance;
Described B. abortus positive quality control standard substance are comprised of the nucleotide fragments that contains 494 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described brucella melitensis positive quality control standard substance are comprised of the nucleotide fragments that contains 732 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described pig kind brucella positive quality control standard substance are comprised of the nucleotide fragments that contains 272 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described kind of dog brucella positive quality control standard substance are comprised of the pMD-18T recombinant plasmid that the nucleotide fragments that contains 272 bases forms,
The pMD-18T recombinant plasmid that the pMD-18T recombinant plasmid that the described nucleotide fragments that contains 494 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 732 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms and the nucleotide fragments that contains 272 bases form all can be bred in the bacillus coli DH 5 alpha competent cell;
The described sequence that contains the nucleotide fragments of 494 bases is:
5′-gacgaacggaatttttccaatcccatcgtttccgtttcacttggcctgccggcaacgtttcagtttggcggaatga?aacggacggacccgatcaccaaatatatcctgcaccatggcgacgtggttgtctggggcgggccgtcgcggcttttctatc?acggtattctaccattgaagtctggcgagcatgagcggctggggccgtttcggcttaatctgaccttccgaaaggcgttcta?gggcgtgtctgcatttaacgtaaccagatcatagcgcatgcgagatggacgaaacccatgaatgcggtcaatgttttctcgc?atcgcagcgcaatacgacgatagcgtttcaacttgttaaaaaagcattcaatctgatggcgttccttgtacagcctccagtcg?attgttggggcactggaacgtgttggattgaccttgatctgagccgttgccttgagatcgctggcaatgaaggcccttaagtg?atcggca-3′，
The described sequence that contains the nucleotide fragments of 732 bases is:
5′-tgccgatcacttaagggccttcattgccagcaatctcaaggcaacggctcagatcaaggtcaatccaacacgtt?ccagtgtcccaacaatcgactggaggctgtacaaggaacgccatcagattgaatgcttttttaacaagttgaaacgctatcgt?cgtattgcgctgcgatgcgagaaaacattgaccgcattcatgggcttcgtccatctcgcatgcgctatgatctggttacgttg?aatgcagacacgccctaggggtgaatctggaaattgtcagaaagacagtgcttcgtcacgctagagcgctcgctgccata?cttgcaacagtgacagcgataattgccgttattggctggtggcagggcgaagattggcgggtaagctattccaatctcgcta?ttgttaatggcgtctattggatattactgctctaccttctgtggattattctgagccgaaacactcctgattttttaggggtgccgc?ttgttaaggcgattcacgacaagaaacttcttatagtcgatggtgcaccatggctaagtttgggtgtcatgaccgcgatttacg?tcaaagacggtgaatatgagcggcttgtgtgcaccggagaggtagtaaacgttcagacaaataagctggttcagattcatat?ccggggttatgaagaaatttataacgatatcgaagctgttggtgaaaaacttaatcagaccagcaaggacgcgattt-3′，
The described sequence that contains the nucleotide fragments of 591 bases is:
5′-ctggtgggtatcgcattactcggcgccctggggctgacggggctgacaagtagcggcatcggggcggtggc?tgccggcgggcttctcggttttgccctgttcaaccgcccgcctgccagcatttttctcggcgattccggaagccccccattg?gggctgatcgtaggaacagccttgctgctgcttgcgcgtgaaacacacatcgtggtcgcgcttgttctgccgctttattatatt?ctggatgcgggcaccacaattgtcatgcgtgcagcccaaggtgagaacatcctcaaggctcactcgaaacatgcctatca?gatagcaaaacgcagtggctggagtgtgccgaaagtggtggcccatgtggcgcttttaaatacaatcctgatcgcctgcgt?ggtggccttgctggcgctggatcatccgctcgcacaactgacatttctgctggtcgcagccgttgccacactcattctgctg?ctcgatttccgcgggcgcttcaggaagctatgacctcgaaaagcaccttgcggaccatttcgatatccttcttttccatggcg?gtccgcagtaccgccaggctttcctgaa-3′，
The described sequence that contains the nucleotide fragments of 272 bases is:
5′-gatggagcaaacgctgaagcccaagcgaggccccgaaatagaccaggaagagcgtcaggagttccggca?gaccgcgaaagatggtggtgtagatattagccgcaagccgcatcgaaggttcgctggactgcttgcccagagcgaccag?aaaaccgaggatgagaccaacgggaagtgtggcgagtgccagactgacggtaacaagaagtcctttggcaatgctgata?ccccagccatcgggaccgaaactcaacaaggtggcgtagtgttcc-3′。
Described PCR reaction solution also comprises and contains dNTPs, Mg
2+, distilled water the PCR damping fluid.
The described nucleotide fragments that contains 494 bases is from B. abortus reference culture 544A, cloning and obtain, the described nucleotide fragments that contains 732 bases is from brucella melitensis reference culture 16M, cloning and obtain, the described nucleotide fragments that contains 591 bases is from B. abortus reference culture 544A, brucella melitensis reference culture 16M, standard substance of cloning simultaneously in pig kind brucella reference culture 1330S and checking order after consistent and therefrom taking out, the described nucleotide fragments that contains 272 bases is from kind of dog brucella reference culture RM6/66, cloning and obtain.
Described negative quality control standard product are aseptic double-distilled water.
The present invention also provides the preparation method who detects simultaneously four kinds of brucellar PCR test kits of cattle and sheep pig dog, and condition and the step of the method are as follows:
(1) preparation PCR reaction solution: described PCR reaction solution is by identifying ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog and containing dNTPs, Mg
2+, distilled water the PCR damping fluid form, described four pairs of primers are respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
(2) set up the positive quality control standard substance:
Described positive quality control standard substance comprise B. abortus positive quality control standard substance, brucella melitensis positive quality control standard substance, pig kind brucella positive quality control standard substance and kind of dog brucella positive quality control standard substance;
Described B. abortus positive quality control standard substance are comprised of the nucleotide fragments that contains 494 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described brucella melitensis positive quality control standard substance are comprised of the nucleotide fragments that contains 732 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described pig kind brucella positive quality control standard substance are comprised of the nucleotide fragments that contains 272 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described kind of dog brucella positive quality control standard substance are comprised of the pMD-18T recombinant plasmid that the nucleotide fragments that contains 272 bases forms,
The pMD-18T recombinant plasmid that the pMD-18T recombinant plasmid that the described nucleotide fragments that contains 494 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 732 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms and the nucleotide fragments that contains 272 bases form all can be bred in the bacillus coli DH 5 alpha competent cell;
The described sequence that contains the nucleotide fragments of 494 bases is:
5′-gacgaacggaatttttccaatcccatcgtttccgtttcacttggcctgccggcaacgtttcagtttggcggaatga?aacggacggacccgatcaccaaatatatcctgcaccatggcgacgtggttgtctggggcgggccgtcgcggcttttctatc?acggtattctaccattgaagtctggcgagcatgagcggctggggccgtttcggcttaatctgaccttccgaaaggcgttcta?gggcgtgtctgcatttaacgtaaccagatcatagcgcatgcgagatggacgaaacccatgaatgcggtcaatgttttctcgc?atcgcagcgcaatacgacgatagcgtttcaacttgttaaaaaagcattcaatctgatggcgttccttgtacagcctccagtcg?attgttggggcactggaacgtgttggattgaccttgatctgagccgttgccttgagatcgctggcaatgaaggcccttaagtg?atcggca-3′，
The described sequence that contains the nucleotide fragments of 732 bases is:
5′-tgccgatcacttaagggccttcattgccagcaatctcaaggcaacggctcagatcaaggtcaatccaacacgtt?ccagtgtcccaacaatcgactggaggctgtacaaggaacgccatcagattgaatgcttttttaacaagttgaaacgctatcgt?cgtattgcgctgcgatgcgagaaaacattgaccgcattcatgggcttcgtccatctcgcatgcgctatgatctggttacgttg?aatgcagacacgccctaggggtgaatctggaaattgtcagaaagacagtgcttcgtcacgctagagcgctcgctgccata?cttgcaacagtgacagcgataattgccgttattggctggtggcagggcgaagattggcgggtaagctattccaatctcgcta?ttgttaatggcgtctattggatattactgctctaccttctgtggattattctgagccgaaacactcctgattttttaggggtgccgc?ttgttaaggcgattcacgacaagaaacttcttatagtcgatggtgcaccatggctaagtttgggtgtcatgaccgcgatttacg?tcaaagacggtgaatatgagcggcttgtgtgcaccggagaggtagtaaacgttcagacaaataagctggttcagattcatat?ccggggttatgaagaaatttataacgatatcgaagctgttggtgaaaaacttaatcagaccagcaaggacgcgattt-3′，
The described sequence that contains the nucleotide fragments of 591 bases is:
5′-ctggtgggtatcgcattactcggcgccctggggctgacggggctgacaagtagcggcatcggggcggtggc?tgccggcgggcttctcggttttgccctgttcaaccgcccgcctgccagcatttttctcggcgattccggaagccccccattg?gggctgatcgtaggaacagccttgctgctgcttgcgcgtgaaacacacatcgtggtcgcgcttgttctgccgctttattatatt?ctggatgcgggcaccacaattgtcatgcgtgcagcccaaggtgagaacatcctcaaggctcactcgaaacatgcctatca?gatagcaaaacgcagtggctggagtgtgccgaaagtggtggcccatgtggcgcttttaaatacaatcctgatcgcctgcgt?ggtggccttgctggcgctggatcatccgctcgcacaactgacatttctgctggtcgcagccgttgccacactcattctgctg?ctcgatttccgcgggcgcttcaggaagctatgacctcgaaaagcaccttgcggaccatttcgatatccttcttttccatggcg?gtccgcagtaccgccaggctttcctgaa-3′，
The described sequence that contains the nucleotide fragments of 272 bases is:
5′-gatggagcaaacgctgaagcccaagcgaggccccgaaatagaccaggaagagcgtcaggagttccggca?gaccgcgaaagatggtggtgtagatattagccgcaagccgcatcgaaggttcgctggactgcttgcccagagcgaccag?aaaaccgaggatgagaccaacgggaagtgtggcgagtgccagactgacggtaacaagaagtcctttggcaatgctgata?ccccagccatcgggaccgaaactcaacaaggtggcgtagtgttcc-3′；
(3) negative quality control standard product: described negative quality control standard product are aseptic double-distilled water.
The described nucleotide fragments that contains 494 bases is from B. abortus reference culture 544A, cloning and obtain, the described nucleotide fragments that contains 732 bases is from brucella melitensis reference culture 16M, cloning and obtain, the described nucleotide fragments that contains 591 bases is from B. abortus reference culture 544A, brucella melitensis reference culture 16M, standard substance of cloning simultaneously in pig kind brucella reference culture 1330S and checking order after consistent and therefrom taking out, the described nucleotide fragments that contains 272 bases is from kind of dog brucella reference culture RM6/66, cloning and obtain.
The present invention also provides the using method that detects simultaneously four kinds of brucellar PCR test kits of cattle and sheep pig dog, and condition and the step of the method are as follows:
(1) to sample extraction sample DNA to be detected;
(2) take the sample DNA extracted is template, sample DNA, positive quality control standard substance and negative quality control standard product are joined respectively in the pipe that contains PCR reaction solution and archaeal dna polymerase, according to the PCR reaction conditions after optimizing, increase: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min;
(3) get 5 μ LPCR products, with 1.5% agarose gel electrophoresis, detect, observe the band that B. abortus amplifies 494bp and 591bp, brucella melitensis amplifies the band of 732bp and 591bp, pig kind brucella amplifies the band of 272bp and 591bp, and the kind of dog brucella amplifies the band of 272bp.
Described sample to be detected is milk, adopt liquid nutrient medium increase bacterium beef, adopt liquid nutrient medium increase bacterium mutton, adopt liquid nutrient medium increase the pork of bacterium and adopt liquid nutrient medium to increase the dog meats of bacterium.
Described PCR reaction solution is by identifying ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog and containing dNTPs, Mg
2+, distilled water the PCR damping fluid form;
Described four pairs of primers are respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 '.
Inventive principle: the present invention is by the disappearance partial design primer after the difference of research B. abortus and brucella melitensis insertion sequence IS711 copy number and ox kind, sheep kind, pig kind, the comparison of kind of dog brucella complete genome sequence, finally differentiates ox kind, sheep kind, pig kind, kind of dog brucella according to the difference of pcr amplified fragment size.
IS711 is distinctive insertion sequence in the brucella genome, this sequence is very conservative in brucella genome not of the same race, but have copy number difference, according to two pairs of primers of different designs of insertion sequence IS711 position in ox kind and brucella melitensis, these two pairs of primers are respectively: P
IS711fAnd P
494r, P
IS711fAnd P
732rA primer that is B. abortus and brucella melitensis is anchored on insertion sequence IS711 simultaneously above as common sequence, another primer is selected in respectively on the monosome DNA be close to insertion sequence IS711, the specificity of representative species, B. abortus and brucella melitensis, as amplification region, are differentiated by the length of pcr amplified fragment in zone between selection common sequence and conserved sequence.
Lipopolysaccharides is brucellar major antigen and virulence factor, the brucellar lipopolysaccharides of smooth type is by lipid A, core oligosaccharide and the genomic constitution of O-side chain, the kind of dog brucella belongs to Rough Anti-Brucella, lack the O-side chain radical because of, by analyze the O-side chain radical because of polymorphism, the significant difference of finding smooth type brucella and Rough Anti-Brucella is wbkF gene and wbkD gene, these two genes that adjoin with induce the O-side chain radical relevant because of the bactoprenol of polymerization.There is excalation in the kind of dog brucella in the wbkF-wbkD zone, gene by amplification wbkF-wbkD zone, and and B. abortus, brucella melitensis and pig kind brucella carry out sequence relatively, learn kind of dog brucella RM6/66(ATCC23365) disappearance 351bp, particular location in wbkF-wbkD zone disappearance is from wbkD Nucleotide 1594(inBMEI1426) to wbkF Nucleotide 918(inBMEI1427), so primer of design in the wbkF-wbkD zone of disappearance, outside the wbkF-wbkD zone of disappearance, design another primer, adopt this a pair of primer of design to identify the kind of dog brucella, this a pair of primer is P
591fand P
591r.
According to ox kind, sheep kind, pig kind, the brucellar complete genome sequence comparison of kind of dog, B. abortus and brucella melitensis above lack 2653bp at abc transport in conjunction with albumen (BR0951-BR0955), and then the design pair of primers is used for identifying pig kind brucella and kind of dog brucella in this disappearance zone, this a pair of primer is P
272fAnd P
272r.
To sum up, the polymorphism that the present invention causes according to the location of the genetic constitution IS711 species specificity in brucella karyomit(e), and B. abortus and brucella melitensis at abc transport in conjunction with the upper disappearance of albumen (BR0951-BR0955) 2653bp, the kind of dog brucella is at wbkF-wbkD zone disappearance 351bp, design four pairs of primers, thereby set up ox kind, sheep kind, pig kind, the brucellar multi-PCR detection method of kind of dog.
According to pcr amplified fragment length, judge that brucella belongs to any type in ox kind, sheep kind, pig kind, kind of dog brucella, wherein expanding fragment length is respectively: B. abortus 498bp and 591bp, brucella melitensis 731bp and 591bp, pig kind brucella 272bp and 591bp, kind of dog brucella 272bp.
The present invention utilizes Prmier5.0 software, and ox kind, sheep kind, pig kind and kind of dog brucella are carried out to design of primers.
The invention has the beneficial effects as follows:
PCR test kit of the present invention is practical, can detect simultaneously ox kind, sheep kind, pig kind, kind of dog brucella, detection can reach the level of brucella kind, and can distinguish is the brucellosis caused by which kind of brucella kind type, and there do not is cross reaction, but the brucellar kind of type polluted in the direct-detection sample, and detection speed is fast, highly sensitive, sensitivity reaches 10
2Cfu/mL, high specificity, favorable repeatability, safe and reliable, fast and convenient, can carry out qualitative detection to brucellar kind of type in sample, can substitute traditional etiology and serological method, in general laboratory, just can operate, be applicable to batch detection, can apply fully, for diagnosis, epidemiology survey and the Epidemic disease prevention of brucellosis, very large pushing effect is arranged.
The accompanying drawing explanation
Fig. 1 is that the repeatability of first batch of PCR test kit detects electrophorogram;
Fig. 2 is that the repeatability of second batch of PCR test kit detects electrophorogram;
Fig. 3 is that the repeatability of the 3rd batch of PCR test kit detects electrophorogram;
In Fig. 1 to Fig. 3: M is DL1000DNAMarker, the 1st, B. abortus S19 is the pcr amplification result for the first time, the 2nd, brucella melitensis M5 is the pcr amplification result for the first time, the 3rd, pig kind brucella S2 is the pcr amplification result for the first time, the 4th, kind of dog brucella RM6/66 is the pcr amplification result for the first time, the 5th, B. abortus S19 is the pcr amplification result for the second time, the 6th, brucella melitensis M5 is the pcr amplification result for the second time, the 7th, pig kind brucella S2 is the pcr amplification result for the second time, the 8th, kind of dog brucella RM6/66 is the pcr amplification result for the second time, the 9th, B. abortus S19 is the pcr amplification result for the third time, the 10th, brucella melitensis M5 is the pcr amplification result for the third time, the 11st, pig kind brucella S2 is the pcr amplification result for the third time, the 12nd, kind of dog brucella RM6/66 is the pcr amplification result for the third time, the 13rd, the 4th pcr amplification result of B. abortus S19, the 14th, the 4th pcr amplification result of brucella melitensis M5, the 15th, the 4th pcr amplification result of pig kind brucella S2, the 16th, the 4th pcr amplification result of kind of dog brucella RM6/66, the 17th, the 5th pcr amplification result of B. abortus S19, the 18th, the 5th pcr amplification result of brucella melitensis M5, the 19th, the 5th pcr amplification result of pig kind brucella S2, the 20th, the 5th pcr amplification result of kind of dog brucella RM6/66, the 21st, negative control.
Fig. 4 is the specific detection electrophorogram of PCR test kit of the present invention;
In figure: M is DL1000DNAMarker, the 1st, the pcr amplification result of B. abortus S19, the 2nd, the pcr amplification result of brucella melitensis M5, the 3rd, the pcr amplification result of pig kind brucella S2, the 4th, the pcr amplification result of kind of dog brucella RM6/66, the 5th, the pcr amplification result of Salmonellas, the 6th, the pcr amplification result of Listeria monocytogenes, the 7th, the pcr amplification result of yersinia entero-colitica O:9, the 8th, the pcr amplification result of Escherichia coli O 157: H7, the 9th, colibacillary pcr amplification result, the 10th, the pcr amplification result of streptococcus aureus, the 11st, the pcr amplification result of streptococcus suis 2-type, the 12nd, negative control.
In the positive quality control standard product of Fig. 5, the sensitivity Detection electrophorogram of the pMD-18T recombinant plasmid formed by the nucleotide fragments that contains 494 bases;
In the positive quality control standard product of Fig. 6, the sensitivity Detection electrophorogram of the pMD-18T recombinant plasmid formed by the nucleotide fragments that contains 732 bases;
In the positive quality control standard product of Fig. 7, the sensitivity Detection electrophorogram of the pMD-18T recombinant plasmid formed by the nucleotide fragments that contains 591 bases;
In the positive quality control standard product of Fig. 8, the sensitivity Detection electrophorogram of the pMD-18T recombinant plasmid formed by the nucleotide fragments that contains 272 bases;
In Fig. 5 to Fig. 8: M is DL1000DNAMarker, and 1～9 is positive recombinant plasmids of 10 times of doubling dilutions, and 1 is 1.0 * 10
1Copies/ μ L, 2 is 1.0 * 10
2Copies/ μ L, 3 is 1.0 * 10
3Copies/ μ L, 4 is 1.0 * 10
4Copies/ μ L, 5 is 1.0 * 10
5Copies/ μ L, 6 is 1.0 * 10
6Copies/ μ L, 7 is 1.0 * 10
7Copies/ μ L, 8 is 1.0 * 10
8Copies/ μ L, 9 is 1.0 * 10
9Copies/ μ L.
Fig. 9 is the sensitivity Detection electrophorogram of PCR test kit of the present invention to bacterium reference material (B. abortus S19);
In figure: M is DL1000DNAMarker, and 0～9 is pcr amplification figure of 10 times of doubling dilution B. abortus S19 genomic dnas, and 0 concentration is 1.1 * 10
0Cfu/mL, 1 concentration is 1.1 * 10
1Cfu/mL, 2 concentration is 1.1 * 10
2Cfu/mL, 3 concentration is 1.1 * 10
3Cfu/mL, 4 concentration is 1.1 * 10
4Cfu/mL, 5 concentration is 1.1 * 10
5Cfu/mL, 6 concentration is 1.1 * 10
6Cfu/mL, 7 concentration is 1.1 * 10
7Cfu/mL, 8 concentration is 1.1 * 10
8Cfu/mL, 9 concentration is 1.1 * 10
9Cfu/mL, the 10th, negative control.
Figure 10 is the sensitivity Detection electrophorogram of PCR test kit of the present invention to bacterium reference material (brucella melitensis M5);
In figure: M is DL1000DNAMarker, and 0～9 is pcr amplification figure of 10 times of doubling dilution brucella melitensis M5 genomic dnas, and 0 concentration is 5.1 * 10
0Cfu/mL, 1 concentration is 5.1 * 10
1Cfu/mL, 2 concentration is 5.1 * 10
2Cfu/mL, 3 concentration is 5.1 * 10
3Cfu/mL, 4 concentration is 5.1 * 10
4Cfu/mL, 5 concentration is 5.1 * 10
5Cfu/mL, 6 concentration is 5.1 * 10
6Cfu/mL, 7 concentration is 5.1 * 10
7Cfu/mL, 8 concentration is 5.1 * 10
8Cfu/mL, 9 concentration is 5.1 * 10
9Cfu/mL, the 10th, negative control.
Figure 11 is the sensitivity Detection electrophorogram of PCR test kit of the present invention to bacterium reference material (pig kind brucella S2);
In figure, M is DL1000DNAMarker, and 0～9 is pcr amplification figure of 10 times of doubling dilution pig kind brucella S2 genomic dnas, and 0 concentration is 3.5 * 10
0Cfu/mL, 1 concentration is 3.5 * 10
1Cfu/mL, 2 concentration is 3.5 * 10
2Cfu/mL, 3 concentration is 3.5 * 10
3Cfu/mL, 4 concentration is 3.5 * 10
4Cfu/mL, 5 concentration is 3.5 * 10
5Cfu/mL, 6 concentration is 3.5 * 10
6Cfu/mL, 7 concentration is 3.5 * 10
7Cfu/mL, 8 concentration is 3.5 * 10
8Cfu/mL, 9 concentration is 3.5 * 10
9Cfu/mL, the 10th, negative control.
Figure 12 is the sensitivity Detection electrophorogram of PCR test kit of the present invention to bacterium reference material (kind of dog brucella RM6/66);
In figure: M is DL1000DNAMarker, and 0～9 is pcr amplification figure of 10 times of doubling dilution kind of dog brucella RM6/66 genomic dnas, and 0 concentration is 2.5 * 10
0Cfu/mL, 1 concentration is 2.5 * 10
1Cfu/mL, 2 concentration is 2.5 * 10
2Cfu/mL, 3 concentration is 2.5 * 10
3Cfu/mL, 4 concentration is 2.5 * 10
4Cfu/mL, 5 concentration is 2.5 * 10
5Cfu/mL, 6 concentration is 2.5 * 10
6Cfu/mL, 7 concentration is 2.5 * 10
7Cfu/mL, 8 concentration is 2.5 * 10
8Cfu/mL, 9 concentration is 2.5 * 10
9Cfu/mL, the 10th, negative control.
Figure 13 is the sensitivity Detection electrophorogram of B. abortus S19 artificial challenge milk sample;
Figure 14 is the sensitivity Detection electrophorogram of brucella melitensis M5 artificial challenge milk sample;
Figure 15 is the sensitivity Detection electrophorogram of pig kind brucella S2 artificial challenge milk sample;
Figure 16 is the sensitivity Detection electrophorogram of kind of dog brucella RM6/66 artificial challenge milk sample;
In Figure 13 to Figure 16: M is DL1000DNAMarker, and 0 concentration is 1.0 * 10
0Cfu/mL, 1 concentration is 1.0 * 10
1Cfu/mL, 2 concentration is 1.0 * 10
2Cfu/mL, 3 concentration is 1.0 * 10
3Cfu/mL, 4 concentration is 1.0 * 10
4Cfu/mL, 5 concentration is 1.0 * 10
5Cfu/mL, 6 concentration is 1.0 * 10
6Cfu/mL, 7 concentration is 1.0 * 10
7Cfu/mL, 8 concentration is 1.0 * 10
8Cfu/mL, 9 concentration is 1.0 * 10
9Cfu/mL, the 10th, negative control.
Embodiment
Experiment material:
B. abortus reference culture 544A(B.abortus544A), B. abortus vaccine strain S19(B.abortusS19), brucella melitensis reference culture 16M(B.melitensis16M), brucella melitensis vaccine strain M5(B.melitensisM5), pig kind brucella reference culture 1330S(B.suis1330S), kind of dog brucella reference culture RM6/66(B.canisRM6/66), Listeria monocytogenes (CMCC54002), streptococcus aureus (CMCC26003), Escherichia coli O 157: H7, yersinia entero-colitica O:9, Salmonellas, streptococcus suis 2-type, intestinal bacteria (CMCC44102) preserve by MILITARY VETERINARY INST ACADE's veterinary microorganism and Immunology Lab,
Pig kind brucella vaccine strain S2(B.suisS2) by the Feng Shuzhang researcher of MILITARY VETERINARY INST ACADE, be so kind as to give;
The PCR primer is synthetic by China large gene company limited;
MultiplexPCRAssayKit, pMD-18T carrier, DNAMakrerDL1000, bacillus coli DH 5 alpha competent cell are all purchased from the precious biotechnology (Dalian) of TaKaRa company limited;
DNA gel reclaims test kit, plasmid extraction kit, bacterial genomes DNA extraction test kit all purchased from liking to pursue progress biotechnology (Hangzhou) company limited;
The TSB substratum is purchased from FLUKA company;
Agarose is purchased from Spain Biowest company.
Laboratory apparatus:
The model of the French VILBERLOURMAT of gel image analysis instrument selection company is the gel image analysis instrument of x-press;
It is the voltage stabilization and current stabilization electrophoresis apparatus of DYY-6B that the voltage stabilization and current stabilization electrophoresis apparatus is selected the model of Beijing Liuyi Instrument Factory;
It is the PCR instrument of ThermalCyclerPX2 that the PCR instrument is selected the model of Thermo company.
(1) to sample extraction sample DNA to be detected, sample to be detected is milk, adopts liquid nutrient medium to increase the beef of bacterium, the mutton that the employing liquid nutrient medium increases bacterium, the pork that the employing liquid nutrient medium increases bacterium, the dog meats that the employing liquid nutrient medium increases bacterium etc.;
(2) take the sample DNA extracted is template, sample DNA, positive quality control standard substance and negative quality control standard product are joined respectively in the pipe that contains PCR reaction solution and archaeal dna polymerase, according to the PCR reaction conditions after optimizing, increase: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min; The pcr amplification effect directly affects susceptibility and the specificity of detection, need the parameters such as the primer concentration in PCR reaction system and reaction conditions, annealing temperature, annealing time, cycle index, extension time are optimized for this reason, prior art is adopted in the optimization of PCR reaction conditions, PCR reaction conditions after final optimization pass is: primer concentration is 7.5 μ mol/L, and annealing temperature is 62 ℃, annealing time 30sec, cycle index 30 times, extend time 30sec;
(3) get 5 μ LPCR products, with 1.5% agarose gel electrophoresis (120V20min), detect, observe the band whether B. abortus amplifies 494bp and 591bp, whether brucella melitensis amplifies the band of 732bp and 591bp, whether pig kind brucella amplifies the band of 272bp and 591bp, whether the kind of dog brucella amplifies the band of 272bp, if amplify the band of 494bp and 591bp, the B. abortus that contains in sample to be detected is described; If amplify the band of 732bp and 591bp, the brucella melitensis that contains in sample to be detected is described; If amplify the band of 272bp and 591bp, illustrate and contain pig kind brucella in sample to be detected; If amplify the band of 272bp, illustrate and contain the kind of dog brucella in sample to be detected.
The assembling of embodiment 2PCR test kit
(1) preparation PCR reaction solution:
1. design four pairs and identify ox kind, sheep kind, pig kind, the brucellar primer of kind of dog: be respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
2. the preparation of PCR reaction solution: the PCR reaction solution is by identifying ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog and containing dNTPs, Mg
2+, distilled water (ddH
2O) PCR buffer reagent forms, and the cumulative volume of PCR reaction solution is 47.8 μ L, and the amount of every primer is that 1 μ L(primer concentration is 7.5 μ mol/L), contain dNTPs, Mg
2+, distilled water (ddH
2The total amount of PCR buffer reagent O) is 39.8 μ L, distilled water (ddH wherein
2O) be 14.8 μ L;
(2) set up the positive quality control standard substance:
1. the cultivation of bacterium: TSA plate streaking recovery B. abortus reference culture 544A(B.abortus544A), brucella melitensis reference culture 16M(B.melitensis16M), pig kind brucella reference culture 1330S(B.suis1330S), kind of dog brucella reference culture RM6/66(B.canisRM6/66), cultivate 48h for 37 ℃, picking list bacterium colony is cultivated 48h, 180r/min in 37 ℃ of TSB liquid nutrient mediums respectively;
2. the extraction of template DNA: respectively DNA is extracted in the bacterium liquid of B. abortus reference culture 544A, brucella melitensis reference culture 16M, pig kind brucella reference culture 1330S, kind of dog brucella reference culture RM6/66;
3. pcr amplification: the DNA extracted of take is template, according to the PCR reaction system: DNA profiling 2 μ L, archaeal dna polymerase 0.2 μ L, PCR reaction solution 47.8 μ L, the PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min, extension increasing sequence fragment on the PCR instrument, the agarose gel electrophoresis with 1.5% (120V20min) is identified PCR purpose product;
4. the bacterium liquid of recombinant plasmid is identified: utilize gel to reclaim test kit PCR purpose product is reclaimed, connect the pMD-18T carrier, transform, bacterium liquid PCR identifies;
5. the order-checking of recombinant plasmid is identified: choose positive bacteria liquid, extract the pMD-18T recombinant plasmid and check order, sequencing result is compared with Blast, and result is in full accord with pre-extension increasing sequence, shows that Insert Fragment is correct in sudden change;
6. the foundation of positive quality control standard substance: the positive bacteria liquid that will check order correct is cultivated and is increased bacterium in 37 ℃ of shaking tables, adopts plasmid extraction kit to extract recombinant plasmid, namely obtains required positive quality control standard substance;
Said positive quality control standard substance comprise B. abortus positive quality control standard substance, brucella melitensis positive quality control standard substance, pig kind brucella positive quality control standard substance and kind of dog brucella positive quality control standard substance;
Said B. abortus positive quality control standard substance are comprised of the nucleotide fragments that contains 494 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms; Said brucella melitensis positive quality control standard substance are comprised of the nucleotide fragments that contains 732 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms; Said pig kind brucella positive quality control standard substance are comprised of the nucleotide fragments that contains 272 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms; Said kind of dog brucella positive quality control standard substance are comprised of the pMD-18T recombinant plasmid that the nucleotide fragments that contains 272 bases forms;
The pMD-18T recombinant plasmid that the pMD-18T recombinant plasmid that the said nucleotide fragments that contains 494 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 732 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms and the nucleotide fragments that contains 272 bases form all can be bred in the bacillus coli DH 5 alpha competent cell;
The said sequence that contains the nucleotide fragments of 494 bases is:
5′-gacgaacggaatttttccaatcccatcgtttccgtttcacttggcctgccggcaacgtttcagtttggcggaatga?aacggacggacccgatcaccaaatatatcctgcaccatggcgacgtggttgtctggggcgggccgtcgcggcttttctatc?acggtattctaccattgaagtctggcgagcatgagcggctggggccgtttcggcttaatctgaccttccgaaaggcgttcta?gggcgtgtctgcatttaacgtaaccagatcatagcgcatgcgagatggacgaaacccatgaatgcggtcaatgttttctcgc?atcgcagcgcaatacgacgatagcgtttcaacttgttaaaaaagcattcaatctgatggcgttccttgtacagcctccagtcg?attgttggggcactggaacgtgttggattgaccttgatctgagccgttgccttgagatcgctggcaatgaaggcccttaagtg?atcggca-3′，
The said sequence that contains the nucleotide fragments of 732 bases is:
5′-tgccgatcacttaagggccttcattgccagcaatctcaaggcaacggctcagatcaaggtcaatccaacacgtt?ccagtgtcccaacaatcgactggaggctgtacaaggaacgccatcagattgaatgcttttttaacaagttgaaacgctatcgt?cgtattgcgctgcgatgcgagaaaacattgaccgcattcatgggcttcgtccatctcgcatgcgctatgatctggttacgttg?aatgcagacacgccctaggggtgaatctggaaattgtcagaaagacagtgcttcgtcacgctagagcgctcgctgccata?cttgcaacagtgacagcgataattgccgttattggctggtggcagggcgaagattggcgggtaagctattccaatctcgcta?ttgttaatggcgtctattggatattactgctctaccttctgtggattattctgagccgaaacactcctgattttttaggggtgccgc?ttgttaaggcgattcacgacaagaaacttcttatagtcgatggtgcaccatggctaagtttgggtgtcatgaccgcgatttacg?tcaaagacggtgaatatgagcggcttgtgtgcaccggagaggtagtaaacgttcagacaaataagctggttcagattcatat?ccggggttatgaagaaatttataacgatatcgaagctgttggtgaaaaacttaatcagaccagcaaggacgcgattt-3′，
The said sequence that contains the nucleotide fragments of 591 bases is:
5′-ctggtgggtatcgcattactcggcgccctggggctgacggggctgacaagtagcggcatcggggcggtggc?tgccggcgggcttctcggttttgccctgttcaaccgcccgcctgccagcatttttctcggcgattccggaagccccccattg?gggctgatcgtaggaacagccttgctgctgcttgcgcgtgaaacacacatcgtggtcgcgcttgttctgccgctttattatatt?ctggatgcgggcaccacaattgtcatgcgtgcagcccaaggtgagaacatcctcaaggctcactcgaaacatgcctatca?gatagcaaaacgcagtggctggagtgtgccgaaagtggtggcccatgtggcgcttttaaatacaatcctgatcgcctgcgt?ggtggccttgctggcgctggatcatccgctcgcacaactgacatttctgctggtcgcagccgttgccacactcattctgctg?ctcgatttccgcgggcgcttcaggaagctatgacctcgaaaagcaccttgcggaccatttcgatatccttcttttccatggcg?gtccgcagtaccgccaggctttcctgaa-3′，
The said sequence that contains the nucleotide fragments of 272 bases is:
5′-gatggagcaaacgctgaagcccaagcgaggccccgaaatagaccaggaagagcgtcaggagttccggca?gaccgcgaaagatggtggtgtagatattagccgcaagccgcatcgaaggttcgctggactgcttgcccagagcgaccag?aaaaccgaggatgagaccaacgggaagtgtggcgagtgccagactgacggtaacaagaagtcctttggcaatgctgata?ccccagccatcgggaccgaaactcaacaaggtggcgtagtgttcc-3′；
7. the preparation of positive quality control standard substance: B. abortus positive quality control standard substance: each the 1 μ L of pMD-18T recombinant plasmid that contains nucleotide fragments the pMD-18T recombinant plasmid formed and the nucleotide fragments formation that contains 591 bases of 494 bases; Brucella melitensis positive quality control standard substance: each the 1 μ L of pMD-18T recombinant plasmid that contains nucleotide fragments the pMD-18T recombinant plasmid formed and the nucleotide fragments formation that contains 591 bases of 732 bases; Pig kind brucella positive quality control standard substance: each the 1 μ L of pMD-18T recombinant plasmid that contains nucleotide fragments the pMD-18T recombinant plasmid formed and the nucleotide fragments formation that contains 591 bases of 272 bases; Kind of dog brucella positive quality control standard substance: the pMD-18T recombinant plasmid 2 μ L that contain the nucleotide fragments formation of 272 bases;
(3) sequence of ox kind, sheep kind, pig kind, kind of dog brucella primers designed:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
(4) negative quality control standard product: negative quality control standard product are aseptic double-distilled water, and volume is 2 μ L;
The replica test of embodiment 3PCR test kit
(1) adopt bacterial genomes DNA extraction test kit to extract respectively brucella DNA to B. abortus S19, brucella melitensis M5, pig kind brucella S2, kind of dog brucella RM6/66, for pcr amplification;
(2) get respectively each 47.8uL of PCR reaction solution, get each 2 μ L of (1) step gained brucella DNA and brucella positive quality control standard substance, and establish negative control, add respectively different PCR reaction tubess, the parallel pcr amplification of doing on the PCR instrument, the PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min.
(3) get 5 μ LPCR products, with 1.5% agarose gel electrophoresis, detect, on the gel image analysis instrument, observe the band that B. abortus amplifies 494bp and 591bp, brucella melitensis amplifies the band of 732bp and 591bp, pig kind brucella amplifies the band of 272bp and 591bp, and the kind of dog brucella amplifies the band of 272bp.
The PCR test kit that extracts three batches carries out replica test, in batch, revision test is 5 times, between batch, revision test is 3 times, result as shown in Figure 1, Figure 2, Figure 3 shows, the gained detected result is all identical, detected result between this explanation different batches has comparability and good repeatability, the repeatability of PCR test kit is better, and adopt the PCR test kit only to need just can complete in 3 hours to the detection of sample (DNA), show PCR test kit of the present invention can rapid detection ox kind, sheep kind, pig kind, kind of dog brucella.
The specific test of embodiment 4PCR test kit
Adopt PCR test kit of the present invention to increase to B. abortus S19, brucella melitensis M5, pig kind brucella S2, kind of dog brucella RM6/66, Salmonellas, Listeria monocytogenes, yersinia entero-colitica O:9, Escherichia coli O 157: H7, intestinal bacteria, streptococcus aureus, streptococcus suis 2-type genomic dna, and establish negative control, the specificity of checking multi-PRC reaction, the PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min.
Result as shown in Figure 4, only have B. abortus S19 to amplify the band of 494bp and 591bp, brucella melitensis M5 amplifies the band of 732bp and 591bp, the band that pig kind brucella S2 amplifies 272bp and 591bp, the band that kind of dog brucella RM6/66 amplifies 272bp, and Salmonellas, streptococcus aureus, streptococcus suis 2-type, intestinal bacteria, yersinia entero-colitica O:9, Escherichia coli O 157: H7, Listeria monocytogenes and negative control are without band, illustrate that the PCR test kit has specificity preferably, and no cross reaction.
The sensitivity test of embodiment 5 positive quality control standard substance
The correct bacterium liquid that will check order is cultivated and is increased bacterium in 37 ℃ of shaking tables, adopt plasmid extraction kit to extract recombinant plasmid, with the nucleic acid concentration determinator, survey plasmid concentration (ng/ μ L), prepared plasmid concentration is converted into to copy number, wherein in every 1 μ L sample, detects gene copy number and calculate according to formula: plasmid copy number concentration (copies/ μ L)=concentration (ng/ μ L) * avogadros constant * 10
-9/ recombinant plasmid base number * 660.After drawing the copy number of recombinant plasmid, release according to 10 times of gradients, plasmid is diluted to 1 * 10
1Copies/ μ L, 1 * 10
2Copies/ μ L, 1 * 10
3Copies/ μ L, 1 * 10
4Copies/ μ L, 1 * 10
5Copies/ μ L, 1 * 10
6Copies/ μ l, 1 * 10
7Copies/ μ L, 1 * 10
8Copies/ μ L, 1 * 10
9Copies/ μ L.Getting 1 μ L plasmid is that template is carried out pcr amplification, and the PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min.
Result such as Fig. 5, Fig. 6, Fig. 7, shown in Figure 8, the lowest detection that contains the pMD-18T recombinant plasmid that the nucleotide fragments of 494 bases forms is limited to: 1 * 10
2Copies/ μ L, the lowest detection of the pMD-18T recombinant plasmid consisted of the nucleotide fragments that contains 732 bases is limited to: 1 * 10
2Copies/ μ L, the lowest detection of the pMD-18T recombinant plasmid consisted of the nucleotide fragments that contains 591 bases is limited to: 1 * 10
2Copies/ μ L, the lowest detection of the pMD-18T recombinant plasmid consisted of the nucleotide fragments that contains 272 bases is limited to: 1 * 10
3Copies/ μ L.
The sensitivity test of embodiment 6PCR test kit
Get respectively the bacterium liquid of B. abortus S19, brucella melitensis M5, pig kind brucella S2, kind of dog brucella RM6/66, utilize colony counting method to calculate bacterial concentration, respectively with 10 times of doubling dilution bacterium liquid, get each 1mL of bacterium liquid of corresponding extension rate, adopt bacterial genomes DNA extraction test kit to extract genomic dna, getting 1 μ L genomic dna is that template is carried out pcr amplification, the PCR reaction conditions is: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min.
Result such as Fig. 9, Figure 10, Figure 11, shown in Figure 12, result determines that the susceptibility of PCR test kit detection B. abortus S19 is 1.1 * 10 by experiment
2Cfu/mL, the susceptibility that detects brucella melitensis M5 is 5.1 * 10
2Cfu/mL, the susceptibility that detects pig kind brucella S2 is 3.5 * 10
2Cfu/mL, the susceptibility that detects kind of dog brucella RM6/66 is 2.5 * 10
3Cfu/mL.
The detection of embodiment 7 artificial challenge's milk
B. abortus S19, brucella melitensis M5, pig kind brucella S2, tetra-kinds of bacterium liquid of kind of dog brucella RM6/66, by colony counting method, are drawn to bacterial concentration, these four kinds of bacterium liquid are mixed with germ-free milk respectively, make bacterium liquid final concentration be respectively 1 * 10
9Cfu/mL, 1 * 10
8Cfu/mL, 1 * 10
7Cfu/mL, 1 * 10
6Cfu/mL, 1 * 10
5Cfu/mL, 1 * 10
4Cfu/mL, 1 * 10
3Cfu/mL, 1 * 10
2Cfu/mL, 1 * 10
1Cfu/mL, 1 * 10
0Cfu/mL, adopt bacterial genomes DNA test kit to extract genomic dna, increases under the multiplex PCR system, and detecting the multi-PRC reaction of setting up is the practicality of PCR test kit.
Result such as Figure 13, Figure 14, Figure 15, shown in Figure 16, four kinds of brucella lowest detection of artificial challenge's milk respectively are limited to: B. abortus S19 is 1.0 * 10
3Cfu/mL, brucella melitensis M5 are 1.0 * 10
3Cfu/mL, pig kind brucella S2 is 1.0 * 10
3Cfu/mL, kind of dog brucella RM6/66 is 1.0 * 10
4Cfu/mL.
Claims (9)
1. detect simultaneously four kinds of brucellar PCR test kits of cattle and sheep pig dog, it is characterized in that,
This PCR test kit is comprised of PCR reaction solution, archaeal dna polymerase, positive quality control standard substance and negative quality control standard product;
Described PCR reaction solution comprises identifies ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog, is respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
Described positive quality control standard substance comprise B. abortus positive quality control standard substance, brucella melitensis positive quality control standard substance, pig kind brucella positive quality control standard substance and kind of dog brucella positive quality control standard substance;
Described B. abortus positive quality control standard substance are comprised of the nucleotide fragments that contains 494 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described brucella melitensis positive quality control standard substance are comprised of the nucleotide fragments that contains 732 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described pig kind brucella positive quality control standard substance are comprised of the nucleotide fragments that contains 272 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described kind of dog brucella positive quality control standard substance are comprised of the pMD-18T recombinant plasmid that the nucleotide fragments that contains 272 bases forms,
The pMD-18T recombinant plasmid that the pMD-18T recombinant plasmid that the described nucleotide fragments that contains 494 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 732 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms and the nucleotide fragments that contains 272 bases form all can be bred in the bacillus coli DH 5 alpha competent cell;
The described sequence that contains the nucleotide fragments of 494 bases is:
5′-gacgaacggaatttttccaatcccatcgtttccgtttcacttggcctgccggcaacgtttcagtttggcggaatga?aacggacggacccgatcaccaaatatatcctgcaccatggcgacgtggttgtctggggcgggccgtcgcggcttttctatc?acggtattctaccattgaagtctggcgagcatgagcggctggggccgtttcggcttaatctgaccttccgaaaggcgttcta?gggcgtgtctgcatttaacgtaaccagatcatagcgcatgcgagatggacgaaacccatgaatgcggtcaatgttttctcgc?atcgcagcgcaatacgacgatagcgtttcaacttgttaaaaaagcattcaatctgatggcgttccttgtacagcctccagtcg?attgttggggcactggaacgtgttggattgaccttgatctgagccgttgccttgagatcgctggcaatgaaggcccttaagtg?atcggca-3′，
The described sequence that contains the nucleotide fragments of 732 bases is:
5′-tgccgatcacttaagggccttcattgccagcaatctcaaggcaacggctcagatcaaggtcaatccaacacgtt?ccagtgtcccaacaatcgactggaggctgtacaaggaacgccatcagattgaatgcttttttaacaagttgaaacgctatcgt?cgtattgcgctgcgatgcgagaaaacattgaccgcattcatgggcttcgtccatctcgcatgcgctatgatctggttacgttg?aatgcagacacgccctaggggtgaatctggaaattgtcagaaagacagtgcttcgtcacgctagagcgctcgctgccata?cttgcaacagtgacagcgataattgccgttattggctggtggcagggcgaagattggcgggtaagctattccaatctcgcta?ttgttaatggcgtctattggatattactgctctaccttctgtggattattctgagccgaaacactcctgattttttaggggtgccgc?ttgttaaggcgattcacgacaagaaacttcttatagtcgatggtgcaccatggctaagtttgggtgtcatgaccgcgatttacg?tcaaagacggtgaatatgagcggcttgtgtgcaccggagaggtagtaaacgttcagacaaataagctggttcagattcatat?ccggggttatgaagaaatttataacgatatcgaagctgttggtgaaaaacttaatcagaccagcaaggacgcgattt-3′，
The described sequence that contains the nucleotide fragments of 591 bases is:
5′-ctggtgggtatcgcattactcggcgccctggggctgacggggctgacaagtagcggcatcggggcggtggc?tgccggcgggcttctcggttttgccctgttcaaccgcccgcctgccagcatttttctcggcgattccggaagccccccattg?gggctgatcgtaggaacagccttgctgctgcttgcgcgtgaaacacacatcgtggtcgcgcttgttctgccgctttattatatt?ctggatgcgggcaccacaattgtcatgcgtgcagcccaaggtgagaacatcctcaaggctcactcgaaacatgcctatca?gatagcaaaacgcagtggctggagtgtgccgaaagtggtggcccatgtggcgcttttaaatacaatcctgatcgcctgcgt?ggtggccttgctggcgctggatcatccgctcgcacaactgacatttctgctggtcgcagccgttgccacactcattctgctg?ctcgatttccgcgggcgcttcaggaagctatgacctcgaaaagcaccttgcggaccatttcgatatccttcttttccatggcg?gtccgcagtaccgccaggctttcctgaa-3′，
The described sequence that contains the nucleotide fragments of 272 bases is:
5′-gatggagcaaacgctgaagcccaagcgaggccccgaaatagaccaggaagagcgtcaggagttccggca?gaccgcgaaagatggtggtgtagatattagccgcaagccgcatcgaaggttcgctggactgcttgcccagagcgaccag?aaaaccgaggatgagaccaacgggaagtgtggcgagtgccagactgacggtaacaagaagtcctttggcaatgctgata?ccccagccatcgggaccgaaactcaacaaggtggcgtagtgttcc-3′。
2. PCR test kit according to claim 1, is characterized in that, described PCR reaction solution also comprises and contains dNTPs, Mg
2+, distilled water the PCR damping fluid.
3. PCR test kit according to claim 1, it is characterized in that, the described nucleotide fragments that contains 494 bases is from B. abortus reference culture 544A, cloning and obtain, the described nucleotide fragments that contains 732 bases is from brucella melitensis reference culture 16M, cloning and obtain, the described nucleotide fragments that contains 591 bases is from B. abortus reference culture 544A, brucella melitensis reference culture 16M, standard substance of cloning simultaneously in pig kind brucella reference culture 1330S and checking order after consistent and therefrom taking out, the described nucleotide fragments that contains 272 bases is from kind of dog brucella reference culture RM6/66, cloning and obtain.
4. PCR test kit according to claim 1, is characterized in that, described negative quality control standard product are aseptic double-distilled water.
5. prepare the method for PCR test kit claimed in claim 1, it is characterized in that, condition and the step of the method are as follows:
(1) preparation PCR reaction solution: described PCR reaction solution is by identifying ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog and containing dNTPs, Mg
2+, distilled water the PCR damping fluid form, described four pairs of primers are respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 ';
(2) set up the positive quality control standard substance:
Described positive quality control standard substance comprise B. abortus positive quality control standard substance, brucella melitensis positive quality control standard substance, pig kind brucella positive quality control standard substance and kind of dog brucella positive quality control standard substance;
Described B. abortus positive quality control standard substance are comprised of the nucleotide fragments that contains 494 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described brucella melitensis positive quality control standard substance are comprised of the nucleotide fragments that contains 732 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described pig kind brucella positive quality control standard substance are comprised of the nucleotide fragments that contains 272 bases the pMD-18T recombinant plasmid formed and the pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms, described kind of dog brucella positive quality control standard substance are comprised of the pMD-18T recombinant plasmid that the nucleotide fragments that contains 272 bases forms,
The pMD-18T recombinant plasmid that the pMD-18T recombinant plasmid that the described nucleotide fragments that contains 494 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 732 bases forms, pMD-18T recombinant plasmid that the nucleotide fragments that contains 591 bases forms and the nucleotide fragments that contains 272 bases form all can be bred in the bacillus coli DH 5 alpha competent cell;
The described sequence that contains the nucleotide fragments of 494 bases is:
5′-gacgaacggaatttttccaatcccatcgtttccgtttcacttggcctgccggcaacgtttcagtttggcggaatga?aacggacggacccgatcaccaaatatatcctgcaccatggcgacgtggttgtctggggcgggccgtcgcggcttttctatc?acggtattctaccattgaagtctggcgagcatgagcggctggggccgtttcggcttaatctgaccttccgaaaggcgttcta?gggcgtgtctgcatttaacgtaaccagatcatagcgcatgcgagatggacgaaacccatgaatgcggtcaatgttttctcgc?atcgcagcgcaatacgacgatagcgtttcaacttgttaaaaaagcattcaatctgatggcgttccttgtacagcctccagtcg?attgttggggcactggaacgtgttggattgaccttgatctgagccgttgccttgagatcgctggcaatgaaggcccttaagtg?atcggca-3′，
The described sequence that contains the nucleotide fragments of 732 bases is:
5′-tgccgatcacttaagggccttcattgccagcaatctcaaggcaacggctcagatcaaggtcaatccaacacgtt?ccagtgtcccaacaatcgactggaggctgtacaaggaacgccatcagattgaatgcttttttaacaagttgaaacgctatcgt?cgtattgcgctgcgatgcgagaaaacattgaccgcattcatgggcttcgtccatctcgcatgcgctatgatctggttacgttg?aatgcagacacgccctaggggtgaatctggaaattgtcagaaagacagtgcttcgtcacgctagagcgctcgctgccata?cttgcaacagtgacagcgataattgccgttattggctggtggcagggcgaagattggcgggtaagctattccaatctcgcta?ttgttaatggcgtctattggatattactgctctaccttctgtggattattctgagccgaaacactcctgattttttaggggtgccgc?ttgttaaggcgattcacgacaagaaacttcttatagtcgatggtgcaccatggctaagtttgggtgtcatgaccgcgatttacg?tcaaagacggtgaatatgagcggcttgtgtgcaccggagaggtagtaaacgttcagacaaataagctggttcagattcatat?ccggggttatgaagaaatttataacgatatcgaagctgttggtgaaaaacttaatcagaccagcaaggacgcgattt-3′，
The described sequence that contains the nucleotide fragments of 591 bases is:
5′-ctggtgggtatcgcattactcggcgccctggggctgacggggctgacaagtagcggcatcggggcggtggc?tgccggcgggcttctcggttttgccctgttcaaccgcccgcctgccagcatttttctcggcgattccggaagccccccattg?gggctgatcgtaggaacagccttgctgctgcttgcgcgtgaaacacacatcgtggtcgcgcttgttctgccgctttattatatt?ctggatgcgggcaccacaattgtcatgcgtgcagcccaaggtgagaacatcctcaaggctcactcgaaacatgcctatca?gatagcaaaacgcagtggctggagtgtgccgaaagtggtggcccatgtggcgcttttaaatacaatcctgatcgcctgcgt?ggtggccttgctggcgctggatcatccgctcgcacaactgacatttctgctggtcgcagccgttgccacactcattctgctg?ctcgatttccgcgggcgcttcaggaagctatgacctcgaaaagcaccttgcggaccatttcgatatccttcttttccatggcg?gtccgcagtaccgccaggctttcctgaa-3′，
The described sequence that contains the nucleotide fragments of 272 bases is:
5′-gatggagcaaacgctgaagcccaagcgaggccccgaaatagaccaggaagagcgtcaggagttccggca?gaccgcgaaagatggtggtgtagatattagccgcaagccgcatcgaaggttcgctggactgcttgcccagagcgaccag?aaaaccgaggatgagaccaacgggaagtgtggcgagtgccagactgacggtaacaagaagtcctttggcaatgctgata?ccccagccatcgggaccgaaactcaacaaggtggcgtagtgttcc-3′；
(3) negative quality control standard product: described negative quality control standard product are aseptic double-distilled water.
6. the preparation method of PCR test kit according to claim 5, it is characterized in that, the described nucleotide fragments that contains 494 bases is from B. abortus reference culture 544A, cloning and obtain, the described nucleotide fragments that contains 732 bases is from brucella melitensis reference culture 16M, cloning and obtain, the described nucleotide fragments that contains 591 bases is from B. abortus reference culture 544A, brucella melitensis reference culture 16M, standard substance of cloning simultaneously in pig kind brucella reference culture 1330S and checking order after consistent and therefrom taking out, the described nucleotide fragments that contains 272 bases is from kind of dog brucella reference culture RM6/66, cloning and obtain.
7. the using method of PCR test kit as claimed in claim 1, is characterized in that, condition and the step of the method are as follows:
(1) to sample extraction sample DNA to be detected;
(2) take the sample DNA extracted is template, sample DNA, positive quality control standard substance and negative quality control standard product are joined respectively in the pipe that contains PCR reaction solution and archaeal dna polymerase, according to the PCR reaction conditions after optimizing, increase: 95 ℃ of 5min, 94 ℃ of 30sec, 62 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min;
(3) get 5 μ LPCR products, with 1.5% agarose gel electrophoresis, detect, observe the band whether B. abortus amplifies 494bp and 591bp, whether brucella melitensis amplifies the band of 732bp and 591bp, whether pig kind brucella amplifies the band of 272bp and 591bp, and whether the kind of dog brucella amplifies the band of 272bp.
8. the using method of PCR test kit according to claim 7, it is characterized in that, described sample to be detected is milk, adopt liquid nutrient medium increase bacterium beef, adopt liquid nutrient medium increase bacterium mutton, adopt liquid nutrient medium increase the pork of bacterium and adopt liquid nutrient medium to increase the dog meats of bacterium.
9. the using method of PCR test kit according to claim 7, is characterized in that, described PCR reaction solution is by identifying ox kind, sheep kind, pig kind, the brucellar four pairs of primers of kind of dog and containing dNTPs, Mg
2+, distilled water the PCR damping fluid form;
Described four pairs of primers are respectively P
IS711fAnd P
494r, P
IS711fAnd P
732r, P
591fAnd P
591r, P
272fAnd P
272r:
Forward primer P
IS711f: 5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ',
Reverse primer P
494r: 5 '-GACGAACGGAATTTTTCCAATCCC-3 ',
Reverse primer P
732r: 5 '-AAATCGCGTCCTTGCTGGTCTGA-3 ',
Forward primer P
591f: 5 '-CTGGTGGGTATCGCATTACTCGG-3 ',
Reverse primer P
591r: 5 '-TTCAGGAAAGCCTGGCGGTACTG-3 ',
Forward primer P
272f: 5 '-GGAACACTACGCCACCTTGT-3 ',
Reverse primer P
272r: 5 '-GATGGAGCAAACGCTGAAG-3 '.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570780A (en) * | 2009-03-20 | 2009-11-04 | 郑秋月 | Detection kit and detection method for brucellae in meat products |
| CN101659995A (en) * | 2009-06-24 | 2010-03-03 | 吉林大学 | PCR method for quickly detecting brucella in milk sample |
-
2013
- 2013-08-06 CN CN201310339187.3A patent/CN103409520B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570780A (en) * | 2009-03-20 | 2009-11-04 | 郑秋月 | Detection kit and detection method for brucellae in meat products |
| CN101659995A (en) * | 2009-06-24 | 2010-03-03 | 吉林大学 | PCR method for quickly detecting brucella in milk sample |
Non-Patent Citations (3)
| Title |
|---|
| LUISA F O等: "Identifcation of Brucella canis Group 2 in colombian kennels", 《REVISTA COLOMBIANA DE CIENCIAS PECUARIAS》 * |
| R MIRNEJAD等: "Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR", 《ASIAN PACIFIC JOURNAL OF TROPICAL MEDICINE》 * |
| 李光辉: "布鲁氏菌荧光定量PCR快速检测方法的建立及检测试剂盒的组装", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
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